Upon reading chapter 7 of the main text book on “Quality Control”, please POST your answers for the following questions in this Discussion Forum.

Your firm, RPX Pharma, has successfully tested a recombinant protein candidate for bone growth indications in clinical trials.  The FDA will send a team to your manufacturing site for pre-market inspection.  As the head in QC, you will work with your team members to prepare for the site visit.  Please address the following questions, so that the inspection will be successful.

1) Describe quality control test schemes that have been used to produce bio-pharmaceuticals of a similar molecular nature.  Identify regulatory guidelines which apply to the QC of such products.

2) Define and justify the attributes of final product as they are based on its known nature and the intended manufacturing scheme for final product.  Once the attributes are listed, draft a Certificate of Analysis, adding analytical methods and specifications to the proposed attributes and tests.

3) Describe in-process samples that will be taken during the manufacturing process, and identify attributes, tests, and possible specifications for each sample.

4) Describe what is known about the specificity, accuracy, precision, range, and robustness of two of the QC assays to be used.  Please justify the need to develop the assays and the control reagents or reference standards for the assays.

5) Identify which analytical tests will be qualified, verified, and validated, and describe the most likely point in the development cycle of each activity. 

Principles and Practices
BIOTECHNOLOGY
OPERATIONS
S E C O N D E D I T I O N

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Principles and Practices
BIOTECHNOLOGY
OPERATIONS
S E C O N D E D I T I O N
John M. Centanni
Michael J. Roy

CRC Press
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Library of Congress Cataloging‑in‑Publication Data
Names: Roy, Michael Joseph, author. | Centanni, John M., author.
Title: Biotechnology operations : principles and practices / John M. Centanni
and Michael J. Roy.
Description: Second edition. | Boca Raton : CRC Press/Taylor & Francis, 2017.
| Michael J. Roy’s name appears first in the previous edition. | Includes
bibliographical references and index.
Identifiers: LCCN 2016034538 | ISBN 9781498758796 (hardback : alk. paper)
Subjects: | MESH: Biotechnology–organization & administration | Biomedical
Technology–organization & administration | Program Development–methods |
Total Quality Management–methods | Planning Techniques
Classification: LCC TP248.2 | NLM W 82 | DDC 660.6–dc23
LC record available at https://lccn.loc.gov/2016034538
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Although our passion is in the expeditious development of biomedical products, it
is also important to recognize the selflessness of research volunteers and patients
who have the compassion and strength to participate in human clinical research
studies for the betterment of others and in the hope of advancing medicine;
without this, the development of new treatments would not be possible.

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vii
Contents
Preface ………………………………………………………………………………………………………xv
Acknowledgments ………………………………………………………………………………… xvii
Authors ………………………………………………………………………………………………….. xix
1. Introduction to Biotechnology Operations: Planning for Success ……. 1
Biotechnology Operations …………………………………………………………………… 1
Marketing, Financial, and Business Considerations for Development …..4
Product Development Planning ………………………………………………………….. 7
Rationale for Product Development Planning …………………………………. 7
The Targeted Product Profile ………………………………………………………… 10
The Product Development Plan …………………………………………………….. 16
Clinical Development Planning ………………………………………………… 18
Project Management Planning ………………………………………………….. 19
Regulatory Planning …………………………………………………………………. 20
Nonclinical Planning ………………………………………………………………… 22
Biomanufacturing Planning ……………………………………………………… 23
Quality Control Planning …………………………………………………………. 24
Quality Systems and Quality Assurance Planning …………………… 26
Additional Elements of Product Planning ………………………………… 26
Summary of Planning for Success …………………………………………………….. 28
2. Project Management ………………………………………………………………………… 29
Biotechnology and Project Management……………………………………………. 29
Background of Project Management ………………………………………………….. 31
Project Management Plan ………………………………………………………………….. 32
The Project Management Environment …………………………………………. 34
Project Objectives and Schedules ………………………………………………….. 36
Sociotechnical Considerations ………………………………………………………. 37
Participants in Project Management ……………………………………………… 37
Project Management in Biotechnology Operations ……………………………. 41
Establishing Project Management …………………………………………………. 41
The Work Breakdown Structure ……………………………………………………. 42
Forming a Project Team and Hands-on Project Management ……….. 46
Team Dynamics …………………………………………………………………………….. 46
Communication and Feedback ……………………………………………………… 49
Project Risk Assessment and Management …………………………………… 51
Metrics and Tracking Progress ……………………………………………………… 53
Resources: Planning and Usage …………………………………………………….. 54
Human Factors in Project Management ………………………………………… 55
Project Completion………………………………………………………………………… 57

viii Contents
Project Management with Contracts and Collaborations …………………… 59
Virtual Teams ……………………………………………………………………………………. 60
Tools for Effective Project Management …………………………………………….. 61
Summary of Project Management in Biotechnology Development ……. 64
3. Regulatory Affairs …………………………………………………………………………… 65
The U.S. Food and Drug Administration: Law and Regulations for
Biopharmaceuticals …………………………………………………………………………… 65
Historical Basis for FDA Regulation ……………………………………………… 65
Regulatory Organization of the FDA …………………………………………….. 66
Food and Drug Law, Regulation, and Guidance ……………………………. 71
FDA-Regulated Products …………………………………………………………………… 71
Biologics ………………………………………………………………………………………… 72
Drugs ……………………………………………………………………………………………. 75
Medical Devices ……………………………………………………………………………. 76
Combination Products…………………………………………………………………… 77
Other Classes of Biotechnology Products and Their Review at
the FDA …………………………………………………………………………………………. 79
Products for Veterinary Use ……………………………………………………… 79
Cosmetics, Food, Dietary Supplements, Homeopathic, or
Nutritional Products …………………………………………………………………. 79
FDA Regulatory Information and Resources: Regulatory Intelligence ….. 81
Regulatory Operations for FDA Applications ……………………………………. 84
Regulatory Planning and the Regulatory Environment ………………… 84
Risk Versus Benefit………………………………………………………………………… 84
Applications Seeking FDA Investigational Use or Marketing
Approval ……………………………………………………………………………………….. 87
Investigational Use Applications. The Investigational New
Drug Application ………………………………………………………………………….. 89
Common Technical Document ………………………………………………….. 91
Electronic Submission of a CTD ………………………………………………… 92
Marketing Applications: BLA and NDA ……………………………………….. 96
Medical Device Applications. 510(k) and PMA ………………………………….. 99
Special Documents, Pathways, or Exemptions …………………………….. 101
Generic Drugs and Biosimiliar or Follow-on Biologics ………………… 104
Other Regulatory Activities …………………………………………………………….. 105
Public Meetings and Advisory Committees ………………………………… 105
Postmarketing Requirements and Activities ……………………………….. 107
Advertising and Promotion …………………………………………………………. 108
Summary of Regulatory Affair Activities in Biotechnology
Operations ……………………………………………………………………………………….. 109
References ……………………………………………………………………………………….. 111

ixContents
4. Regulatory Compliance ………………………………………………………………….. 113
Regulatory Compliance …………………………………………………………………… 113
Quality Systems to Meet Regulatory Compliance ……………………………. 113
Compliance and Quality Systems ……………………………………………….. 113
Current Good Manufacturing Practices for Manufacture and
Quality Control …………………………………………………………………………… 114
Current Good Laboratory Practices for Nonclinical Laboratory
Studies ………………………………………………………………………………………… 117
Current Good Clinical Practices for Clinical Studies …………………… 117
Compliance for Biopharmaceuticals: Other Regulations of
Importance ………………………………………………………………………………………. 119
Compliance for Import of Biopharmaceuticals into the
United States ……………………………………………………………………………….. 119
Compliance for Medical Devices …………………………………………………. 120
Inspection and Enforcement ……………………………………………………………. 120
Inspections ………………………………………………………………………………….. 121
Enforcement Actions …………………………………………………………………… 123
Product Liability………………………………………………………………………….. 125
Compliance with Non-FDA Regulations: International, National,
State, and Local ……………………………………………………………………………….. 126
International and Foreign National Regulatory Authorities for
Medical Biotechnology Products …………………………………………………. 126
Transporting Infectious or Otherwise Hazardous Materials……….. 132
Importing, Possessing, or Transferring Controlled
Biotechnology Materials ……………………………………………………………… 134
The Public Health Security and Bioterrorism Preparedness and
Response Act of 2002 …………………………………………………………………… 136
Importation or Exportation of Biotechnology Products for the
Purpose of Treatment of Diseases in Humans …………………………….. 137
Occupational Health and Safety ………………………………………………….. 140
Environmental Regulations in Biotechnology ……………………………… 141
Genetically Modified Organisms or Molecules ……………………………. 142
International Diligence in Biotechnology Operations ………………….. 144
Summary of Regulatory Compliance ………………………………………………. 146
Summary of Non-FDA Compliance …………………………………………….. 147
References ……………………………………………………………………………………….. 148
5. Quality Systems ……………………………………………………………………………… 149
Overview of Quality in Biotechnology…………………………………………….. 149
History: Evolution of Quality Concepts and Practices ……………………… 150
Quality Systems Approach to Product Development ………………………. 153
Planning a Quality System ………………………………………………………………. 156
Defining Objectives and Ensuring Management Support……………. 156
The Quality Manual ……………………………………………………………………. 156
The Quality Plan …………………………………………………………………………. 157

x Contents
Hallmarks of Quality: Fundamental Criteria for Building Effective
Quality Systems ………………………………………………………………………………. 159
Management Responsibility ………………………………………………………… 160
Defined Quality System ………………………………………………………………. 162
QbD and Design Control …………………………………………………………….. 163
Quality by Design …………………………………………………………………… 163
Design Control ………………………………………………………………………… 164
Design Change ……………………………………………………………………….. 169
Contractor, Vendor, and Consultant Control ……………………………….. 169
Product Identification and Traceability ……………………………………….. 171
Process Control ……………………………………………………………………………. 172
Environmental Controls ………………………………………………………………. 173
Inspection or Testing (Quality Control) ……………………………………….. 173
Release of Material, Service, or Product ………………………………………. 174
Change Control and Corrective or Preventive Actions ………………… 175
Packaging and Labeling ………………………………………………………………. 176
Preservation, Storage, and Handling …………………………………………… 176
Servicing ……………………………………………………………………………………… 178
Customer Concerns and Adverse Event Reports …………………………. 178
Document Control ………………………………………………………………………. 178
Training ………………………………………………………………………………………. 178
Auditing ……………………………………………………………………………………… 179
The Quality Assurance Unit ……………………………………………………………. 180
Manage the Quality Assurance Function…………………………………….. 181
Control Documents and Manage the Documentation System ……… 182
Investigate Situations: Manage and Control Change …………………… 184
Ensure Qualified and Trained Staff …………………………………………….. 184
Perform Audits ……………………………………………………………………………. 185
Initiate a Quality System for a Biotechnology Operation …………………. 188
Unique and Effective Approaches to Quality Management …………….. 190
Risk-Based Approaches to Quality Systems ………………………………… 190
Total Quality Management …………………………………………………………. 190
Six Sigma …………………………………………………………………………………….. 191
Statistics in Quality Assurance ……………………………………………………. 191
Quality Systems for Research ……………………………………………………… 191
Resolving Quality Issues or Problems …………………………………………. 192
Summary of Quality Systems ………………………………………………………….. 193
References ……………………………………………………………………………………….. 194
6. Biomanufacture ………………………………………………………………………………. 195
Overview of Biomanufacturing Requirements ………………………………… 195
Design in Biomanufacture ……………………………………………………………….. 196
Technical Considerations for Biomanufacture …………………………………. 200
Phases and Scale-up: The Biomanufacturing Life Cycle ………………….. 201
Raw Material Considerations …………………………………………………………… 205

xiContents
Compliance and Quality in Biomanufacture: Current Good
Manufacturing Practices ………………………………………………………………….. 207
Biomanufacturing Processes for Biotechnology Products ………………… 209
Expression of Recombinant Proteins and Nucleic Acids ……………… 209
Production of Recombinant Molecules from Expression
Vectors …………………………………………………………………………………….. 209
Genes, Vectors, and Host Cells ………………………………………………… 210
Bacterial Cell Expression Systems …………………………………………… 212
Yeast Cell Expression Systems ………………………………………………… 213
Mammalian or Insect Cell Expression Systems ………………………. 213
Production of Master Cell Banks and Working Cell Banks ……… 216
Biomanufacture of Recombinant Proteins …………………………………… 217
Planning Production of a Recombinant Protein ………………………. 217
Upstream Process: Production by Bacterial or Yeast Cell
Fermentation …………………………………………………………………………… 218
Upstream Process: Production by Mammalian or Insect
Cell Culture …………………………………………………………………………….. 220
Upstream Process: Recovery …………………………………………………… 221
Downstream Process: Purification ………………………………………….. 222
In-Process Testing and Analysis of Bulk Substance …………………….. 230
Production of Bacterial Plasmid DNA …………………………………………. 231
Production of Live Recombinant Organisms: Bacteria and Virus … 232
Production of Products Composed of Mammalian Somatic
Cells or Tissues ……………………………………………………………………………. 234
Production of Cellular Products Derived from Pluripotent
(Stem) Cells………………………………………………………………………………….. 236
Production of Biological Molecules by Transgenic Animals or
Plants …………………………………………………………………………………………… 238
Production of Biologically Active Lipids, Glycolipids, and
Complex Carbohydrates ………………………………………………………………. 245
Production of Biologically Active Peptides ………………………………….. 245
Production of Combination Products: Biopharmaceutical with
a Drug or Medical Device ……………………………………………………………. 247
FP: Formulation, Fill, Finish, and Labeling ………………………………………. 248
Biomanufacturing Facilities, Utilities, and Equipment …………………….. 253
Facility Design Considerations ……………………………………………………. 253
The Facility and Utilities: A Controlled Environment …………………. 254
Operation of Clean Work Areas for Biomanufacture …………………… 255
Biomanufacturing Equipment …………………………………………………….. 257
Contract Manufacturing Options …………………………………………………….. 257
Validation of Biomanufacturing Facilities, Utilities, Equipment,
and Processes ………………………………………………………………………………….. 259
Summary of Biomanufacture …………………………………………………………… 261
References ……………………………………………………………………………………….. 262

xii Contents
7. Quality Control ………………………………………………………………………………. 263
Quality Control Overview ……………………………………………………………….. 263
Definition of Product Attributes ………………………………………………….. 265
Analytical Methods to Measure Attributes ……………………………… 266
Traits of Analytical Methods …………………………………………………… 267
Drafting a Certificate of Analysis (Bulk Substance) …………………….. 267
Selection of Analytical Methods ………………………………………………….. 270
Development of Specifications …………………………………………………….. 277
Entering Test Results …………………………………………………………………… 282
Certificate of Analysis for Drug Product ………………………………………….. 282
In-Process Testing ……………………………………………………………………………. 285
Analytical Methods …………………………………………………………………………. 286
Additional Analytical Tools and Concepts ………………………………………. 295
Quality Control of Cell Banks ………………………………………………………….. 297
Samples and Sampling …………………………………………………………………….. 298
Analytical Controls and Reference Standards …………………………………. 299
Test Failures, Out-of-Specification Results, and Retesting ………………… 300
Testing for Product Stability …………………………………………………………….. 302
Quality Control Testing of Raw Materials ……………………………………….. 308
Quality Control and the Manufacturing Environment ……………………. 310
Qualification, Validation, and Verification of Analytical Methods …… 312
Application of Statistics in Assay Performance and Validation………… 317
Summary of Quality Control …………………………………………………………… 318
References ……………………………………………………………………………………….. 319
8. Nonclinical Studies ………………………………………………………………………… 321
Nonclinical Studies and Risk Assessment ……………………………………….. 321
Biopharmaceutical Delivery, Pharmacokinetics, and
Pharmacodynamics …………………………………………………………………………. 323
Product Delivery to the Body ………………………………………………………. 323
Adsorption, Distribution, Elimination, and Metabolism (ADME) .. 325
Absorption………………………………………………………………………………. 325
Distribution …………………………………………………………………………….. 325
Metabolism and Biotransformation …………………………………………. 327
Excretion …………………………………………………………………………………. 328
Pharmacokinetics and Pharmacodynamics …………………………………. 328
Application of Pharmacokinetics and Pharmacodynamics in
Biopharmaceutical Development …………………………………………………. 333
Safety Assessment of Biopharmaceuticals ……………………………………….. 336
Toxicology …………………………………………………………………………………… 336
Design of a Safety Assessment Program ……………………………………… 337
In Vitro Screens: Surrogate Measures of Toxicity ………………………… 340

xiiiContents
In Vivo Safety Testing of Biopharmaceuticals ……………………………… 342
Animal Model Development …………………………………………………… 342
Test Product Formulations, Routes of Delivery, and Dosing
Designs …………………………………………………………………………………… 344
Protocols and Performance of Biopharmaceutical Safety Studies
in Animals …………………………………………………………………………………… 346
Elements of a Nonclinical Study Design ……………………………………… 347
Nonclinical Safety Testing …………………………………………………………… 351
Acute Toxicity Testing …………………………………………………………………. 351
Subchronic and Chronic Toxicity Testing ……………………………………. 356
Reproductive, Developmental, and Teratogenicity Toxicity
Testing …………………………………………………………………………………………. 359
Carcinogenicity Testing ………………………………………………………………. 360
Immunotoxicology ………………………………………………………………………. 361
Genetic Toxicology ………………………………………………………………………. 363
Tissue Binding or Local Tissue Tolerance ……………………………………. 367
Quality of Nonclinical Studies: Current Good Laboratory Practices ….. 368
Summary of Nonclinical Studies …………………………………………………….. 369
References ……………………………………………………………………………………….. 370
9. Clinical Trials …………………………………………………………………………………. 371
Introduction to Clinical Trials………………………………………………………….. 371
Background of Clinical Research …………………………………………………….. 373
Introduction ………………………………………………………………………………… 373
Historical Information on Clinical Trials …………………………………….. 374
Organization of Clinical Research …………………………………………………… 375
Phases of Clinical Trials ………………………………………………………………. 375
The Science of Clinical Research …………………………………………………. 376
Quality in Clinical Research and Current Good Clinical Practices …… 377
Clinical Development Planning …………………………………………………… 377
Infrastructure for a Clinical Trial: Individuals, Documents, and
Investigational Product ……………………………………………………………………. 378
Design of Clinical Trials and the Clinical Protocol ………………………. 378
Human Subjects, Patients, and Volunteers …………………………………… 388
The Sponsor ………………………………………………………………………………… 388
The Principal Investigator and His or Her Study Staff ………………… 391
Institutional Review Boards, the Process of IC, and IC Form ………. 392
Investigational Product ……………………………………………………………….. 394
Collection of Clinical Data: Case Report Forms and the Patient
Diary …………………………………………………………………………………………… 395
Clinical Testing Laboratories ……………………………………………………….. 396
Reporting Results of Clinical Trials: Clinical Summary Reports …. 397

xiv Contents
Clinical Trial Operations …………………………………………………………………. 397
Activities Leading to a Clinical Trial …………………………………………… 398
Phase 1 Clinical Trial: First-In-Human Study ………………………………. 400
Clinical Pharmacology Studies of Biopharmaceuticals
in Human………………………………………………………………………………..404
Phase 2 Clinical Trial: Proof-of-Concept Study ……………………………. 405
Phase 3 Clinical Trial: Therapeutic Confirmatory ……………………….. 406
Phase 4 Clinical Study and Risk Evaluation and Mitigation
Strategy ……………………………………………………………………………………….. 407
Clinical Trials for New Populations or Indications ……………………… 408
Global Clinical Trials …………………………………………………………………… 409
Quality Systems for Clinical Trials: Current Good Clinical
Practices ………………………………………………………………………………………. 409
Quality and cGCP in Clinical Trial Operations …………………………… 410
Integrity of Clinical Study Data and Documents …………………………. 413
Monitoring and Auditing Clinical Trials …………………………………….. 414
Ethical Behavior and the Well-Being of Clinical Trial Subjects ……. 415
Summary on Clinical Trials …………………………………………………………….. 416
References ……………………………………………………………………………………….. 417
Additional Readings …………………………………………………………………………….. 419
Glossary ………………………………………………………………………………………………… 425
Appendix ………………………………………………………………………………………………. 451
Index ……………………………………………………………………………………………………… 459

xv
Preface
This book resulted from the authors’ experiences gained while working
in biotechnology development at industry, government, and academia,
and while teaching a graduate course titled biotechnology operations.
This course is offered to graduate students in the master of science (MS)
in Biotechnology Program at the University of Wisconsin-Madison (http://
www.ms-biotech.wisc.edu/). In this course, we examine the undertaking of
developing biotechnology products, focusing on the scientific and manage-
ment skills of biomanufacturing, clinical trials, nonclinical studies, project
management, quality assurance, quality control, and regulatory affairs. The
course emphasizes both operational planning for success and integration
of plans and efforts in these seven functional areas. The instructors real-
ized from their experience in the biotechnology industry the great need to
carefully plan and fully integrate biotechnology development projects. The
course is taught in that manner and this book reflects that philosophy; thus,
this book is a practical guide for students and for those employed or inter-
ested in biotechnology.
This book is intended to meet a need and to fill a gap. Despite the wealth
of experience with operations in the biotechnology industry, there was no
single comprehensive and practical, yet fundamental, guide available. Many
books and most individual scientific or trade publications are highly techni-
cal and focused on a specific aspect of biotechnology. They do not empha-
size the themes of planning and integrating the seven operational endeavors.
Biotechnology Operations: Principles and Practices is written with the objec-
tive of presenting a roadmap and reference for biotechnology operations,
integrating these functional areas through the processes of product plan-
ning and design, and the practice of project management. It applies lessons
learned in the biotechnology industry over past decades as novel products
have been developed from emerging scientific discoveries. The lessons high-
light development principles that could help the industry to bring to market
more efficiently and quickly the safe and effective biotechnology products
of the future. While focused largely on biopharmaceuticals, this book also
reflects development of other biotechnology products. It is anticipated that
this book will provide the reader a clear understanding of basic principles
and practices, and assist in reducing risks and in resolving problems as
future biotechnology discoveries are developed into products.
In preparation of the 2nd edition of this book, and at the request of the
readers, we have enhanced our use of examples by including additional text
boxes, diagrams, and figures. This 2nd edition now includes up-to-date meth-
odologies associated with current biotechnology industry practices; incor-
porated are examples of tissue engineering, stem cell technologies, and the

homepage

homepage

xvi Preface
use of alternative bioreactors. Chapter 2 now includes additional schematics
to better depict abstract concepts. Chapter 3 is expanded to include current
thinking of the FDA on various topics, and now includes specific infor-
mation on submission formats and processes such as Common Technical
Document format and electronic submissions. Chapters 2, 5, and 6 contain
additional illustrations and examples of design and change control, man-
agement responsibilities, quality audit process, biomanufacturing facilities,
whole animal bioreactors, and stem cell manufacturing processes. Chapter 7
is updated and includes depictions of testing equipment, figures outlining
new concepts, and examples of trending and trend analysis. Chapter 8 now
includes specific study design examples that have been used successfully to
support translation of new biopharmaceutical products into human clinical
trials. Finally, Chapter 9 includes an emphasis on the practical use of Good
Clinical Practice (GCP) and how it directly applies to human clinical study
management.
The target audience for this book is advanced undergraduates or postgrad-
uate students pursuing an advanced degree in biotechnology and individu-
als working in any aspect of biotechnology product development. This book
should be particularly relevant to students interested in biotechnology, bio-
pharmaceutical product development, and those already working in biotech-
nology. The information presented in this book can be used to expand upon
one’s current experience while providing an additional level of appreciation
and overview of the product development process. For those in the biotech-
nology industry, this book provides guidance on planning a new develop-
ment program or managing an ongoing program. Noting that irrespective
of the nature of the new biomedical product, the principles and practices
outlined in this book are essential for the success of developing and market-
ing of a new product.

xvii
Acknowledgments
The authors sincerely hope the experiences, ideas, and examples related in
Biotechnology Operations: Principles and Practices will inspire the reader to
plan and implement meaningful strategies and thereby expedite the devel-
opment of desperately needed new medical products. Many of the examples
and suggestions in this book represent challenges and successes that we’ve
experienced throughout our careers. It is our passion to contribute ways that
facilitate the transition of new therapies from the discovery or research envi-
ronment into the clinic.
Special thanks go
• To the many students in the Master of Science in Biotechnology
Program for helpful discussions and feedback on the best way to
present this wealth of information.
• To Eric Schmuck and Derek J. Hei for their assistance with develop-
ing ideas and materials for this 2nd edition.
• To our many colleagues (especially Anthony [Tony] Clemento,
Natalie Betz, and Edmund J. Elder Jr.) who have contributed to dis-
cussions and suggestions that made writing this book possible and
also for their continued support and encouragement.
Our special thanks go to Kurt Zimmerman, program director of the Master of
Science in Biotechnology Program at the University of Wisconsin-Madison,
for his continuing support and for providing a program in which the stu-
dents are trained and encouraged to become industry leaders.

http://taylorandfrancis.com

xix
Authors
John M. Centanni, MS, has a faculty associate appointment in the School of
Medicine and Public Health at the University of Wisconsin-Madison, in the
Master of Science in Biotechnology Program. He has firsthand experience
of leading functional groups in biotechnology firms as a project manager.
His strong scientific background has allowed him to serve multiple scientific
R&D roles in the biotech industry contributing to the development of pre-
clinical safety studies, quality control assays, and animal models. Centanni
has participated in the development and implementation of quality systems
to meet regulatory compliance in both the industry and academic envi-
ronments. He has directly overseen the regulatory and clinical operations
associated with a number of early phase, multicenter, human clinical trials.
Centanni has worked in the pharmaceutical and biotechnology industry
since 1987 and has more than 20 years of product development experience
for drugs, biologics, and devices. He has instructed and trained basic sci-
entists and clinical researchers in regulatory compliance and expectations
associated with clinical product development (Good Laboratory Practice
(GLP), Good Manufacturing Practice (GMP), and Good Tissue Practice
(GTP), and Good Clinical Practice). Centanni is experienced in preclinical
research, regulatory, quality, clinical development, and project management,
and has been involved in the development and registration of pharmaceuti-
cal products across a number of therapeutic categories.
Centanni is the director of the Investigational New Drug (IND)/
Investigational Device Exemption (IDE) Consultation Services, where he
leads a team of consultants at the University of Wisconsin-Madison, Institutes
for Clinical and Translational Research. In this role, he provides campus-
wide support to clinical investigators advancing their investigational prod-
uct from the research environment into the clinic. This support ranges from
strategic support for the selection of viable product development candidates
to characterization of products and design and implementation of human
clinical trials.
Centanni is also an active participant in the Stem Cell and Regenerative
Medicine Center and Cardiovascular Regeneration Focus Group at the
University of Wisconsin. He serves on a number of grant review panels that
include California Institute for Regenerative Medicine (CIRM), Institute for
Clinical and Translational Research (ICTR) Novel Therapeutics Pilot Awards,
and American Burn Association (ABA) Multicenter Clinical Trials Group.
Before joining the University of Wisconsin, Centanni directed the regula-
tory, quality, and clinical efforts of a small biotechnology firm in Madison,
Wisconsin. As an accomplished molecular and cellular biologist, Centanni
has successfully directed multimillion dollar translational and clinical

xx Authors
research projects as principal investigator. Additional professional attributes
of Centanni include a notable patent portfolio as an inventor on more than
a half dozen intellectual property filings and authorship of several scientific
journal articles and book chapters.
Centanni is a graduate of Hood College, Frederick, Maryland, with a mas-
ter’s degree in biomedical sciences supported by a thesis and defense. Prior
to graduate school, Centanni earned a BS in biology at the University of
Wisconsin-Oshkosh, Wisconsin. In his free time, Centanni enjoys saltwater
fishing, snorkeling, traveling, and playing racquet ball.
Michael J. Roy, PhD, is an emeritus professor at the University of
Wisconsin-Madison, where he previously taught in the Master of Science in
Biotechnology Program in the School of Medicine and Public Health. He has
successfully developed biopharmaceutical products and medical devices for
private and publically held firms, nongovernmental organizations, and the
federal government for more than 27  years, serving as a consultant in bio-
technology development over the past decade. Many of his efforts, including
product development planning, regulatory affairs, quality systems, project
management, biomanufacturing, quality control, and clinical studies, focus
on early development of novel biotechnology products, notably vaccines and
antimicrobial agents.
He is a graduate of the University of Wisconsin-Madison with a PhD in
pathology, of Louisiana State University Medical Center, New Orleans, with
an MS in tropical medicine and medical parasitology, and of the University
of Wisconsin-Platteville with a BS in biology. Colonel Roy is retired from
the U.S. Army Reserves, where he was involved in developing in vitro diag-
nostic devices and vaccines and in establishing quality systems at the U.S.
Army Medical Research Institute of Infectious Diseases, Fort Detrick. He
also enjoys hiking, raising hardwood trees in southwestern Wisconsin, and
has archeological interests of that region.

1
1
Introduction to Biotechnology
Operations: Planning for Success
Biotechnology Operations
Biotechnology encompasses a wide variety of scientific, business, and
operational endeavors in life sciences. It is applied across a broad range
of specific disciplines, for example, plant, animal, medical, microbiologi-
cal, biopharmaceutical, agricultural, and environmental to name just a few.
Biotechnology is practiced worldwide and at many institutions: small pri-
vate firms, large public corporations, nonprofit organizations, universities,
and research institutes. Those practicing biotechnology include individuals
with diverse skills and backgrounds: entrepreneurs, scientists, business-
persons, managers, product developers, and other highly educated and
motivated specialists. As seen by the inexperienced and at the macro level,
biotechnology appears to be a vast three-dimensional matrix, broad and
oftentimes baffling in scope and operation. However, to those experienced in
biotechnology, there is organization and rationale. The keys to successfully
managing a biotechnology firm are a focus on carefully crafted plans and
efforts to accomplish a specific objective and to integrate operational activi-
ties within the operational matrix. This is especially true for biotechnology
product development operations, where the objective is to increase the value
of specific products by moving them through sequential phases and to the
marketplace.
Virtually every aspect of biotechnology has two common themes: (1)
to extend  our knowledge of life sciences and (2) to produce a product or
service that someday will improve the condition of humankind. In the
commercial sector of biotechnology, there is also the objective to profit
financially. There are subplots to every biotechnology endeavor as well.
Developing a novel biotechnology product, especially a biopharmaceutical,
is an extremely technical, highly regulated, complex, expensive, and long
process. Biopharmaceuticals are in development for more than 5 years, and
it is not unusual for schedules to extend, from research to market approval,
beyond 10 years. The risks associated with biotechnology are tremendous,

2 Biotechnology Operations
since most biopharmaceuticals fail at some point in development. Yet, there
are compelling reasons to undertake biotechnology product development.
The profits can be substantial, and there are needs and markets for useful
products. For some individuals, it is not financial incentives, but altruistic
purposes or the challenge of pursuing an ambition and lifelong dream.
This provides a stream of bright individuals willing to labor at bringing
biotechnology products to market. So, biotechnology development contin-
ues to grow in importance, size, and scope, and is highly regarded by the
public.
Biotechnology has its own jargon as evidenced by terms used in this book
and other references listed in the Additional Readings, and a great amount
of operational information, notably regulatory, is available at websites and
some of these are identified in this book.
Words, some considered jargon, have developed to describe certain aspects
of the biotechnology operational trades, and these can be confusing, even
counterintuitive, to the uninitiated. The reader may refer to Glossary for
definitions used in this book.
This book focuses on biotechnology product development, specifically the
scientific skills commonly applied worldwide to move in an ordered man-
ner a product from concept at the laboratory bench to the marketplace. It
emphasizes product design, development planning, project management,
and elements of each major operational function applied to the development
process. These combined activities we refer to as biotechnology operations.
The seven major functional areas of biotechnology operations, identified in
Box 1.1, are further described in individual chapters of this book. Additional
functional areas, such as business development and finance, also directly
impact biotechnology operations, and these are recognized because they are
keys to success.
The focal point of a biotechnology operation is the product, and at the
heart of product development are the user and intended use. The opera-
tional team of professionals works together to add value, bring the product
to market, and ultimately to the end user. Hence, a key to building a suc-
cessful biotechnology operation is to maintain this focus on product and its
intended use and the user. In biopharmaceutical development, the intended
use is the product indication, a word that will be used repeatedly in this book.
In medicine, an indication is defined as the reason a product is used to diag-
nose, prevent, or treat a specific disease or condition. An indication also
identifies, to a great extent, the intended user of a biotechnology product.
This is especially true for biopharmaceuticals. In addition to having an indi-
cation, biotechnology development is also based on an understanding of
the molecular or cellular nature of a product and on the product’s safety,
strength, purity, and potency.
Seven major areas of biotechnology operations are listed in Box 1.1. In
addition, there is a need to integrate and coordinate each of these skills in

3Introduction to Biotechnology Operations: Planning for Success
an effective and timely manner, focusing on making operational headway,
and moving the product toward market approval. Given the complex-
ity of biotechnology operations, the need for careful planning is intui-
tive. Planning is an activity that results in a written strategy. Together
they establish the objectives and also map out a means of integrating the
skills and events that lead to success. Indeed, a product development plan
(PDP) allows a development program to be successful. Without a carefully
crafted and functionally integrated plan, biotechnology operations typi-
cally fail.
To begin our journey through biotechnology operations, this chapter intro-
duces the planning process for product development. Think of the plan as a
skeleton and each element of the plan a bone that gives structure to the over-
all operation. Chapters 3 through 9 describe individual functional areas that
execute or flesh out the plan and provide operational activities (Box 1.1). The
functional areas do the heavy lifting, so to speak, in an operation, and six of
them are considered the muscles of an operation. Chapter 2 describes proj-
ect management, the operational function that serves as the neural system to
a biotechnology operation, coordinating movement of operational elements
according to the plan.
BOX 1.1 SEVEN MAJOR AREAS OF BIOTECHNOLOGY
OPERATIONS
Operational Area Definition Chapters
Project
Management
Lead the planning, organization, and management of the
overall development project and associated resources.
2
Regulatory Affairs Advise on regulatory aspects and climate for product
development, coordinate activities with regulatory
agencies, and ensure regulatory compliance.
3 and 4
Quality Assurance Provide support to ensure that all efforts and the product
are of highest quality through quality management,
audits, documentation, and other quality functions.
5
Biomanufacture Produce the highest quality product through phased
manufacturing development and final commercial
production.
6
Quality Control Ensure quality product through laboratory testing. 7
Nonclinical Develop pharmacology and toxicology laboratory and
animal studies and reports to ensure the safe and
proper use of the product.
8
Clinical Determine the safety and effectiveness of the product
when used to treat human subjects.
9

4 Biotechnology Operations
Marketing, Financial, and Business Considerations
for Development
Biotechnology products in general and biopharmaceutical products in particu-
lar, with their stringent regulatory guidelines and strict need for a high benefit-
to-risk ratio, are particularly expensive to develop. So expensive, in fact, that
investment capital and public funding often provide insufficient resources to
support the complete product development cycle. Today, the total development
cycle costs for a biopharmaceutical can reach or exceed one billion U.S. dollars.
Although somewhat less expensive to develop, other types of biotechnology
products, such as those in the agricultural or environmental sectors, might still
cost in excess of one-half billion U.S. dollars. Indeed, some biotechnology firms
never even enter the development arena because of high cost and inability to
raise capital to meet projected expenses.
Biotechnology firms rely on both public or private financing and partner-
ships with traditional pharmaceutical firms to provide capital needed to reach
their development goals. Of course, money always comes with tradeoffs and
an investor or partner may hold definite ideas and opinions regarding how
the biotechnology firm should develop the product. In the end, some biotech-
nology firms are acquired by the partner during the development cycle and
well before a product comes to market. Raising capital is not a subject of this
book, but one must consider expenses and budgets during development plan-
ning and again at every milestone.
Once a project has begun, financing and budgets continue to have an impact
on decisions made both in planning and in executing a project. Indeed, they are
often the primary consideration regarding a decision on whether or not to con-
tinue a product development project. There are tradeoffs for the biotechnology
firm. Development of a specific product may necessitate the sacrifice of other
endeavors, such as pursuing promising lines of research. The philosophy of a
company may have to be changed to pursue development, with hiring of devel-
opment staff offset by the loss of research scientists. Facilities inevitably must
be added or modified to suit development efforts and, as noted later, this can
be extremely resource intensive. Once these resources have been committed,
there is no turning back without incurring significant loss of time and money.
No wonder biotechnology executives typically refer to the decision to embark
on development as betting the farm or entering the valley of death.
Given all these warnings, what is the prudent way for a biotechnology firm
to enter product development? The answer is simple: one step at a time, with
a market analysis, a carefully defined product and indication and a well-
considered PDP.
Earlier in this chapter, a metaphor—skeletal, muscular, and neural
systems—was used to introduce the concepts of biotechnology development
plans, operational elements, and integration by project management, respec-
tively. This metaphor is further explained and developed in Box 1.2. Further

5Introduction to Biotechnology Operations: Planning for Success
BOX 1.2 A BIOLOGICAL METAPHOR FOR
PLANNING BIOTECHNOLOGY OPERATIONS
A metaphor to planning a biotechnology operation is taken from the
organized development of the mammalian neural, muscular, and skel-
etal systems. This metaphor seems relevant, given the biological nature
of our professional work.
An organism is composed of individual organs and tissues, and as
they develop and function they work together in harmony and allow
the animal to function and survive. The skeletal, muscular, and neu-
ral tissues provide functions, respectively of support, movement, and
perception of or reaction to stimuli. Each tissue arises in an exact man-
ner, shaped according to a plan programmed in the genetic code. The
developing skeletal system is composed of bones, logically arranged
and able to provide the outline of a unique organism.
To begin the metaphor a biotechnology operation functions, or
should function, in the manner of a healthy organism, with the indi-
vidual organs and tissues coordinated and working in harmony. An
operational plan, the PDP, is the skeleton of that operation. It provides
shape to the overall project. Although the individual bones of an ani-
mal form a strong framework, they must move in an integrated and
coordinated manner. For this to happen in an organism, muscle is the
organ system holding bones in a particular manner, yet moving them
so they are useful structural elements. In a like manner, the PDP is moti-
vated by the various functional areas of biotechnology development—
clinical, manufacture, nonclinical, quality assurance, quality control,
and regulatory affairs—that implement the plan, providing outcomes,
yet allowing movement and flexibility of operation.
Returning to the organism in this metaphor, a neural system signals
the bones and muscles to work together in a timely and effective man-
ner. The neural system makes the bones and muscles useful to the body
by coordinating endeavors, both as affecters and effectors. Thus the
bones and muscles achieve specific objectives. In biotechnology prod-
uct development, the neural system is represented by project manage-
ment, a key function that ensures the various elements work together in
harmony, sensing the operating environment, and reacting accordingly.
Perhaps the most important part of this metaphor is to imagine an
organism deficient in one of these three elements: skeleton, muscle, or
neural. Indeed, there are diseases for which this is the case. The result
is illness and eventual death. Here the metaphor carries to the biotech-
nology operation, because without each of the functional elements, a
PDP to bring them all together, and a system to integrate and manage
(Continued)

6 Biotechnology Operations
to this metaphor, consider that these three organ systems would not function
properly in any animal without support provided by other organs: the heart,
liver, and kidneys for example. So, it is in biotechnology development, where
support from research, marketing, business development, management, and
other areas is essential to the life of the operation. An important element of
any good development program is the need to consider the advice, exper-
tise, and support of individuals with skills that do not apply directly to the
technical agenda of an operation but have great impact nonetheless. We have
mentioned financing and now consider input from the business and market-
ing professionals. While these professionals might seem at times to perceive
situations and issues differently from operational staff, their skills and judg-
ment are indeed important throughout the development process and their
input is especially critical to success at the planning stage.
They sit on the product development team, advising and planning from
business, finance, and marketing standpoints. The product development
team members, often referred to as a project team, should pose to them criti-
cal questions from the outset of the planning process. Is there a market for
the biotechnology product as it is currently designed and, if so, is the market
large and extensive enough to generate a profit and is it open to introduc-
tion of this new or improved product? Or should another product design
be chosen? Is there competition and, if so, is it prohibitive to the intended
market? Will it be necessary for the firm to develop or further develop the
market and, if so, how long might this take? Are there advantages and dis-
advantages to the market due to regulatory pressure, not just the U.S. Food
and Drug Administration (FDA) but any regulatory agencies? How might
we price this product in the current market? Here, the business and finance
elements of the entity become especially important, and a well-considered
business plan provides valuable information for development planning pur-
poses. At this time, it may be difficult to exactly identify business advan-
tages of a particular product, but certain elements can be considered. At a
strategic level, several questions are posed. In theory, is money available to
develop products in this market sector and, if so, is there precedent? What
are the potential sources of funding and are partnerships with larger firms
possible? Alternatively, might competitors try to impede our progress in an
effort to retain their market share? At this early stage of predevelopment, it is
BOX 1.2 (Continued) A BIOLOGICAL METAPHOR FOR
PLANNING BIOTECHNOLOGY OPERATIONS
their operation, the product development program does not function
properly and eventually does not survive. Alternatively, if skeletal,
muscle, and neural systems are healthy and carry their weight, then
the organism, and by analogy the biotechnology operation, prospers.

7Introduction to Biotechnology Operations: Planning for Success
be impossible for even the most seasoned business experts to have all the
answers; indeed, meetings at this time may generate more questions than
answers. Yet, such discussions are critical to the planning process.
Product Development Planning
Rationale for Product Development Planning
Biotechnology operations have borrowed many concepts and operating prin-
ciples from the drug industry. Indeed, both drug and biopharmaceutical devel-
opment projects often focus on preventive and therapeutic biopharmaceuticals
intended for use in humans. Drug development, a phased or step-wise process
well established by the drug industry and regulatory authorities, is commonly
applied to biopharmaceutical development. Figure 1.1 outlines functional ele-
ments involved in a phased scheme for a biopharmaceutical or drug develop-
ment project and the approximate schedule for each. It represents a project
beginning with discovery or engineering of a novel biologic and ending with a
product entering the marketplace. It is an idealized and simplified cartoon, but,
in reality, the process is much more complex than depicted and may be abbre-
viate or lengthened. Nonetheless, such schemes are developed and applied as
planning and operational management tools, thus providing visual representa-
tion of the major events, processes and milestones, and facilitating communica-
tion and understanding by project teams and upper management.
In Figure 1.1 the stages are defined as: Stage I—Research, the molecular or
cellular entity is discovered, isolated, engineered, and characterized. This ini-
tial stage begins with the discovery of a novel product. The research labora-
tory assists in the characterization and perhaps reengineering or refinement of
the product to meet design criteria. In Stage II—Development, a host of activi-
ties are described throughout this book that led to marketing approval in year
2011. The design of a targeted product profile (TPP) and product development
plan (PDP) are prepared under the guidance of formal project management
(Figure 1.2), which initiates the development pathway. Project management and
quality  assurance are active throughout the development life cycle, whereas
formulation, analytical, pharmacology, biomanufacture, toxicology, clinical,
and regulatory activities are staggered within the development life cycle.
Discovery research is the foundation on which most biotechnology products
are based. Some refer to it as Phase 0 in the development process because dis-
covery must happen before Phase 1 or early development may begin. It is sci-
entists in laboratories who discover, sometimes serendipitously and, in other
instances by plan, the information on which biotechnology product develop-
ment is based. Gene cloning, propagation of stem cells, engineering a drought-
resistant trait into plants, and a monoclonal antibody directed against a tumor
protein are but a few of the thousands of the proven discoveries that have been

8 Biotechnology Operations
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9Introduction to Biotechnology Operations: Planning for Success
the foundations for important and useful products. In most instances, these dis-
coveries are patented, which legally ensures that the discoverer, or the affiliated
institution, receives proper credit for any worthwhile product that might be
developed from their invention. Patents ensure that the patent holder, the dis-
coverer, reaps a monetary reward if the technology is licensed or the product
is marketed. As product development requires substantial resources, typically
tens to hundreds of millions in U.S. dollars, only a few biologic discoveries are
taken through the development life cycle to become a product. Most biotech-
nology products are therefore based on a unique discovery that either has a pat-
ent or is patentable. However, few discoveries or inventions in biotechnology
are themselves marketable products; they must first be developed.
What then can we do with an exciting, patented biological innovation that
holds potential value to humankind and in the marketplace? What must we
do to develop that product? First, we carefully and exactly define the prod-
uct. Although this may sound simple, reaching a definition is no easy task
and, unfortunately, many discoveries enter product development without an
exact definition of what the intended product is or what is expected of it. In
such cases, the PDP, and hence product development operations are unfo-
cused, wasteful, and far too often unsuccessful. A biotechnology product,
and product develop pathway, must be planned, it cannot simply evolve.
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FIGURE 1.2
Planning backward: Targeted product development. A product development plan (PDP) is
written in a reverse manner (arrow on the left) from what one might imagine. Working from
the targeted product profile (TPP) (lower left), label claims are the first step in the planning
process. Then, working backward, or from lower left to upper left in the figure, the planner
develops a PDP, planning each part of the project, listed in the middle of the figure. The plan
is then implemented (shown on the right side, reading top to bottom) in a forward manner,
through each stage of development (provided in Figure 1.1) to produce a final product with
approved label claims. (Courtesy of Anthony Clemento, 2008.)

10 Biotechnology Operations
There is not enough time and money to take any product development route
other than that of a well-considered PDP.
How does planning work? Let us consider another metaphor to explore tar-
geted product planning. Suppose we had the power to design and then develop
a new species of mammal. Our first step would be to define, in various ways, the
purpose of this desired mammal. Specifically, we begin by asking how it would
meet needs of the user. Let’s say that we wish our new animal to pick fruit from
trees in orchards. Then our design, based on this user need, would be bipedal
and tall, with long arms and dexterous fingers. It should have the strength to
stand for hours, and muscles that allow it both to stretch and to rapidly pluck
fruits from a tree. It should have intellect: an ability to differentiate oranges from
apples and to discern ripe oranges from immature fruit and for a brain to signal
the muscles and skeleton to pick that fruit. Hence, we have defined a creature
intended to pick fruits from trees. In planning the bone structure of this animal,
would we borrow the design of a dog or a meadow vole? Certainly not, instead
we would shape our plan, the bones if you will, around a bipedal creature, per-
haps a primate. But we would design especially long bones in the arms and
legs, a vertical or erect and strong vertebral column and lengthy arm bones with
many digits. Indeed, we might include bones for four arms, one to grasp the tree
branch, one to pluck the fruit, one to catch the fruit and yet another to transfer it to
a basket. Hence, our bone structure forms a framework for the intended creature.
The plan for the muscular system would make these bones useful to the
creature’s intended purpose. Would we link these bones with muscles that
allow our creature to run fast, like a cheetah? Probably not; we would instead
give it muscles that allow those bones to stand all day, to continually grasp
limbs, and to carefully pick, grasp, and transfer fruits.
We would plan a neural system that coordinates these musculoskeletal func-
tions, one that achieves the primary objective of picking fruits, but also allows
the grasp to rapidly change as the creature reaches for a new branch, to pluck
the fruit with one arm even as the grasp is changed with another arm and the
fruit is transferred with a third arm, and to discriminate a ripe from unripe fruit,
immediately before it is plucked.
Biotechnology product development, to be successful, follows a specific plan-
ning process in much the same way as we designed the fruit-picking creature.
However, in biotechnology, the long process of discovery research and economic
realities do not allow us the luxury of millennia, the time needed for evolutionary
processes in nature. In biotechnology operations, products are developed rapidly
and efficiently from innovations. We begin with a clear understanding of what
the product is and what it must do and how the product will be used. This is writ-
ten in a targeted product profile (TPP).
The Targeted Product Profile
Product development planning is said to happen in a backward manner
because the process begins with generation of a TPP, which in fact is a draft

11Introduction to Biotechnology Operations: Planning for Success
of the product label with product claims. The planning process is outlined in
Figure 1.2. In biopharmaceutical development, the TPP has in the past been
referred to as draft or concept product labeling. The FDA strongly encour-
ages sponsors, defined as the entity responsible for developing a biopharma-
ceutical, to prepare and use a TPP to support communication with regulatory
authorities. More recently, investors in biotechnology have asked petitioners
to provide them with a TPP along with the business and technical plans.
Simply stated, the TPP is a clear and detailed description of what a product
should be, how it will appear, and, most importantly, what it must do. The
term TPP says it all, establishing a target of or focus on the product and profil-
ing or summarizing characteristics of the product. Box 1.3 lists the elements
of a typical TPP for a biopharmaceutical product. Generation of a TPP is truly
the first step in managed product development. A TPP is written by a lead
author, someone familiar with both the product and with various aspects of
biopharmaceutical development. Teamwork is essential for a successful start,
and this means appointing a product development team and project man-
ager (PM) and holding team meetings at regular intervals (Chapter 2). Team
members review and recommend changes to the draft TPP. Members of the
product development team, each representing a functional area, investors,
and upper management are involved in this process with a final TPP as the
BOX 1.3 ELEMENTS OF A TPP
• Trade name and chemical name: A draft trade name or interim
designation, such as a compound number, is developed for the
product.
• Warnings: Warnings might be added for this product, based on
its class of product and previous experience. Messages to the
prescriber or user for this product are noted.
• Description: The product’s nature and classification are devel-
oped. The formulation in the final container, with excipients,
is included.
• Clinical pharmacology: The mechanism of action, pharmaco-
dynamics, and pharmacokinetics that are known to date or
should be explored are presented. Drug interactions are given.
• Clinical studies: Identified are pivotal clinical studies to include
patient populations, endpoints, and outcomes.
• Indications and usage: The expected indication is given as is the
intended patient or user population.
• Contraindications: Situations in which the product should not
be used (e.g., pregnancy or congestive heart failure) are stated.
(Continued)

12 Biotechnology Operations
team’s first goal. These early interactions set the context and tone for later
discussions (team members bond, agree, or disagree), and leadership skills
become evident. The need for additional professional skills is recognized, and
so teams are filled out to meet management and technical objectives and,
early on, thought is given to the nature, scope, and possible general sched-
ule of the development project. Thus, preparation of a TPP provides a critical
guidance document and solidifies the new product development team.
Of all elements attributed to a TPP, the first, most important and, often
times, the most contentious, is establishing the indication, also referred to
as the label claim or simply the claim (Figure 1.2). Note similarities between
information in a TPP and in an actual product label. Any differences are
largely because of the fact that a TPP is an expectation in nature and tone,
BOX 1.3 (Continued) ELEMENTS OF A TPP
• Warnings and precautions: The users or physicians become aware
of events or reactions to the product and the more serious or
common of these are given in a warning. They are written for
the physician and user, or composed as information for patients,
specifically written for the user. Instructions for special situa-
tions are also placed here, and specific items are highlighted as
paragraph headings. Recommendations may be given to stop
using a product, for example, if a disease progresses or if certain
symptoms are noted. Drug interactions, use in nursing mothers
or in pregnancy, pediatric and geriatric use, or use in other spe-
cial populations are generally included in this section.
• Adverse reactions: Types of adverse reactions that might be
acceptable, given the intended use and user profile, are
identified.
• Overdosage: This describes reactions or remedies, should a
patient take more than the prescribed amount.
• Dosage and administration: This provides a statement on how
the product will be provided as dosage form to a patient in
final format. The intended final container or delivery system
is described.
• How supplied: This describes the packaging format that will be
produced and marketed.
• References: A few key scientific publications regarding the
product and indication are included.
• Patient information: This expands special instructions that
might be required for proper handling, storage, or use by the
patient.

13Introduction to Biotechnology Operations: Planning for Success
whereas the actual labeling of a marketed product is FDA approved, the
real thing, based on data. For a biopharmaceutical, an indication might be
defined as a treatment or prevention for a disease or condition that has a
specific cause and symptom. Let’s begin by demonstrating poorly worded
indications. One, for a peptide therapeutic product, is to lower blood pressure
in benign hypertension. Another vague example of a vaccine composed of a
recombinant protein is to prevent malaria. It is critical that an indication be as
specific as possible and that it be matched with a proper biopharmaceutical.
Here the biotechnology development team must set aside bias and grand or
long-range projections of safety or efficacy (e.g., this biopharmaceutical is so
great it will cure every type of cancer and never result in a side effect) and instead
focus on the research data. The key is to settle on an indication for which
the product would likely reach the market in short order. Having said this
and returning to our examples, the indication for the peptide might be more
rationally stated as lowering blood pressure in individuals with uncomplicated
benign hypertension and between the ages of 60 and 82  years, where blood pres-
sure has remained elevated above 140/90 mm Hg despite the use of other common
drugs and where there are no symptoms of congestive heart failure. The example
of the malaria vaccine might better be stated as indicated for use in infants and
children between the ages of 6 months and 5 years for prevention of serious disease
and death from falciparum malaria in endemic regions of Africa, Asia, and South
America. The TPP also makes claims for safety parameters, and so it is impor-
tant to consider the safety profile that would be acceptable for the product
and include this in the final profile.
A claim is a contention or assertion that something will be achieved.
Biotechnology products are all accompanied by printed labeling, and it is here
that claims on product safety and efficacy are made. Claims are also reflected
in advertisements and labeling, not just for medicines but for all types of
products. Consumers read these claims (or should read them) when making
purchases and before use. A TPP is draft labeling with predicted claims, and
as such the TPP is used to guide the planning and actual development of all
biotechnology products, not only biopharmaceuticals. Some examples follow.
First, a recombinant bacterium is indicated for remediation of crude oil spills
in salt water where the air and water temperatures are more than 40°F, wave
action is not severe, and the spill is contained to a geographic region of area
less than 100  km2. Or a genetically engineered soybean has the indication to
increase yields by more than 20% in comparison with other varieties when
grown in zones 3 or 4, and where rainfall averages between 12 and 20 inches
per year, there is no irrigation, and the soil is slightly acidic or neutral.
Returning to biopharmaceutical development, a TPP discusses the other
objectives, and these are listed in Box 1.3; note the various claims identi-
fied on the TPP, such as indication and safety profile. It is worth noting that
results from research or early development completed to date, market driv-
ers, and perhaps the experiences and ingenuity of individual team members
are the basis for deciding on each claim. The target population is identified

14 Biotechnology Operations
as part of the claim. For example, a product is to be used only in adults of
more than 50 years of age and in otherwise good health. Often, experts, such
as physicians highly regarded in a specialty area, are consulted before the
team reaches a consensus on a target indication and population.
The next step in developing a TPP for a biopharmaceutical is to determine
the target dosage and route of administration best suited to the product and
the population identified in the indication. The peptide example might be best
administered by the parental route, such as subcutaneous, because peptides
might not be amenable to the hostile environment of the gut. The vaccine
might be preferred as an intramuscular injection from a disposable syringe.
In each example, the dosage may need to be 1 mL or less. Dosage forms and
strength refer to the formulation of the product and how it might be presented
in a marketed or final container, such as a vial or syringe. The peptide might
only be stable in a buffer of low pH. In the case of the malaria vaccine, the
product profile includes a preservative, so it can be used in the tropics. This,
in turn, necessitates a formulation that allows it to be shipped and stored
with breaks in refrigeration. For the microbes used to remediate an oil spill
of more than 100  square miles, it might be necessary to consider a product
that could be disbursed from large mechanical sprayers on aircraft. The seeds
of a drought-resistant soybean plant might need to be planted further apart
from each other, as compared to current soybeans. This type of information is
agreed by the product development team and included in the TPP.
The TPP also considers dosage form and strength. For medical products,
there will be limitations as to the mass of product, peptide, or recombinant
protein that can be held in 1  mL of solution. The optimal formulation, one
that is simplest and least expensive, may not be feasible, and the product team
could decide a special formulation was necessary, for example instructions to
keep a protein product from aggregating and thus preventing loss of activity.
In these examples, the TPP prompts the team to consider manufacture, formu-
lation, and quality control issues and highlights the need for additional steps
in development and, perhaps most importantly, identifies both complexities
and costs of actually developing the individual product.
Contraindication refers to those times when the team recommends that
the product simply should not be used, when it might be unsafe, for exam-
ple. Basic contraindications should be considered, and here again it might be
helpful to consult a physician with experience of treating the disease in the
indicated patient population. Warnings and precautions, on the other hand,
are more difficult to define at this very early stage of development and in the
absence of any safety information on the product. However, warnings and
precautions from products similar in nature, treatment indication, and target
patient populations may be instructive as to what may or may not be accept-
able for this product. The contraindications, warnings, and precautions often
narrow the indication, and this is important information to consider in prod-
uct development. For the peptide used to treat hypertension, it might be con-
traindicated to use the drug in patients with certain other cardiovascular

15Introduction to Biotechnology Operations: Planning for Success
diseases as known from experience in cardiovascular medicine and phar-
macology of similar products. The malaria vaccine might be contraindicated
when the patient was already infected with the parasite. The remedial bacte-
rium might be contraindicated when other petrochemicals, such as gasoline
or diesel fuel, were present. The drought-resistant soybean plant might not
be used within a kilometer of other soybean fields. The main point is that a
knowledgeable product development team confronts these issues during the
process of developing a TPP and well before development begins. This facili-
tates early planning to resolve, if possible, each potential problem or issue.
Identification of undesirable and product-related adverse reactions, risk of
overdose, and interactions of the biopharmaceutical with other drugs are, to a
great extent, items that must be addressed during clinical studies (Chapter 8).
However, it is possible during TPP preparation to consider the limits of
adverse events or precautions the team might allow for a product. With the
peptide antihypertensive, serious illness or death resulting from therapeu-
tic doses, no matter how infrequent, might pass the acceptable threshold for
adverse events. The malaria vaccine for children should not cause local reac-
tions and discomfort that are of great concern to the child or a parent. In the
case of the remedial bacteria or the soybean plant, one might respectively
establish limits regarding how extensively the bacteria could multiply in the
environment in the absence of crude oil or how far the soybean could spread
to neighboring fields. The process does identify, to the development team,
certain limits that might be applied to the development program.
Use in special populations further defines when and how one might use
the product; it extends the indication by considering individuals of certain
age groups, such as adolescents or the elderly, or of physiologic status, such
as nursing mothers or pregnant women. Drug abuse and dependence is typi-
cally not an issue with biopharmaceuticals but can be important with certain
types of drugs.
Adding a description of the product to the TPP would seem, on the surface,
to be a simple task but product development teams often find it to be a chal-
lenge, especially in regard to describing all intended physical, chemical, and
biological characteristics. This is discussed in greater length in Chapter  7.
Any biotechnology product that will be used in man, animals, or the envi-
ronment will need to be very well characterized in all respects but, at this
juncture, product characteristics are unknown. Preparation of the TPP forces
the team to consider what types or classes of characteristics must be exam-
ined for in the product during development. For any of the examples we
have used, biological characteristics should include potential toxicity or half-
life and description of any living cells. Chemical characteristics may include
the molecular nature of a product and also any impurities or contaminants.
Physical characteristics are size or shape or the ability to withstand adverse
conditions of an acceptable molecule or organism.
Clinical pharmacology may also be unknown at this early stage of
development. The term takes into consideration the distribution of the

16 Biotechnology Operations
biopharmaceutical in the body, the kinetics of distribution from the time of
dosing through the time of clearance, and the dynamic properties while it is
in tissues (Chapter 8). But there should be some information, from laboratory
or animal studies, on which the team can develop desirable parameters or
acceptable limits. The antihypertensive peptide should clear itself from the
body before another dose being given, and the malaria vaccine should not
remain in a subcutaneous tissue indefinitely. The remedial bacterium should
be cleared from the environment and not be present long after the crude oil
has been eliminated, and there may be limits on how long the soybeans can
self-reproduce under field conditions.
Nonclinical toxicology testing (Chapter 8) is very important because the tox-
icity profile of a product in animals is often a predictor of toxicity in humans.
Clinical studies follow nonclinical toxicology and, as discussed in Chapter 9,
they are designed with the results of nonclinical studies in mind. The nonclin-
ical and clinical toxicology profiles are certainly unknown at this early stage,
but the development team does have the opportunity and obligation to set lim-
its for safety and efficacy parameters, even if they are general, for each product
in its TPP. Here the history and labels of competitive products or good medical
judgment come into play, along with scientific and medical experience. Would
one consider developing and marketing the example peptide antihypertensive
if it consistently caused rats to die of hypotensive shock at the intended human
dose? Might the malaria vaccine be advanced to clinical trials in children and
infants or would it even be marketable if it caused severe local reactions in
both rabbits and nonhuman primates? For the remedial bacterium, would one
wish to put it into a field trial in an ocean lagoon if the bacterium itself led to
the death of fish or invertebrates in an aquarium setting? Would it be prudent
to place the drought-tolerant soybeans into field trials if they were found, in
greenhouse studies, to spread the resistance gene to other species of legumes?
Limits for nonclinical and clinical toxicity can and must be established.
The preparation of a TPP not only motivates the team to discuss potential
issues early on, even before a PDP is written, it also forces members of the team
to consider limits to the technology well before a major investment is made in
developing the product. Hence, the value of a TPP goes far beyond internal
use by the sponsor. Once completed, a TPP often becomes the technological
extension of a business plan and is invaluable for business development and
helps to raise working capital from investors. It is a foundation for communi-
cating the technology, potential benefits, and possible risks to the public and
to regulatory agencies, simply because it clearly demonstrates that the sponsor
has considered implications, good and bad and known and unknown, of the
technology.
The Product Development Plan
A PDP, also called a product development strategy, extends the TPP, pro-
viding a roadmap to reach the stated goals. Further, it defines how issues

17Introduction to Biotechnology Operations: Planning for Success
and unknowns identified in the TPP will be addressed and thus become
the basis for scheduling activities and budgeting resources over that
schedule. The PDP is shared by everyone on the product development
team as a common narrative understanding of what has to be done and
how it will be accomplished. Project managers use the PDP for exact task
integration, scheduling, and tracking. The PDP may be shared, in confi-
dence, with potential investors or partners and regulatory agencies so as
to demonstrate that the sponsor has the will and a valid strategy to take
the product to market and thus make a return on investment.
Consider again that planning is a process and a plan is a written document.
Typically, the planning process is managed by a PM, although the plan itself
is written by individual members of the project team. There is no established
order to preparing the individual chapters, discussed later, but most organi-
zations find it is quite helpful to develop a draft or at least an outline of three
sections, clinical, regulatory, and project management, before beginning the
others. The draft project management plan establishes the project team and
provides guidelines, early on, as to the planning process itself. The planning
process requires much discussion, and this comes at team meetings or tele-
conferences. So having a project management approach established early on
facilitates communication and preparation of each section of the PDP. Since
the planning process works backward in a development scheme (Figure 1.2)
and since a Phase 3 clinical study is critical to achieving market approval of a
biopharmaceutical, it is very helpful to draft a clinical plan before other sec-
tions are prepared. Also, having regulatory input upfront provides important
guidelines, especially for biopharmaceutical products.
Once the project management, regulatory, and clinical plans are in outline
or early draft format, a designate from each functional area drafts the appro-
priate section of the plan. The planners each apply the method of working
backward to prepare at least a solid outline. They can then expand the plan,
adding detail while working forward through early, mid, and late phases.
Integration of the elements is important, and the process is facilitated by
effective project management, frequent meetings, and cooperation on the
part of every team member.
The contents of any one PDP are difficult to predict because every product
is unique. Yet experience provides suggestions to ensure any PDP is under-
standable. The PDP has a clearly stated purpose and objective, focused on
the product as described in the TPP. It considers each one of the seven func-
tional areas. It identifies significant risks and foreseeable difficulties and
makes arrangements in the plan to address them. The risk-to-benefit discus-
sion is real and not overly optimistic. The plan is comprehensive by provid-
ing precise technical descriptions. Important steps or stages are not avoided
or omitted, and adequate resources are committed for each functional area.
Finally, the PDP offers a realistic schedule.
Provided next are elements found in most PDPs, written as statements that
should, along with the writings in this book and other publications, stimulate

18 Biotechnology Operations
thought and focus on planning your product. The order of sequence includes
the following:
1. Clinical
2. Project Management
3. Regulatory
4. Nonclinical
5. Manufacturing
6. Quality Control
7. Quality Systems and Quality Assurance
Additional planning considerations are discussed in the various chapters in
this book.
Clinical Development Planning
Overall Clinical Development Planning
• Prepares a broad overview plan for clinical development and con-
firms intended label claims and intended medical outcomes follow-
ing treatment.
• Lists, by phase of development, all clinical trials that are anticipated.
• Defines the most challenging aspects of clinical development for
this product and indication.
• Identifies safety, tolerability, or toxicity factors that are of concern for
the investigational product.
• Describes what has been learned from previous clinical studies with
products of this type and for similar indications.
• Defines pharmacoeconomic and marketing issues related to the
product should it be approved for the stated indication and patient
population.
• Identifies key decision points in the clinical development scheme.
Clinical Development Planning by Phase
• Describes the Phase 3 clinical trial design and elements of a Phase 3
concept protocol to include hypothesis, objectives, outcomes, end-
points, and measurements.
• States the regulatory guidance or precedence needed to develop the
Phase 3 clinical approach.
• Identifies a study or studies to be performed in Phase 2 and given
the intended design and outcomes of Phase 3.

19Introduction to Biotechnology Operations: Planning for Success
• Describes how Phase 2 studies are to be temporally staggered?
• Provides a brief concept design for each Phase 2 study, indicating
the outcomes, endpoints, measurements, and number and nature of
subjects tested.
• Lists Phase 1 studies to be completed before beginning Phase 2.
• Provides a brief concept design for each Phase 1 study, indicating the
objectives, endpoints, measurements, number and nature of subjects
tested, and most likely outcomes.
A General Clinical Development Plan at Each Phase or Study
• Identifies criteria for patients or subjects enrolled.
• Describes unique designs, such as adaptive or crossover, contem-
plated for any study.
• Lists the resources required to perform the study and describes
requirements for clinical study centers or sites.
• Identifies multicenter studies to be performed in late stage and includes
foreign clinical trials considered at early- and mid-stage studies.
• Describes logistical considerations and management of multicenter
trials.
• Names the most likely opportunities for clinical study sites and iden-
tifies studies to be outsourced and the sponsor’s roles and responsi-
bilities to be delegated to outside consultants or contractors.
• Identifies analytical or medical tools or procedures that will be
developed to measure clinical endpoints and describes how and
when each is to be developed.
• Names the internal staff requirements at each stage of development.
• Provides the general statistical approach and lists requirements for
data handling, statistical analysis, and report preparation.
• Outlines the clinical study’s monitoring and auditing plans and
describes how clinical quality will be ensured for each study.
• Identifies clinical trial material (product) requirements at each phase
of development based on the concept protocols and number of sub-
jects and doses per protocol.
Project Management Planning
• Defines the overall objectives and scope of the project.
• Identifies the overall policy for project management applied to the
development project.
• Defines requirements for support from upper management.

20 Biotechnology Operations
• Performs a general work breakdown structure of the major areas of
effort known to date, provides an estimated schedule for the project,
and illustrates this in a chart (e.g., Gantt or PERT).
• Defines roles, responsibilities, and authority of the PM.
• Defines team composition in all areas over the course of the project.
• Defines team communication methods along with anticipated fre-
quency of each type of communication and identifies special com-
munication requirements because of distances or international
participation on the project team.
• Identifies methods to involve contractors, consultants, or vendors
with the team.
• Identifies responsibilities of the team and of the PM for risk assess-
ment and risk management.
• Identifies methods to be used by the team to solve problems.
• Identifies methods for the team’s decision-making process.
• Defines responsibilities and processes for risk analysis, mitigation,
and management.
• Defines tracking and metrics procedures to be applied and indicate
their frequency of use.
• Discusses budget and human resource responsibilities of the PM.
• Develops a project schedule.
• Provides, in general, the objectives and schedule for project closure.
Regulatory Planning
Planning the regulatory approach and operational elements requires sev-
eral skills. First, regulatory intelligence is conducted. Next, a draft plan is
formulated. Finally, all other sections of the PDP are reviewed to ensure that
each is consistent and compliant with the current regulatory environment.
Regulatory Intelligence
• Describes what is known about this product or a similar product
(predicate) from the regulatory literature.
• Describes how predicate products were designed, mentions their
origin and history, and identifies the methods and technologies
used in their discovery and development.
• Identifies potential regulatory routes of approval, both U.S. and for-
eign, used to develop similar or predicate products.
• Lists the technical (e.g., manufacture, control, nonclinical, or clinical)
and regulatory successes and failures for each predicate product and
explains why each succeeded or failed to gain market approval.

21Introduction to Biotechnology Operations: Planning for Success
• Discusses the impact this technical and regulatory intelligence
might have on the intended PDP.
• Discusses how the national political environment may or may not be
supportive of this product and lists state, local, or cultural practices
or laws that might be unfavorable to such a product or indication.
• Discusses how the public might perceive the relative benefits and
risks of this product during the investigational phases. Will public
opinion matter, one way or another, to regulatory agencies in regard
to this product and indication? It also mentions outstanding safety
issues that might concern regulatory agencies and any regulatory
precedent for handling these issues?
• Identifies how FDA regulations might be expected to change before
approval in any given market.
Regulatory Planning
• Identifies regulatory objectives such as Investigational New Drug
Applications and Biologics License Application.
• Defines any special regulatory pathways, activities, or options that
will or might be considered.
• Prepares a regulatory risk-to-benefit analysis for this product.
• Provides one or more possible regulatory outlines or roadmaps with
proposals to overcome perceived or real regulatory hurdles.
• Identifies and proposes means to manage regulatory risks in the U.S.
• Defines global, or ex-U.S., regulatory strategies, primary and alterna-
tive, considers each major market separately, and explains unique
regulatory guidance and country-specific regulatory hurdles.
• Proposes responses to some possible regulatory changes that could
occur before market approval.
• Identifies likely postmarketing regulatory activities and anticipated
advertising and promotion guidelines and restraints for the product
and the labeling claims.
• Identifies methods that would most effectively facilitate regulatory
communication with each agency or office within an agency, defines
each means of communication with a regulatory agency and at each
phase—early, mid, and late—of development, and discusses the
most challenging aspects of the regulatory communication plan.
• Provides an estimate of the number of investigational documents
and market applications that must be filed and the temporal rela-
tionships of each.
• Provides answers to questions, What are alternative regulatory routes
to approval, such as orphan product or fast-track status that might

22 Biotechnology Operations
apply to this product and indication? Have any of these routes been
tried with this class of product and, if so, what were the outcomes?
• Provides an answer to the question, How will compliance be accom-
plished under current Good Manufacturing Practices, current Good
Clinical Practices, and current Good Laboratory Practices and at
which phase of development will they be needed?
• Provides answers to questions such as, If compliance activities are
managed in house, what are the internal programs and guidelines
for handling FDA inspections? Based on risks to the user associated
with the product, is an FDA inspection likely during early investiga-
tional phases of development?
Nonclinical Planning
• Identifies precedence and regulatory guidance for pharmacokinetic
and pharmacodynamic studies performed at each phase of develop-
ment for this class of biopharmaceutical and any predicate products.
• Identifies safety, tolerability, or toxicity factors that are of concern for
the investigational product.
• Refers to intended human dose, dosing regimen, length of dosing,
and route and method of administration in the clinical plan.
• Defines the most challenging aspects of nonclinical development for
this product and indication.
• Outlines objectives, concept study design, and relative schedule for
all intended studies:
• Pharmacokinetic and ADME
• Pharmacodynamic
• In vitro toxicology
• Acute toxicology
• Subchronic toxicology
• Chronic toxicology
• Specialty toxicology in animals
• Identifies analytical or clinical evaluation tools or procedures that
will be developed or used to measure endpoints in animals and
describes how and when each is to be developed.
• Names the internal staff requirements at each stage of development.
• Provides the general statistical approach for these nonclinical stud-
ies and give requirements for data handling, statistical analysis, and
report preparation.
• Outlines the nonclinical study’s monitoring and auditing plans and
describes how clinical quality will be ensured for each study.

23Introduction to Biotechnology Operations: Planning for Success
• Identifies nonclinical study materials (product) requirements based
on the concept protocols and number of animals and treatment doses
per protocol.
• Defines each concept study design once a clinical plan has been
drafted, putting them into perspective with the overall development
scheme, schedule, precedence, and guidance; examines all require-
ments to achieve the objective: scientific, material, and time and
monetary limitations; and proposes budget and schedule for each.
Biomanufacturing Planning
• Identifies and describes the product’s type or class and summarizes
information on the biomanufacture of predicate or similar products
after considering the TPP and research results on the product.
• Outlines a biomanufacturing design, including overall objectives
and goals for each phase of development, early, middle, and late,
after referring to the draft product design, and considers product
risks and hurdles for the biomanufacturing plan.
• Drafts or outlines a biomanufacturing plan based on this design
and considers product quality attributes both from the standpoint
of process control and for quality control testing. In drafting the
plan, it considers the ultimate objective, biomanufacture of commer-
cial product upon market approval, and works backward: it begins
with commercial manufacture of product and proceeds in plan each
phase in reverse order.
• Provides a plan for scale-up of biomanufacture to produce required
amounts of product at each phase and also considers purity and
potency requirements at each phase.
• Defines plans for application of current Good Manufacturing
Practices at each phase of biomanufacturing development.
• Considers each raw material or component that will be used in
production and identifies potential quality criteria and the sources,
and any regulatory guidelines on the quality of proposed raw
materials.
• Identifies and reviews the history of any expression system or host
cell line that will be used and determines if there is precedent for
using the proposed production system and, if so, considers issues
revealed in previous biomanufacturing efforts.
• Identifies any genetic engineering or other biological manipulations
that might be required of the product or a host cell line before the
product enters biomanufacture after reviewing the research back-
ground on the product and its current status in research or early devel-
opment; for example, the need to develop or modify an expression

24 Biotechnology Operations
vector, to evaluate a construct for a particular trait, or to characterize
or do further research on a gene, a vector, or a host cell line.
• Plans the production and in-process and quality testing of any cell
banks.
• Defines early, middle, and late stage development production
schemes for this product, focusing on quality specification and quan-
tity requirements and the chosen processes; considers upstream pro-
duction and downstream purification processes for bulk substance
and formulation, fill, and finish for the final product; and applies
objectives and criteria for quality and quantity, yield, and scale-up
at each stage.
• Identifies requirements for in-process testing after defining the
processes.
• Considers once again risks associated with the chosen processes,
raw material requirements, and unique aspects of production.
• Identifies special requirements for formulation, fill, and finish and
labeling of the final product.
• Defines the containers or delivery devices to be used and storage
conditions and requirements.
• Defines facility requirements for each stage of biomanufacture, con-
siders both quantity and quality, and discusses approaches for meet-
ing these facility requirements or for utilizing contract manufacturers.
• Discusses the need to provide aseptic manufacturing environments
and requirements for clean work areas with classified air supply,
segregation of product, potential for campaign manufacturing or
shared manufacture, and flow of product within a facility.
• Identifies equipment and utility requirements at each stage of bio-
manufacturing development and considers special environmental
issues that are relevant to production of this product.
• Provides an overview of validation requirements and plans for
the biomanufacturing facilities, utilities, equipment, and pro-
cesses proposed in the manufacturing scheme and at each stage of
development.
Quality Control Planning
• Understands from the TPP and draft manufacturing plan any
requirements for quality control testing of product for both product
release and stability.
• Identifies product, bulk substance, and final product attributes (e.g.,
safety, purity, and potency) as they will be considered for testing,
and identifies one or more analytical requirements for each attribute.

25Introduction to Biotechnology Operations: Planning for Success
• Designs a quality control assay for each analytical requirement and
considers a hypothetical specification for each.
• Designs the remainder of the assay development life cycle and har-
monizes this with phases of manufacturing, nonclinical, and clini-
cal development for each quality control assay.
• Provides a plan to identify how and where the assay will be performed
and estimate resource requirements both for assay development and
to perform the assay on expected samples, release, and stability for
each assay.
• Identifies analytical methods that will be developed or used to mea-
sure the quality of each cell bank that is to be tested under quality
control. For each method, it describes how and where the assay will be
performed and estimate resource requirements both for assay devel-
opment and to perform the assay on each sample, and harmonizes
them with the manufacturing plan.
• Identifies analytical controls and reference standards and describes
how and when they will be developed or otherwise obtained and, in
general, give qualitative and quantitative requirements for each assay.
• Outlines the initial (early phase) stability test requirements for bulk
substance, final product, and cell banks or other intermediates. It
describes the attributes that will be tested and identifies one or more
tests for each attribute and outlines the stability test criteria that will
be applied at later phases of development. It also describes any sta-
bility indicating assays that must be developed beyond those con-
sidered and planned for release of bulk substance and final product
and outlines the frequency of testing under stability protocol.
• Describes any requirements for quality control to measure the qual-
ity of the manufacturing environment or output of utilities.
• Identifies quality control tests that will be verified to ensure compli-
ance with compendial methods and the phase of development for
each verification.
• Identifies quality control tests that are candidates for qualification
and state the requirements, expected outputs, and phase of develop-
ment for each assay qualification.
• Identifies quality control tests that should be validated and give
expected outputs and phase of development for each assay qualifi-
cation and describes the resources that might be required for assay
validation and harmonizes the analytical validation with manufac-
turing development and the manufacturing process validation plan.
• Describes the program that will be developed to investigate test fail-
ures or out of specification test results and investigations and har-
monizes this with quality assurance plans.

26 Biotechnology Operations
• Describes efforts planned to ensure quality control is in compliance
with current Good Manufacturing Practices (cGMP) and harmo-
nizes them with plans of regulatory affairs and quality assurance.
Quality Systems and Quality Assurance Planning
• Considers the appropriate quality systems (e.g., current Good
Manufacturing Practices, current Good Clinical Practices, current
Good Laboratory Practices) that must be in place for compliance.
• Provides answers to questions such as, How and at which stage of
development will each quality system be developed and instituted?
Will they be performed in-house or by a contractor or partner?
• Identifies the hallmarks of quality that must be established for each
quality system that will be instituted in-house or at a contract site.
• Identifies applicable U.S. and ex-U.S. regulations that drive the
requirement for each quality system.
• Describes the requirement for in-house quality activities, and dis-
cusses requirements for a quality assurance unit, quality manual,
and quality plan.
• Outlines the elements of the quality manual.
• Outlines the elements of the quality plan.
• Describes the roles for quality in quality by design and at each stage
of design control.
• Identifies requirements for the quality assurance unit and specifically
refers to needs for quality management, document control, auditing,
and training.
• Identifies needs for quality agreements with contractors or
collaborators.
• Identifies any special quality management requirements, such as,
Total Quality Management, Six Sigma, or risk-based approaches.
• Identifies any requirements for quality assurance support in research
activities.
Additional Elements of Product Planning
In addition to these seven functional area plans, three additional elements
of project planning and implementation deserve mention. These are product
design, project risk management, and the risk-to-benefit ratio of the product
itself. Failure to apply these concepts and practices can result in delay or fail-
ure of product development; hence, they are considered during the product
development planning process and identified in the PDP.

27Introduction to Biotechnology Operations: Planning for Success
Product design immediately brings to mind an engineering endeavor,
something that is applied to medical devices but not to a biological prod-
uct. In fact, product design is an important aspect of the planning process
and, in this book, the concepts and practices of design, design control, and
quality by design are discussed at length in Chapter 5. Design is a process
that focuses on the product itself and, as one might expect, design begins
with product criteria and attributes listed in the TPP. Design focuses on criti-
cal quality attributes that are often realized only after a certain amount of
product development planning has taken place, and the product team has
had an opportunity to review in detail the candidate product and the pro-
posed development plan. In a practical sense, this means that design activi-
ties, which are described in Chapter 5, often interrupt the planning process
and require the team to revisit the nature of the product. This can even mean
returning to the research laboratory bench and changing or tweaking the
candidate product to improve it before it enters, or reenters, the develop-
ment arena. Quality by design goes one step further, building quality into
the product and hence into the product’s design process. In effect, this means
that, in addition to user and performance requirements, there is a conscious
effort to design quality criteria into the product. Of course, this means the
quality criteria must be available as product development planning begins
and suggests they be included in the TPP. Design also introduces the idea
of design controls, steps in a formal design process, as discussed further in
Chapter 5. Design is applied to development processes and, in Chapter 6, an
example is given with the design of the manufacturing scheme.
Project risk assessment, mitigation, and management strategies involve
application of procedures and practices to identify potential or actual risks
and to reduce their chance of occurring or, should they do happen, their
impact on a project. Risk management has a significant impact on improving
product quality, safety, and effectiveness and hence is of direct consequence
to the user. It should be part of every PDP, considering both the product and
the development processes. Since risk management activities are often the
purview of the project team, this subject is discussed in Chapter 2.
Risk-to-benefit evaluations are related to risk management and bring with
them other connotations. The term and its concept were developed in the health
products industries and by regulatory agencies as a means to convey a specific
idea: Any product must deliver more benefit than risk to the user. Immediately
one realizes this concept carries with it philosophical as well as practical and
technical implications. We specifically ask how on earth do we weigh risk ver-
sus benefit for any given biotechnology product. The simple answer is that
somehow we do this for every biopharmaceutical before it reaches the mar-
ketplace. A biopharmaceutical intended to treat cancer as a terminal disease is
allowed to have significant associated risk, usually seen as side effect. A vac-
cine intended to prevent a nonlife threatening infection in infants is allowed
to have a low incidence of risks, and these side effects must be considered

28 Biotechnology Operations
mild. We make these choices, relying upon the judgment of experts with input
from the public since as the user they are the object of risk or benefit. We are
not always correct in these analyses, but overall our record is excellent. A PDP
always considers risk-to-benefit of the product and, because it is driven in large
part by regulatory authorities, it is discussed further in Chapter 3.
Summary of Planning for Success
The primary theme of this book, one that is ingrained into each chapter, is to
carefully plan biotechnology operations. This demands that, once a product
vision has been established, a long-range plan, the PDP, is produced to guide
development, manage resources, and reveal to upper management and inves-
tors the progress (or lack thereof) of development during this long period.
However, to produce a meaningful PDP, it is first necessary to write a clear defi-
nition of the product to be developed. Hence, a TPP precedes the PDP. The TPP
is in fact a draft of the intended product label and states the product description,
intended indication, and patient population allowable warnings or contraindi-
cations, and dosage or route of administration. Hence, the TPP and PDP are key
planning documents for the beginning, and they guide development through
the products life cycle. This process has been referred to as planning backward,
since we first identify, in the TPP, where we wish to be once the development
is finished, then fill in all those tasks that must be accomplished to reach those
objectives. Once the planning is finished, the actual development moves for-
ward, following plans outlined in the PDP. Project teams, composed of those
subsequently responsible for the product development, write a TPP and PDP.

29
2
Project Management
Biotechnology and Project Management
Market demand for biotechnology products encourages biotechnology firms
and large, well-established companies to engage in product development, a
process intended to move discoveries from the laboratory through the devel-
opment cycle to the marketplace. Successful development adds value to a
product and ultimately yields profit. Hence, the process of transitioning can-
didate product out of discovery research and into and through development
must be carefully managed. Yet, examples of mismanagement of biotechnol-
ogy product development projects are noted regularly in trade and business
publications.
What then might a biotechnology firm do to manage stress points and
challenges, avoid development failures, and increase the chances of suc-
cessful product development? One answer is a simple and inexpensive rec-
ommendation: apply principles and practices of project management to the
product development endeavor throughout the product development cycle.
In other words, use project teams to apply principles of project management
to integrate the six functional areas of biopharmaceutical development—
manufacture, quality control, regulatory affairs, quality assurance, nonclini-
cal studies, and clinical research—and coordinate these efforts under a team
leader, the Project Manager (PM). When done correctly, and given a robust
technology, it results in a successful outcome.
Project management is the discipline of applying tools, techniques, and
skills to plan, organize, and manage resources through the various phases of
a project to accomplish project goals. Project management has strategic plan-
ning, operational, and closing phases as shown in Figure 2.1. In the planning
phase, a group composed of functional area managers, the PM and corporate
executives, consider the objectives and scope of the technical program out-
lined in the targeted product profile (TPP) and Product Development Plan
(PDP). This product development planning process, described in Chapter 1,
is based on the biopharmaceutical product characteristics and indication,
resulting in a TPP and a PDP. It is important to have the TPP and PDP com-
pleted and agreed before writing a full Project Management Plan (PMP).

30 Biotechnology Operations
Indeed, these technical roadmaps are a requirement if the newly formed
project management team is to draft a useful PMP. In effect, the PMP is
incorporated into the PDP.
The PMP is transparent to the team and to upper management, and it
applies concepts of project management such as team composition, com-
munication, risk analysis and mitigation management, tracking, human ele-
ments, project completion, project management tools, and resources, both
human and monetary. These concepts are discussed later in this chapter.
Project management planning greatly increases the chances of project suc-
cess of meeting the objective of getting a product developed and to market,
on budget and on schedule.
During the operational phase of project management (Figure 2.1), a project
team, led by a PM, follows the project plan, always reflecting upon the tech-
nical tasks, milestones, and schedule of resources and activities provided in
the overall PDP. PMP provides a foundation and charter for the project team,
individuals who will work together over several years to bring their product
from laboratory to market.
A history of success is the reason that project management has proven so
popular and effective in the biotechnology industry and why it is almost
universally applied to biotechnology product development. There are other
reasons. First, project management is very malleable and it allows a firm to
customize project teams and a management structure for development of
each product, no matter how unique the product or the project. Second, it
relies upon team leadership to meet objectives, keep schedules, and move
products to the marketplace. The cost of a single day’s delay in the biophar-
maceutical industry can be one million U.S. dollars! Third, project man-
agement is goal oriented. By its very nature and definition, a project team
has clear goals, enabling the team to focus on larger product development
Initiate
and
operate
Monitor
Output:
Managed project
Development operation
Output:
Completion
Closing
Plan
Output:
Project Management Plan
Project budget
Project schedule
Project objective and scope
Draft targeted product profile and product development plan
Identify stakeholder and management expectations
Agree to project and corporate governance
Apply project management and performance tools
Technical functional areas; financial; communication and
feedback; risk and mitigation; team dynamics;
resolving problem; and metrics
Clinical + nonclinical + biomanufacturing +
QC + QA + regulatory
FIGURE 2.1
Stages of project management and elements of project management plan.

31Project Management
objectives. Fourth, projects are structured, and this structure is in a written
PDP, described in Chapter 1. Structure provided in a PDP and PMP assists
a project team to consistently achieve tangible and profitable results. Fifth,
a project team applies management principles. Each project has a defined
beginning, an end, a schedule for completion, and tools such as task lists
and schedules that assist the PM and team. Project teams are consistent
and resilient. A team stays with the program, from start to finish, pursuing
the objective no matter if individuals leave or new persons join the team.
Another tool is the shared budget for resources: human, fiscal, and capital.
Resources are allocated per plan and according to schedule as PMs strive
to maintain a balance in resources, as they are expended toward a common
goal. Sixth, a team is diverse and professional. People with various  skills
coming  from  all  backgrounds— contractors, employees, consultants, and
clients—are intimately involved and work together on the team. Finally,
there is synchronization, as phases and activities of the project are sequenced
to balance resources, time, and performance against the objectives and the
plan. This sounds a bit idealistic, but, for many well-led teams, it is a reality.
In this chapter, we review the field of project management as practiced in
biotechnology operations, notably by biopharmaceutical firms. It discusses
the history of project management, the construction of a project team, and
selection of a PM, reviews operational principles and practices of leading a
team, and it provides information on project management tools and tech-
niques. Since project management involves social and technical elements,
this chapter touches on both areas. Although not intended to be a com-
plete review of the subject, it should serve as an introduction and provide
some idea of how a biotechnology operation can be successfully managed.
Hopefully, the reader will appreciate the value of project management and
also understand how he or she could apply these skills in his or her work
environment.
Background of Project Management
Project management evolved within the engineering industry. Specifically,
it was first used on large, high-cost, and complex projects that applied cut-
ting edge technology. Examples are projects to build the first atomic weap-
ons, to construct large bridges, to put a man on the moon, or to build any
major defense system or novel automobile. Advances in technology drove
the need for project management. Projects became larger and more complex.
Consider a feat like construction of the Panama canal, completed more than
100 years ago. It was so technically complex and grand for that time (and per-
haps even for our generation) that the project begged for organized manage-
ment. Further, individual  workers brought to the workplace special skills,

32 Biotechnology Operations
and these individuals, and their work, had to be integrated and scheduled.
Choreographed might be a better word. Two hundred years ago shipbuilding
required woodworkers, blacksmiths, sail makers, and perhaps a few other
skills. Today, designing and building new aircraft depend on the integration
of individuals with thousands of skills and subskills. Costs of shipbuilding
are managed in part by careful scheduling of parts and labor. In summary,
project management appeared because it was needed in a technological soci-
ety, and it has evolved to meet demands of cost, quality, and schedule.
In the 1960s and 1970s, as pharmaceutical development technologies
became more complex and regulation of the drug industry further compli-
cated this endeavor, the largest pharmaceutical firms began to adapt, from
other industries, the principles of project management. At first, these prin-
ciples were applied to pharmaceutical manufacture, as engineers, trained
and practiced in project management, brought skills to increasingly more
complex pharmaceutical plants. They were successful in managing teams
and complex technologies, and this was noted by upper management. By the
1980s, project management was being applied to the full scope of pharma-
ceutical development, from discovery to postlicensure activities. Also at this
time, the new industry called biotechnology was just beginning to emerge.
Not surprisingly, as scientists and engineers migrated from pharmaceutical
to biotechnology firms, they transferred project management knowledge
and skills to biopharmaceutical companies. Today, project management has
been adopted by pharmaceutical, medical device, and biotechnology firms
worldwide.
Project Management Plan
The process of planning project management itself begins once an objec-
tive has been established for a development program and both TPP and
PDP have been drafted (Chapter 1). A project may be a 3-month process to
produce a recombinant DNA molecule for sale as a laboratory reagent, or it
can be a complex, 10-year biopharmaceutical effort to develop a monoclonal
antibody to treat a life-threatening disease of children. No matter the com-
plexity or length of a project, both management and technical aspects must
be carefully planned.
Hence, a project management plan establishes goals or objectives for the
life cycle management of a project, recognizing hurdles and providing a
long-range framework to minimize risks and to achieve goals and rewards.
It also puts into place procedures and processes for management. Project
management planning simply takes elements of good planning practices—
planning for success, looking at the ultimate objective and defining goals
along the way, and incorporating quality systems—and formalizes them

33Project Management
into a document or set of documents that can be shared by all team members
throughout the life cycle of the product. If this is achieved and the objective
is clear and shared by all team members, then transition from strategic proj-
ect and management planning to operational project management is easy for
the PM and the development team.
A project management plan may have five basic elements. Each reflects
a phase in the life cycle of project management and indeed in the life of a
technical project:
1. Initiation: Starting the project in a positive manner and formation of
a team
2. Planning: The subject of much of this chapter
3. Executing: The technical and management aspects according to the
plan
4. Monitoring and controlling: Functions that ensure the project is meet-
ing objectives
5. Closing
Further to the five basic elements are a host of other considerations for a
project management plan, outlined in Figure 2.2. These will be discussed
individually in this and other chapters. It is worth noting that the project
planning process, those meetings and discussions that seem to take forever,
and the operation and execution of the development project itself include the
Team and dynamics
Contractors and collaborators
Project
planning
Resources
Metrics and tracking
Objectives
Initiation and completion
Schedule
Scope and complexity
Communication
Social considerations
Technical aspects
Risks and constraints
FIGURE 2.2
Inputs for project management planning.

34 Biotechnology Operations
same technical and management elements. Thus, considering each element
in the project management plan is key to operational success of the project.
The Project Management Environment
The environment in which a biotechnology product is developed mat-
ters to project management planning almost as much as the TPP and PDS.
Biotechnology firms come in all sizes and with various types of structure or
organization. These factors matter to effective project planning and manage-
ment. For this discussion, firms are stratified and considered based upon size
and complexity, with virtual biotechnology firms at one end of the spectrum
and large, experienced companies at the other.
Virtual firms have few full-time employees. A project management team at a
virtual firm might be composed of from one to a very few employees and
in addition include outside partners, consultants, or contractors. A key rep-
resentative, perhaps the titular CEO or a key investor, could lead this small
team. Although there is little formal training or experience on the part of the
PM or project team members and despite the fact that each team member
may be responsible for two or more functional areas, small teams at virtual
firms often outperform their counterparts at much larger biotechnology or
pharmaceutical companies.
Small biotechnology firms normally have little project management infra-
structure at the time their first product enters development. To establish
project management at a small firm, the technology should have reached a
level of maturity and the pathway forward must be clear. Specifically, five
elements must be in place because they form the foundation for successful
product management:
• Management decision and support: A business decision to move for-
ward, made by executive management or a board of directors based
upon project benefit or attributes, risk (technical and commercial),
and resources. There is the intention to apply project management
to product development, and there are or will be resources available.
• Planning: A written PDP and TPP or their equivalent provide ade-
quate information, stating objectives and spelling out a clear route
forward.
• Feasibility to begin to move forward: All elements of the plan are feasi-
ble in the current financial, technical, and regulatory environments.
• Estimate of completion: A realistic schedule based on estimates of
experienced professionals.
• Decision points or milestones: Milestones and decision points are evi-
dent in the plan. For example, Go/No Go criteria to advance the proj-
ect to the next phase are established at the beginning of the project
and revisited at the start of each phase.

35Project Management
Once a decision has been reached to apply project management to a product
development pathway, management intent is best demonstrated by appoint-
ing the initial or core project management team and a PM and, most impor-
tantly, relegating authority and responsibility for product development to
this manager and team. Supporting a development project can be particu-
larly difficult for the product discoverer, company founder, or executive of
an entrepreneurial firm who has, for years, focused on discovery research
or business development. To many founders, a seemingly easier route to suc-
cess may be continuation of discovery research, where perceived risks are
lower than those in the route of product development. For some executives, it
is difficult to let a project team take control of functions considered essential
to success of the firm. These are emotional decisions that must be made for
the small biotechnology firm.
Established biotechnology firms may have it a bit easier when they begin
a new development project because they have the experience and infrastruc-
ture. Indeed, their existing and often mature project management programs
provide experienced and highly trained staff dedicated strictly to building
and managing teams. Many issues related to start-up operations—build-
ing the first team and introducing employees to the principles and practices
of project management—may not apply to the larger biotechnology firm.
However, the established firm has other hurdles to productive project man-
agement. A few are as follows:
• Complex organizational structure and rules confound efforts to
complete any one project on time. For example, merger or acquisi-
tion, a common occurrence in larger firms, results in changes to a
major contractor, disrupting continuity of operations and schedules.
• Priorities change frequently and without clear direction from upper
management. For example, the clinical indication for a product
changes radically because of revised market objectives.
• Upper management is far removed from project teams.
• A large organization may be slow to respond to opportunity or to
change, when these are necessary or desirable.
• Projects are abandoned in midcourse and without explanation to the
development team.
• Problems, incurred in one project, spill over to another project.
• Elements of a project must be reworked because they are considered
unsatisfactory to someone outside the team.
• Communication breaks down due to change in mode of communica-
tion. For example, a new videoconferencing system is required for
all project team meetings, but it does not work properly.
• Corporate politics impact PMs.
• Team membership changes during corporate reorganization.

36 Biotechnology Operations
These examples can complicate efforts toward successful product develop-
ment in a larger biotechnology firm, and they have led some PMs to wish
they were employed by a smaller organization.
Project Objectives and Schedules
Biotechnology firms, especially small and midsized companies, often suffer
from malignant optimism and fail to recognize that few firms successfully
complete projects planned under highly optimistic or unrealistic schedules.
Hence, the PM is responsible for ensuring that a realistic schedule is com-
posed and communicated to the team and to the management. History sug-
gests this is a difficult task, since the common story in the biotechnology
industry goes something like this:
• Project development team is formed.
• Senior management provides the ultimate objective for product
development.
• Project development team prepares a product development strategy
with schedule.
• Senior management demands that work be completed in one-half
the time allotted by the team.
• Project begins under the accelerated schedule.
• Within 1 year, the project is offtrack, management is angry and team
members are discouraged.
• Pessimism or outright failure in the face of a sometimes promising
technology.
What is the solution to artificial compression of schedules, an issue that
constantly plagues small and midsized biotechnology firms? First, upper
management must recognize that the product management team is com-
posed of individuals who, together, have years of experience in estimating
development times. These individuals have been responsible for meeting
schedules in the past. Second, members of the product development team
must recognize the need to expedite development, but not at great risk to
delaying development. Moving quickly along the development pathway
is a hallmark of the industry and provides the biotechnology firm with a
competitive advantage. Although it is inappropriate for upper management
to establish unrealistic schedules or to diminish resources below a certain
level, PMs should take reasonable risks in establishing optimistic time lines.
This is balanced against three very real project constraints: scope, time, and
budget. Managers periodically weigh these constraints while advancing the
project through each phase; balancing these constraints is a major challenge,
while also taking into account quality and performance, in the product

37Project Management
development planning process; yet it is a key to success. Communication is
key to ensuring every team member is aware of the constraints.
Project scope and complexity are important considerations to planning.
Simply stated, there are simple and easy projects, and then there are complex
and difficult projects. Yet others are somewhere in between these extremes.
Project difficulty and complexity, if they exist, become apparent upon
reviewing the PDP and have a great impact on the project management plan.
Complex projects call for more involved and extensive project management.
Sociotechnical Considerations
To be effective, project management focuses on two critical areas: one tech-
nical and the other social. First, it must apply project management skills to
the plan with due consideration to implementation. Examples are establish-
ing objectives, developing work breakdown structure (WBS), and monitor-
ing resources. Project management must also influence individuals whose
cooperation and help is needed to complete the project successfully, for
example, establishing buy-in from the supervisor of a key team member.
This need to use both technical and social skills for effective project man-
agement has resulted in the realization that this trade is a sociotechnical
endeavor. Executives of small biotechnology firms, though successful at
influencing outsiders, such as investors or the scientific community, some-
times lack the ability to influence technical aspects of product development.
Hence, they call upon PMs to play this role. PMs must therefore have strong
interpersonal skills, notably the ability to influence others to achieve project
goals and to interact with upper management. Thus, it is critical for upper
management to understand this and to follow-through by identifying and
retaining experienced individuals, those with both technical background
and social skills.
Participants in Project Management
A biotechnology project team is composed of many individuals, and they
are led in this regard by an appointed PM. Teams vary greatly in size and
scope, depending upon the complexity of the project and the size of the
biotechnology firm. Each individual on a project team has a vested interest
in reaching the same objective no matter what his or her technical skills,
employment rank, or title. Team members may not be employees; instead
they are consultants, contractors, or investors. Individuals who might be
considered for inclusion on a biotechnology product development team
are given in Box 2.1. Individuals who serve on teams have roles, both pro-
fessional and managerial, and some have, shall we say, special status. It
would be nice to think that everyone involved in a biotechnology project
is equal in the eyes of the project and upper management. Unfortunately,
this is seldom the case. A PM and the team recognize key participants in

38 Biotechnology Operations
a project, referred to as stakeholders. These  individuals have a significant
vested interest in the project, even though they are often not, from a prod-
uct development standing, the most active members of the team. Indeed,
some stakeholders, such as major investors or executive level management,
seldom if ever participate in routine team functions. Their interests are
nonetheless held above those of others on the team, and the PM pays spe-
cial attention to their opinions and desires. Even though they sit apart from
the team, stakeholders have great influence on team activities, and each
stakeholder expects regular and often direct communication from the team,
usually by way of the PM. From this, it can be inferred that the PM, in addi-
tion to managing the team, is responsible for communicating with, indeed
for influencing, stakeholders. This can be a stressful and time-consuming
task in itself. Experience suggests that stakeholders often hold the positions
described as follows:
• Project champion: This person, sometimes referred to as the project
leader (as distinguished from the PM), is capable of influencing bio-
technology projects based on scientific expertise, organizational
power, or responsibility for a critical resource (e.g., a patent). In other
firms, the project champion is an executive manager. In either case,
they may be a figurehead (e.g., the historic founder of the firm or
discoverer of the product or technology) and may or may not serve
on the project team or even have a specific technical role. But they
may also consult directly with project team members. They are often
accountable only to upper management, such as a board of directors
or president, and not to a PM or to the team.
BOX 2.1 SKILLS OF INDIVIDUALS ASSIGNED TO A
BIOTECHNOLOGY PRODUCT DEVELOPMENT PROJECT TEAM
1. Project Manager
2. Project leader or project champion
3. Finance
4. Legal and contracts
5. Research
6. Business development
7. Marketing
8. Quality Control
9. Quality Assurance
10. Clinical studies
11. Nonclinical studies
12. Manufacture
13. Regulatory affairs

39Project Management
• Major investor: The golden rule is stated to be: He who has the gold
makes the rules. This has great meaning to a biotechnology firm,
where cash flow is always an issue. Today, investors are very proac-
tive. Few attend project meetings, but most major investors expect
to be frequently informed by the project team manager on techni-
cal successes or failures, news about reaching or missing milestones,
and updates on expenditures.
• Chief executive or board member: Executive officers in small biotech-
nology firms are very hands-on with project teams. Most do not
micromanage their teams, but instead stay in constant contact with
the team leader and key team members. They are often scientifically
astute, interested, and inquisitive. Keep in mind that they are an
important bridge for your firm, communicating the good, bad, and
ugly to analysts, investors, and the public.
Individuals actually serving on the project team may or may not be consid-
ered stakeholders.
• Functional area manager or director: These individuals, and there may
be many, are key architects of the project, responsible for decisions
about strategy, plan, resource requirements, and determining status.
Although they may not have authority to allocate resources, their
influence looms large in other ways. They might direct key technical
or administrative aspects for project support and frequently main-
tain a commanding presence in the smaller biotechnology firm. They
are accountable in two directions: to corporate executives on project
matters, but to line management for functional responsibilities.
• PM: Individuals responsible for leading a team are influential as well.
In some instances, they are or soon become stakeholders themselves,
even though they may be subordinate to executives and directors.
PMs often have great responsibility, but without direct authority. We
refer to roles of a PM throughout this chapter. The way a project
is managed and executed are keys to a project’s success or failure.
Hence, it stands to reason that selection of the appropriate PM is
an important decision. The manager should be experienced with a
project of this scope and nature, although it is certainly not neces-
sary for a candidate to have great technical knowledge in that area.
Ideas for correctly matching a PM with a project are listed in Box 2.2.
Attributes of excellent PMs is given in Box 2.3.
But, where do we find a PM with these attributes? In selecting a PM, the
small biotechnology firm, with a staff of perhaps 20–100 individuals some
of whom have previously served on project management teams, may have
qualified applicants already on staff. Although the firm many not have a

40 Biotechnology Operations
BOX 2.3 ATTRIBUTES OF EFFECTIVE PM
General management Conflict resolution Leadership
Team building Planning and scheduling Resource
allocation
Anticipation of change Acceptance of change Adaption to
change
Execution of change Effective communication Team building
Negotiation Leading decisions Risk analysis
Risk mitigation Risk management Organization
Technical knowledge People and team skills Critical thinking
Facilitation Begging, nagging, and playing devil’s
advocate
BOX 2.2 CONSIDERATIONS FOR SELECTION OF A PM
• What are the objectives and what is the anticipated length of the
project?
• What is the scope of the project management function and
hence the PM? Is it an individual project, a nested project, an
integrated project, or a series of projects?
• Has the project team been previously led by someone, and if
so, what was the outcome and what are the lessons learned from
that leadership?
• Is strategic and operational planning involved?
• Will he or she allocate resources, human or monetary, and
make priority decisions?
• Is the project at more than one location, or in more than one
country?
• To whom will the PM report and at what level within the
organization?
• On what criteria will project management staff and team mem-
bers be selected?
• How will their performance be evaluated?
• What are the roles and responsibilities of project champions
and functional directors?

41Project Management
seasoned and full-time PM, there could be an employee who, through expe-
rience at another firm, has basic skills and thus qualifies to lead a product
development team. The midsized biotechnology firm will have experienced
project management processes and, like a large firm, have project manage-
ment staff with individuals willing and available to move to a new project.
Selection must be rigorous no matter the situation. Individuals designated to
the project team should have an opportunity to interview candidates. Final
selection is influenced by those who understand and, preferably, have prac-
ticed project management in the biotechnology industry.
Project Management in Biotechnology Operations
Establishing Project Management
It is important to decide exactly when to begin the formal process of project
management. Some guidelines and common practices are instructive in this
regard. Project management, as described in this chapter, is seldom used in
discovery research, and so the concept is often foreign to the management of
a small  company. Certain  scientists further argue that formal project man-
agement inhibits good research because a highly structured environment is
not conducive to discovery. Others suggest that it inhibits direct management
of projects by executives. However, most would also agree that an organiza-
tion developing a product through application of more than one functional
operational area must institute at an early stage of product development some
method to coordinate and integrate activities and participants. In the end,
most biotechnology firms elect to apply project management principles and
practices to their projects.
Given that projects, and hence project management, have a defined begin-
ning, when should the biotechnology firm make the transition and establish
formal project management? The best answer may be: whenever planning,
coordination, and scheduling activities will, in some way, help the team and
the stakeholders achieve a common goal.
The process is not difficult at experienced, typically large or medium sized,
biotechnology or pharmaceutical firms. They begin project management at
the outset of technical efforts, immediately on approving an operational
concept and even before a PDP is prepared. These firms have professional
project management staff to draw from or the resources to hire new staff.
Larger firms have significant infrastructure in project management headed
by a vice president dedicated to the task. Also, institution of a project team
usually follows internal guidance, instructions from upper management,
and established corporate guidelines.

42 Biotechnology Operations
The process is not often seen at the inexperienced and smaller biotech-
nology firms. Despite any recommendation to make a conscious decision
and begin project management at a defined point, the fact is that project
management usually evolves at smaller biotechnology firms, with little
conscious effort on the part of executive management. Executives may real-
ize, perhaps after witnessing a failure or set back, that, to develop a product
within the allotted time, a team leader is needed to manage the project,
lead the team, ensure a smooth and timely sequence of events, and care for
mundane items, such as setting an agenda, preparing minutes, communi-
cating with stakeholders and preparing formal project management tools
such as Gantt charts and reports. This process is project management by
evolution, and it is a characteristic of less experienced biotechnology firms.
Often in this situation, executives draw the project management designate
from within the ranks of the project team, even if that person has other
duties.
Another challenge to establishing a managed project in small firms is an
open wariness of any managed development process and thus hesitation to
appoint a PM. At the start, team members may voice many and varied ideas
and opinions concerning the scope, purposes, and strategies for the project
and disagree on management guidelines and styles. This is often the first
sign that a new team is embarking on a sociotechnical endeavor and, in the
absence of a full-time PM, early conflicts must be handled gently so as to
avoid delay or disruption to the new project. Even more reason for executive
management to complete project management planning, appoint a PM and
organize a project team as soon as the requirement is identified.
The Work Breakdown Structure
In planning a new biotechnology project and preparing a project manage-
ment plan, it is important to devise a WBS. The process that results in a WBS,
itself a narrative document or chart or both, is based on an understanding of
the deliverables, the materials, service, or product that the project is intended
to produce. To begin, it is necessary to have completed the TPP, a PDP, and the
intended scope of work. At this stage of planning, these documents are often
rough draft documents. The WBS simply breaks the intended project into
smaller, subcomponents that are more manageable. In a WBS for develop-
ment of a biopharmaceutical product, the organization looks somewhat like
an organization chart with branches representing the functional areas such
as clinical and regulatory affairs. In practice, this is done first on a large sheet
of paper or it can be organized using project management software, a tool that
will be discussed in the section “Tools for Effective Project Management.”
Work is broken down first by deliverables or milestones, then into subhead-
ings referred to as tasks, then into subtasks, and so on. Examples are shown
in Figures 2.3 through 2.5. Each figure shows work breakdown of the same
project but using a different project management tool.

43Project Management
ID Task Name Duration Start Finish
1 Research 50 days Mon 3/14/11 Fri 5/20/11
2 Confirm Efficacy 5 wks Mon 3/14/11 Fri 4/15/11
3 Test Blood Levels 5 wks Mon 4/18/11 Fri 5/20/11
4 Quality Control 40 days Mon 4/18/11 Fri 6/10/11
5 Certify M9 Blood Assay 8 wks Mon 4/18/11 Fri 6/10/11
6 Develop M9 Tissue Assay 6 wks Mon 4/18/11 Fri 5/27/11
7 Assays Completed 0 days Fri 6/10/11 Fri 6/10/11
8 Manufacturing 135 days Mon 1/17/11 Fri 7/22/11
9 Manufacture Pilot Lot 40 days Mon 1/17/11 Fri 3/11/11
10 Manufacture Clinical Lot 40 days Mon 5/30/11 Fri 7/22/11
11 Product Available 0 days Fri 7/22/11 Fri 7/22/11
12 Quality Assurance 70 days Mon 3/14/11 Fri 6/17/11
13 Audit Preclinical Site 3 wks Mon 3/14/11 Fri 4/1/11
14 Review Assay Certification Documents 5 days Mon 6/13/11 Fri 6/17/11
15 Preclinical 156 days Mon 4/25/11 Mon 11/28/11
16 Write Protocol 8 wks Mon 4/25/11 Fri 6/17/11
17 Perform Study 86 days Mon 6/20/11 Mon 10/17/11
18 Order Animals 20 days Mon 6/20/11 Fri 7/15/11
19 Treat Animals 1 day Mon 7/25/11 Mon 7/25/11
20 Observe Animals 60 days Tue 7/26/11 Mon 10/17/11
21 Write Report 6 wks Tue 10/18/11 Mon 11/28/11
22 Preclinical Report Completed 0 days Mon 11/28/11 Mon 11/28/11
23 Clinical 84 days Mon 3/14/11 �u 7/7/11
24 Design Clinical Protocol 30 days Mon 3/14/11 Fri 4/22/11
25 Finish Clinical Protocol 14 days Mon 6/20/11 �u 7/7/11
26 Regulatory 41 days Tue 11/29/11 Tue 1/24/12
27 Draft IND Sections 4 wks Tue 11/29/11 Mon 12/26/11
28 Finish IND 21 days Tue 12/27/11 Tue 1/24/12
29 Submit IND to FDA 0 days Tue 1/24/12 Tue 1/24/12
6/10
7/22
11/28
1/24
rte 1st Quarte 2nd Quart 3rd Quarte 4th Quarte 1st Quarte
Task
Split
Progress
Milestone
Summary
Project summary
External tasks
External milestone
Deadline
Page 1
Project: Fig 2.1 MS Proj TU
Date: Fri 5/21/10
FIGURE 2.3
Gantt chart format for biopharmaceutical development project. Schedule of events for a project
shown in Gantt chart format including the work breakdown by task and task ID number. The
start and finish dates and the duration of each task are given in the narrative listing. The right-
hand panel depicts the project in chart format using solid bars to summarize the duration of a
set of tasks (e.g., research) and shaded bars to represent individual tasks. Diamonds represent
milestones and arrows interconnect tasks to reveal dependencies. Special computer software
is used to compose complex Gantt charts.

44 Biotechnology Operations
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45Project Management
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46 Biotechnology Operations
Forming a Project Team and Hands-on Project Management
However, or whenever, formal project management enters the picture, proj-
ect management planning will begin with a meeting of project management
team members led by the designated PM. Note that this first activity is project
management planning, not operational or product development planning. To
be successful, upper management must wholeheartedly and visibly support
the institution of formal project management whether or not there is dis-
sention regarding the need for a project team. Indeed, a way to open the
first meeting is to have the president or CEO review for team members the
development strategy and the initial or draft project management plan. This
empowers the team and its leader and clearly demonstrates the intended
objectives, deliverables, and measurement criteria of the project; it gets every-
one on the same page as a team. Network building, as team members form
interpersonal relationships, is another important objective of the first meet-
ing, as networks form links and bonds between individual team members.
These first steps have been referred to as the forming stage of a project team.
Once the strategic objective has been reviewed, the PM might focus the
team on critical elements of a project management plan to ensure that all
team members share the same vision. This stage has been referred to as the
storming phase, perhaps because such discussions can seem quite unsettled.
One of the most important tasks is development of the team charter, and this
is expanded in Box 2.4. If nothing else, this first meeting establishes, through
the outline of a team charter, team identity both internally, among team
members, and externally, to senior management or to investors. Finally, the
team agrees to certain attributes that have been stressed by or seem impor-
tant to upper management. Some examples (and these may be very different
depending on project objectives, the firm, and the team) are that the team
must meet expectations of stakeholders, be on or under budget, and keep on
schedule. Note that the team, not specific individuals, now meets goals and
objectives. This has been referred to as the norming stage as team members
agree to norms or standards. For some members new to project management
teams or the concept of teamwork, this may be a new concept. Thus it may
require explanation by and patience on the part of the PM.
With this accomplished, the fourth, longest, and final stage of a project
team may begin and this is referred to as performing.
Team Dynamics
Team dynamics refers to various team activities and interactions, often
defined as communication. As one might guess, social interactions, especially
those identified in Figure 2.6, are at the heart of team dynamics. Of greatest
concern to the PM team are identification of risks, resolution of problems, and
the decision process, three highly related and very dynamic responsibilities.

47Project Management
Decisions are often required when problems or issues arise and must be
addressed by the team. In a high-technology industry such as biotechnol-
ogy, we frequently encounter problems. These may be technical in nature,
but often they are based in regulatory, quality, or management and admin-
istrative areas. Our problems are also derived from the fact that this indus-
try is competitive and therefore fast paced and dynamic. Recommendations
BOX 2.4 GUIDELINES AND ELEMENTS OF
A CHARTER FOR PROJECT TEAMS
• Establish team work rules:
• Identify means of selecting milestones and deliverables
since these are key to project development and tracking.
• Agree on means to identify and track tasks. These basic
building blocks, often technical, must be pursued in
sequence.
• Discuss means of team communication.
• Establish milestones:
• Points in the project, actually major events, at which time
progress can be measured against an objective.
• It may be necessary to initiate planning in various func-
tional areas before this begins.
• Define required skills: Who must sit on the team and when in the
project cycle will their input and skills be needed?
• Reveal constraints: Identify boundary conditions, notably as
resources.
• Outline a team charter and include key elements:
• Our team goals
• The team’s members and what each represents
• The roles of each team member
• Our project team manager, leaders, and stakeholders
• The team quantity (size)
• Our team’s qualities (skills)
• The team’s identity
• Set the network: Draw out an organization chart for the team.
• Share important technical information: Even use the first meeting
to begin technical cross-training and discussions.

48 Biotechnology Operations
for addressing problems typically encountered by project teams are listed
in Box  2.5, where problems are classified as one of two types: adaptive or
technical.
Risks are related to both problems and decision making and are related to
solving problems and making decisions. Risks impact product development,
adding to the already complex nature of projects and their management.
PMs are often called upon (or volunteer) to take the lead in risk identification
and mitigation; or at least lead the team in these functions. Risk management
is discussed in the section “Project Risk Assessment and Management.”
PMs cannot and should not attempt to measure risk or resolve problems by
themselves. Instead their role is to motivate team members to find the best
resolution in the shortest period of time and to lead the team in the problem-
solving process. The PM also communicates the problem, and its resolution,
to team members, upper management, and stakeholders.
The most important and challenging team dynamic for which PMs provide
a leadership role is the decision process. Decisions face the team at virtually
every meeting. Someone once said that the only reasons for having a team
were to solve problems and to make decisions! Although this might not be
completely true, it makes an important point. An agenda would seem barren
without the need to make at least one decision. Experienced PMs have likened
the process of leading the decision process within a biotechnology team to the
activity of herding cats. To avoid unsettled outcomes, the prudent PM estab-
lishes, at the first team meeting, decision-making guidelines. The process itself
should be identified and recorded, so that it is followed in the future. Criteria
for reaching a decision (e.g., by majority rule and by consensus) and time lim-
its are established. The PM needs the support of every team member, even
those who argued against the chosen course of action, once a decision has been
reached. Meeting minutes reflect each decision taken by the team. Finally, the
Respect
M
ak
e
Resolve
Focus
Engage
D
em
ea
no
r
IdentifyCom
municate
FIGURE 2.6
Team dynamics. Positive team dynamics result from an engaged team, where members engage
each other respectfully and focus upon relevant issues, all in a positive demeanor.

49Project Management
team should consider how decisions will be communicated to stakeholders and
how failure to reach a decision might be quickly and amicably be resolved.
Communication and Feedback
Once a team has been established, the charter written and the PM appointed to
lead the team, it is necessary to consider communication issues. Communication
for the project team requires interactions between team members, the project
sponsor (typically senior management), functional area managers, executive
management (e.g., president and chief financial officer), and business users, the
likes of sales, marketing, legal, contracts, and business development. Although
BOX 2.5 MEANS OF TEAM COMMUNICATION
• Formal meetings: Even if the team is scattered in several coun-
tries, there is a need to meet face to face at regular intervals.
Formal meetings always have an agenda and result in formal
minutes.
• Informal meetings: Yes, there are times when some, but not all,
members of the team must meet. Informal meetings are best
summarized by e-mail messages or memoranda that identify
the purposes and outcomes for the meetings and are shared
with all members of the team. The PM may be responsible for
gathering these messages and providing them to the larger
team. Video or telephone conferences may be used for infor-
mal meetings and, for some participants, for formal meetings.
• E-mail, Telephone, and FAX: Today, e-mail is a primary means
of day-to-day communication, especially when individuals are
separated, even by short distances. Telephone conversations
remain very important to the professional. How and when is
the content or outcome of these communications to be copied
to the PM or to other members of the team? Often this is done
periodically, perhaps at meetings, where important elements of
previous conversations or messages are revealed to members
of the larger team. In other cases, important communications
between two team members are transmitted directly to the
PMwho then ensures that they are disseminated to other team
members on a need to know basis.
• Project management tools: Today the PM has at his or her dis-
posal computer-based tools, such as MicroSoft Project® and
Share-Point, and document sharing tools, such as Box®. These
may be used to identify and outline tasks, define resources,
track goals and tasks, and support preparation of reports.

50 Biotechnology Operations
these individuals may not attend each project management team meeting,
they are by nature team members. Indeed, team activities and decisions will
affect them directly. Their membership and participation are encouraged, and
effective two-way communication, in whatever format, is essential to success.
The PM is kept informed of all current events by each team member.
The PM outlines a communication plan with the purpose, frequency, and
format for communication between team members and with executive man-
agement and stakeholders. Examples of project messages are a corporate-
wide or public announcement, upon reaching a milestone, or the financial
information, first reviewed in a budgetary meeting with finance before the
close of a fiscal quarter. Today, there are many methods of communication
from which to choose, but proven methods used by the biotechnology indus-
try are listed in Box 2.6.
BOX 2.6 STEPWISE APPROACHES TO RESOLVING PROBLEMS
1. Identify the problem. Be very specific.
2. Classify the problem. Is it adaptive or technical?
3. Assess the problem in relation to the team and team members.
Is it the team’s or the project’s problem or does it belong to
another entity? If it does not belong to this team, then refer it to
the proper authority.
4. Identify the importance of the problem to operations and objec-
tives of the team.
5. A PM ensures all team members are aware of the issue and
remain engaged and informed throughout the remainder of the
problem-solving process. Keep the team informed, focused, and
involved and maintain an urgency to find a cause and resolution.
6. Look for causes and identify the nature and source of each pos-
sible cause.
7. Identify approaches to resolve the problem.
a. For technical problems, consider technical solutions.
Engage technical experts and apply scientific methods.
b. Adaptive problems are often more difficult to address or
resolve. They may be complex, occur in a changing envi-
ronment, and lack predictability. It may be necessary for a
team composed largely of individuals with technical or sci-
entific backgrounds to resolve an adaptive problem. Seek
help of experts.
8. Once resolved, document the problem, the process, and the
resolution. Implement the solution.

51Project Management
Project Risk Assessment and Management
Risk identification and management, important assets to a biotechnology
project, are often project team functions, at least for projects under the team’s
purview, and are led by the PM. Processes inherent to project management
lend themselves to identification and control of risks. The PM is assisted in
risk management efforts by others on the team, often including representa-
tives of quality assurance and regulatory affairs.
Each biotechnology operation faces a number of risks, as exemplified in
Figure 2.7. Team meetings provide a forum to discuss each risk, perceived
or actual. Yet, it may be difficult for the project team to hold open and frank
discussions of risks, even those with a high probability to occur and then
confound, delay, or impede a project. Perhaps this stems from the nature
of biotechnology itself, an entrepreneurial endeavor operating in an envi-
ronment with many inherent financial, technical, and operational  risks.
Common
project risks in
biotechnology
operations
Unacceptable safety
profile
Low efficiency
Unacceptable quality
Regulatory
disapproval
Unable to meet
schedule
Excessive cost
FIGURE 2.7
Examples of project risks in biotechnology operations. Which of these are adaptive, which are
technical, and which may have elements of both? Which are often considered together, proving
that we seldom encounter a single risk?

52 Biotechnology Operations
Yet boards of directors and a biotechnology firm’s investors expect that
significant risks be revealed, at the time of investment and throughout the
operational phases of a project. Regulatory agencies increasingly demand
that risk analysis be part of any product development program since
such efforts result in safer and more efficacious biopharmaceutical prod-
ucts. Hence, project teams in biotechnology development are increasingly
becoming the clearinghouse for risk assessment and mitigation and, with
this, the PM becomes a leader in risk identification, assessment, and mitiga-
tion activities.
The practical aspects of project team risk management require consider-
able time and effort. A project team is aware of their role in risk management
and ensures defined processes are in place so each response is appropriate
and all responses are consistent. The process allows for the initial assess-
ment of potential risk elements and for prioritization of the risks as well as
for implementation of mitigating actions and periodic reassessment of risks.
A project team performs an initial risk assessment at the outset of a proj-
ect and renews and revises this assessment at predetermined milestones or
whenever significant changes are made on a project. For example, product
and process risks are reexamined prior to clinical trials, in the case of medi-
cal products, or before field studies, for agricultural products. Biotechnology
operational projects establish milestones, and risk assessment is considered
a milestone-related task. The PM ensures that risk assessment programs
are established with milestones and scheduled for completion. Whenever a
project team is involved, risk management is part of the project management
plan. At an early meeting, the team agrees to the most likely possible risks
proposed by individual team members. Next, each risk is subjected to one of
three types of risk assessment—fault-tree analysis, informal assessment, or
failure mode and effects analysis (FEMA)—by the appropriate professional
or committee. Informal risk analysis is used whenever the likelihood of a
risk is low, if it is a highly technical problem or if the risk has minor conse-
quences. One individual or a few people may review the issue and report
back to the project team.
Fault-tree analysis is a complex process often used in engineering sys-
tems, but can be quite helpful with some biotechnology operational
endeavors, such as biomanufacturing. FMEA is most commonly applied
to projects in the biotechnology, drug, and medical device industries. A
team usually performs this analysis. Every step of a process or feature of
a product is listed, and possible failure modes are identified. Then each
possible failure is assigned a score, and the team makes plans to measure
and address risks that exceed a certain score. FMEA can be time consum-
ing upfront but, under the guidance of a project team, it ultimately saves
considerable time and resources. The PM must drive these processes and
ensure they are brought to completion. Finally, it is often the PM, speaking
on behalf of the team, who communicates risk information to executive
management.

53Project Management
Metrics and Tracking Progress
Once the project is underway, the PM is responsible for tracking prog-
ress of each task against the plan and schedule. Metrics demand the team
have a method to measure progress of each task and of the overall project.
Although some tracking is done informally, using memory and communi-
cation with colleagues, most is performed using written schedules and lists
of interrelated tasks and milestones prepared with computer programs
such as Microsoft Project®. A scheduled list of tasks forms a track or road-
map for the project; this can be visualized in Figure 2.3. Such tools are
introduced later in this chapter. The purposes of tracking are both to moni-
tor progress against objectives (e.g., milestones) and to control the process.
Tracking allows the PM to predict if any piece of the project (e.g., a task)
seems to be at risk of failure or is heading offtrack.
Specific activities in tracking include collecting actual work and cost per-
formance information and estimates to completion of milestones. The PM
then compares actual performance with the plan and, if necessary, revises
tasks, working with project team members in an effort to bring the proj-
ect back ontrack. Indeed, a diligent PM uses tracking to identify problems
before they delay or limit progress. Tracking methods are also used to com-
municate issues to the project team. Reports to senior management and
to investors are based upon data obtained by timely and accurate project
tracking.
Additional guidelines for effective project tracking by the PM are to
• Review all aspects of a project at regularly scheduled meetings.
• Make changes between meetings and notify team members.
• Use various tracking tools, timelines, and charts, to communicate
with the project team.
• Keep management and team members informed through meeting
summaries and minutes.
• Consider that changes in project/schedules necessitate both reas-
sessment by the team and changes in resources.
Since biotechnology development projects frequently encounter issues that
must be addressed by the team and since potential delays are not uncommon
to product development operations, tracking is an intensive but important
aspect of project management.
Another metric tool available to PMs and teams is earned value manage-
ment (EVM), which is a means of identifying variances. This, in turn, allows
teams to make more accurate forecasts, which management greatly appre-
ciates, and to recommend changes whenever necessary. Its use depends
upon application of proper metrics. EVM compares work performed to work
planned, as shown in Figure 2.8.

54 Biotechnology Operations
Resources: Planning and Usage
Resources are the people, facilities, equipment, raw materials, and money
applied to a biotechnology development project. The process of allocating
or reallocating resources is referred to as budgeting. Budgets themselves are
negotiated during the project’s planning stage, and executive management
provides a team with a specified amount of resources. Each team member,
having outlined the pathway and schedule to the objective has, for his or
her functional area, identified the resources his or her department needs
to achieve objectives. This process requires great time and effort, some
negotiation, and full justification. The PM is responsible for preparing an
overall budget, by task, year (or quarter), and functional area manager. The
PM tracks progress of the project against consumption of resources and is
responsible for identifying budgetary risks and overages. This is done peri-
odically, and reports are provided to team members and executive manage-
ment. Project management programs, such as Microsoft Project®, are very
helpful, as they provide a means of entering and tracking resource usage as
compared to completion of tasks or achievement of milestones. The result-
ing charts and graphs allow the PM and team members to visualize this
information.
0
20
40
60
80
100
120
5 10 15 20
Ea
rn
ed
v
al
ue
v
s.
ex
pe
ct
at
io
ns
(%
)
Time (wks)
Task 1
Task 2
Task 3
FIGURE 2.8
Earned value versus time. Three tasks, each represented by a distinct line in the graph, are
expected to complete by week 20, a major milestone, of our project. The PM has measured
progress for each task, as a percent of anticipated progress at 5, 10, and 20 week time points in
the project. Now, at week 15 and using this earned value chart, he or she projects an outcome
for each task at 20 weeks, projecting how the curve might appear between weeks 15 and 20.
Task 1, marked by diamonds, has progressed very well, is on schedule, and should complete
by week 20. Task 2, marked by squares, is making progress and may be completed by week 20,
but, then again, it might be delayed. Task 3, marked by triangles, has lagged behind schedule
throughout the project and, without immediate assistance and perhaps some luck, is unlikely
to complete at week 20.

55Project Management
Finances are important to a project, but people are the primary resource and
so a PM considers the many facets of human resource planning and use. The PM
is aware of several human resource factors. First, most staff assigned to a proj-
ect actually report to functional area supervisors. Ultimately, it is the respon-
sibility of functional area managers or supervisors, not of the PM, to manage
staff and performance issues on a day-to-day basis. Indeed, many members of a
project team will be responsible to more than one project, and each of their staff
has additional responsibilities, as well. Furthermore, since human resources
are typically the greatest expense at any company, salaries have a tremendous
impact on the overall project budget. The PM pays particular attention, through
tracking, to utilization of staff and supervisors assigned to their project. Also,
if not enough staff are assigned to a particular technical task, then the project
is at risk of failure. Even if a staff member is assigned to a team full time, their
time is defined and limited. Planning and tracking of human resources are
ultimately and routinely the responsibility of functional area supervisors, but,
for the overall team effort, they are always major concerns to the team’s PM.
Budgeting monetary requirements is a process referred to as costing by
PMs. As noted earlier, there are people costs (internal employees), but there
are also external (e.g., consultant), capital, revenue operating, raw material,
energy, and other project costs. Accurate costing may be beyond the train-
ing or experience of some PMs. Hence, a prudent PM will ask for assistance
from the finance department to plan cost budgets and to serve on or advise
the project management team. Since the financial department is ultimately
responsible for the annual corporate budget and since financial staff have
experience in estimating costs, they can be immensely helpful, even indis-
pensable, to a PM and the team. Indeed, a financial officer is a great asset to
any project team, no matter how technical the objective. Yet even with the
assistance of financial staff, the PM, representing the team, has continuing
responsibilities for planning and tracking a project’s resources. Project plan-
ning tools provide the PM with a means of accomplishing these objectives
throughout the life cycle of a project.
Human Factors in Project Management
A newly appointed PM either adopts a preexisting team, builds, or rebuilds a
project team. We have already discussed administrative aspects of building
teams and human resource functions, but there is another side to the issue.
Even though the PM does not directly supervise team members, they still take
on numerous responsibilities related to coordinating activities of team mem-
bers and they do this in both the planning and performance stages of a project.
A short list of a PM’s human resource duties might include the following:
• Integrating human resource planning and incorporate strategic
planning into the project
• Structuring teams

56 Biotechnology Operations
• Fostering working relationships between team members
• Interacting with supervisors and human resource professionals
• Respecting dual roles or career paths (e.g., discovery scientist and
development leader) of team members
• Leading creative and innovative people
• Remaining open to new ideas from team members
• Managing unique personalities
• Anticipating and then managing change in teams and team members
• Managing conflict within the team
• Trusting team members with scientific and technical expertise
Interestingly, most of these responsibilities match skills that were listed for
effective PMs (Boxes 2.2 and 2.3). Although managing a team can be a daunt-
ing task for anyone, it may quickly overwhelm a new PM, the individual
with little experience or training in interpersonal relationships or in human
resource management. For those with no supervisory authority, human issues
can be the leading cause of anxiety, frustration, and stress. This is particularly
true in smaller biotechnology firms, where the PM may have limited experi-
ence and little or no authority but the responsibility to manage team mem-
bers, some of whom might be brilliant, opinionated, scientifically experienced,
extremely busy, or hold lofty titles. Certainly this is a challenge for any PM.
The PM also ensures a balanced team, one composed of members suit-
able for achieving the objective. Balance must be established at the project’s
initiation and maintained through the project’s lifetime, even as the need for
certain skills varies from phase to phase. The PM spells out roles and expec-
tations for each team member, and, since executive management may not
agree on every proposed position, filling out a team may involve explanation
and negotiation.
The most effective teams have a project champion, sometimes called a
project leader, in addition to the appointed PM. The PM must ensure that the
roles of project champion, a proponent of the technology and often a stake-
holder or influential scientist, do not conflict with theirs. It is important for
executive management and everyone on the team to recognize that the role
of PM differs from that of project champion or from those of each functional
area director serving on the team. These roles are based, to a great degree,
on each organization’s philosophy, organization, and policies. The PM must
understand this and adapt his or her team leadership style accordingly.
Thus, there is a need for the PM to manage egos, the team, and the project.
Not long after project teams are formed, the PM and functional area manag-
ers or directors may come to view each other as different and even difficult
individuals in each other’s minds. Yet, in a successful biotechnology opera-
tion, each person will realize the importance and the contributions of others
on the team.

57Project Management
However, there are instances in which true emotional hostilities break out
between team members or the PM. Dealing with difficult people or difficult
situations is a critical issue in project management. Indeed, some argue that
conflict is inevitable on any project team, even a well-managed one. However,
the PM is well advised to never ignore conflict, but to recognize it, identify
the sources, and work with the team to manage disputes, overt or hidden,
and simmering. There are many sources of conflict, a few of which are listed
in Box 2.7. Conflict is recognized by the PM in many ways and some, such
as body language or facial expressions, are subtle. Conflict must be differ-
entiated from disagreements, which can actually be a positive for team per-
formance as it engages team members in healthy debates and discussions.
However, a real conflict is never to be ignored because it can get out of control
and disrupt progress. More often than not, executive management expects
the PM to resolve conflicts that involve the team or the project and to lead the
team down a pathway paved in productive behaviors. It is often necessary
for the PM to mediate conflicts and negotiate resolutions. There are many
approaches and tools, some of which are shown in Figure 2.9. In summary,
people skills are essential for effective project management and leadership.
Project Completion
Yes, projects are actually completed, perhaps not always on time or within
budget, but, like a movie, each project does have an ending and some are
happy and others sad. Wrap-up is an important part of any project and
BOX 2.7 COMMON HUMAN SOURCES OF
CONFLICT ON PROJECT TEAMS
• Team size or composition not suited to project
• Lag in schedule or task or project priorities
• Technical failures
• Inherent tensions (especially true with matrix organizations)
• Disagreements, often longstanding, on results or decisions
• Individual background or developed styles of team members
(e.g., communication)
• Difficult individual behaviors (e.g., divisive, passive, and
aggressive)
• Senior management intervention or micromanagement of the
team or project
• Personal work styles
• Scarce resources

58 Biotechnology Operations
should be considered by the PM as a separate task. What better way to indi-
cate to the team that an end is in sight than to reveal, early on and in the proj-
ect schedule, a date for the final project meeting! The last meeting includes
a project wrap, simply a review of what has happened: the good, the bad,
and the ugly aspects of the project. The final meeting is a learning experi-
ence as well as cause for celebration. Paperwork, such as reports, and other
outstanding responsibilities are assigned and scheduled. From the meeting,
a lessons learned document is produced for management. It need not be long
or detailed, but it must be honest and reflect actual team performance and
project outcomes. Whether technical aspects of the project succeed or fail,
the team is recognized by the PM and executive management for a job well
done, a team effort.
Lessons learned reports and meetings need not wait for total completion of
a project. They can also be used whenever major milestones are reached or
when a significant risk or issue has been resolved. Such a look-back exercise
allows for team members to share experiences and better prepare for future
challenges.
PM facilitates
communication
Compromise
Problem
solve
Accommodate
ConfrontDirect
Reconcile
Collaborate
FIGURE 2.9
Preventing or resolving conflicts. The project manager facilitates commu ni cation and hence pre-
vents or resolves conflicts by applying various social skills.

59Project Management
Project Management with Contracts and Collaborations
Outsourcing of technical efforts, such as manufacturing, quality testing, and
nonclinical animal or clinical studies, is a common, indeed an important,
practice in the biotechnology industry. Managers from functional areas are
responsible for their contractors, and they assign one individual the respon-
sibility for outsourcing a piece of work and managing the agreement. He or
she has specific technical, project management, and contractual experience
and can best ensure a successful outcome to partnerships and agreements.
The PM seldom has direct responsibility for a contractor or consultant, but
they do consider all contractual efforts as integral to the overall project.
Hence, the budgets and schedule of tasks and milestones for a contractual,
collaborative, or consultant’s efforts are, in all respects, part of the project
and considered in the PDP, WBS, and schedules and reports.
Numerous outsourcing models are available to the PM. Some examples are
as follows:
• Competing several vendors with similar and acceptable capabilities
• Selecting vendors from a list of prequalified contractors
• Partnering with a particular vendor or sole-source contracting with
an established vendor
Virtually any service or material may be outsourced by a firm. Vendors pro-
vide functional and technical services, such as manufacture and regulatory
affairs. Although not generally recommended, the virtual biotechnology
firm may even use a consultant or contractor to provide project management
services and to manage their other contractors, consultants, and vendors.
In addition to managing contractors or vendors, biotechnology firms often
collaborate and codevelop products. These business arrangements are usu-
ally between two biotechnology companies or between a biotechnology firm
and a large pharmaceutical firm. Another business model is partnership
between a biotechnology firm and a contract research organization (CRO).
Some CROs provide services in part for equity in the product and sponsoring
firm. Whatever the business arrangement, the project team at a biotechnol-
ogy firm must follow progress of each and every aspect of codevelopment.
Biotechnology firms often enter into partnerships with larger biotechnol-
ogy or pharmaceutical companies. Here, interfaces may be quite broad and
also have depth, extending well into highly technical endeavors. In a partner-
ship example, one party manufactures product, whereas both parties provide
quality control testing services. Each partner must understand the nature of
the product and the full scope of manufacturing and control. In addition,
they both must have a clear understanding of all technical details in these
areas. Teams can become large, with many functional specialists, up to 50

60 Biotechnology Operations
residing at several locations, comprising a single team. This in itself presents
a challenge to the PM. When developing partnerships in operational areas,
individuals in business may not appreciate future needs for professional
project management to guide the relationship. To ensure success in highly
technical partnership, there is often a need to meld two different technical
and business cultures (e.g., the culture of a large pharmaceutical firm with
the culture of the biotechnology firm), and this requires much coordination
between many individuals at both organizations. It also involves contract
or legal specialists for both parties. PMs are often responsible for forming
integrated project teams and ensuring effective project leadership. Business
development must work with PMs before a partnership deal is consum-
mated, and PMs are well advised to include business developers on their
teams. Alternatively, arrangements with partners may have collaborating
parties working quite independently from each other. In such cases, interface
between the parties is more commonly one on one: between respective PMs
and between like functional area managers from each organization.
No matter what the business or management relationship, strong proj-
ect management experience, leadership, and negotiating skills are absolute
musts if these arrangements are to succeed. Hence, the project team and
manager are fully aware of all contracts and collaborations and the state-
ments of work, roles, and responsibilities of each one.
Virtual Teams
Today many teams can be described as virtual, and this is especially true in
biopharmaceutical development. Twenty-five years ago team members were
typically collocated, but today individuals are often separated by distance,
time zones, and organizational boundaries, and work together as a virtual
team. Add to this the international nature of many teams, complicating mat-
ters with very different time zones, native languages, and cultural practices.
Electronic communication and digital technologies facilitate such project
teams, and many PMs are experienced at working in such venues. The criti-
cal success factors for virtual teams are many; indeed, books have been writ-
ten on just this subject. A few issues are worth highlighting. Cross-cultural
understanding is a must, and it is often the PM’s responsibility to facilitate
cultural understanding and to ensure that confusion and division do not
arise from such matters. It is most critical that every team member has a
well-defined role, one that is understood and appreciated by all other mem-
bers. Cultural differences across organizations, even when they are located
in one country, must be considered. Electronic collaboration and communi-
cation technology must be current, rapid, and dependable. The latest gadget
may not be the best, but modern, proven technologies must be available to

61Project Management
each team member. Face-to-face meetings, to include at least key members
of the team, are essential at least in the beginning and periodically thereaf-
ter. Stakeholders and senior management and supervisors of team members
may be numerous, widely scattered, and reflect the cultural makeup of the
team. Standard team procedures and guidelines are most essential with vir-
tual teams, and it helps to have them written. Despite these issues, virtual
teams have proven to be very effective in the biopharmaceutical industry,
especially when there is excellent and experienced team leadership.
Tools for Effective Project Management
How is it possible to put together a project and then communicate and track
it over several years especially given the complexity, size, and inevitable
changes to many biotechnology efforts? Today, PMs have at their disposal
and at reasonable cost, comprehensive and powerful project management
tools to help in these efforts. Microprocessors and project management soft-
ware provide four areas of project support. They are used to define plans
and schedules, identify resources, track tasks and milestones, and produce
reports. Software will assist the PM in establishing a WBS, listing each task
and placing under it any number of subtasks. This organizes the project so
that the reader discerns project structure and definition.
The process itself is rather simple. The user first prepares a list of what
must be accomplished, breaking the list down in outline format. The list, a
breakdown of the project by tasks and milestones and estimates of the sched-
ules for each, becomes the input for entering each task and subtask into a
project management computer software program. Tasks and milestones are
also linked to each other as a means of identifying and demonstrating depen-
dencies and interrelationships. The software then presents this information
in both graphical and written format to the user; this is the draft or initial
output of a WBS (Figures 2.3 and 2.5). It is then shared by the PM with other
members of the team for review and comment. With a little training, each
team member may now visualize both the overall project plan and WBS and
they then visualize their designated role and responsibilities for the project.
Task and milestone relationships and integration into the overall schedule
are also clear. Potential risks and problems of the intended project become
apparent to each team member when witnessed in graphical format; hope-
fully these weak points can be corrected. The next step in using this tool is for
the reviewer to identify specific steps that might be taken to resolve issues
over the course of the project. The draft WBS, referred to as output, may now
be revised based on recommendations of project team members. This review-
to-revision process is repeated several times before project tasks, milestones,
integration, and schedules are finally established to the team’s satisfaction.

62 Biotechnology Operations
The computer software or program most widely used by PMs today is
Microsoft Project®, but other good programs are on the market. A Gantt
chart, shown in Figure 2.3, depicts the output from MS Project and pro-
vides an example of how this chart demonstrates a WBS. For this example,
the project was divided into functional areas and each was entered as a
line into Gantt format as research, quality control, manufacturing, qual-
ity assurance, preclinical, clinical, or regulatory affairs. Then tasks, each a
technical or administrative step in a project, were entered under a respec-
tive functional area. A task is a piece of work, clearly definable in terms
of technical requirements and schedule. In the example, the tasks Write
Protocol, Perform Study, Write Report, and Report Completed were listed
under the functional area Preclinical. Tasks were further broken down into
subtasks, and this is shown in the example (Figure 2.3) under the area of
Nonclinical and task Perform Study. Here, three subtasks were entered as
Order Animals, Treat Animals, and Observe Animals. As each task or sub-
task was entered, it was assigned an identification number, to the left of the
name, a start and finish date or duration and predecessor tasks, to the right
of the name. For the example of the Preclinical task ID number 21, Write
Report, it began on October 18 and took 6 weeks, until November 28. It had
one predecessor task, ID #20, and successor task #22, Report Completed.
A milestone, which is a major event in the project schedule, is completion
of the report; this was added to indicate the date on which this and other
related tasks were completed.
The software produces a visual in Gantt chart format, to the right of these
entries. Horizontal clear bars identify each task against the schedule or cal-
endar, whereas the overall schedule for each functional area is shown in a
dark horizontal bar. Milestones are visualized in the Gantt chart as dark
diamond shapes. Vertical lines in the chart outline the dependencies of tasks,
shown as arrows leading from a predecessor to a successor task. Other infor-
mation, such as notes and resources, may be inserted by adding columns. If
resources are included, budgets are then calculated by task or subtask and
by specific time period. The integrated nature of each task and milestone is
readily apparent from the chart.
Once a project is underway, tracking functions of the program allow the
PM to compare actual to planned progress. It is also a simple matter to enter
proposed changes and determine how any given change or set of changes
will impact the overall timeline or any other task. Once a change is made
to one task, the hierarchy and schedule are automatically recalculated and
the outcome is made immediately obvious on the revised Gantt chart. For
example, let us suppose the animals do not arrive on July 15, as scheduled in
Task ID #18, but instead arrive 2 weeks later on July 29. By simply changing
this date, from July 15 to July 29, it is possible to learn if this will delay any
other task or even delay or otherwise impact the project completion date.
The project manger is able to demonstrate this to the project team and this

63Project Management
allows the team to consider alternative arrangements or risk mitigation strat-
egies. Indeed, PMs often project Gantt charts at team meetings to demon-
strate to the whole team exactly how a change or delay might impact the
overall sequence of events or schedule. This tool is a very powerful means of
communication.
Project management software also assists the PM with preparation
of reports, outlining various elements and adding to the report-specific
examples and illustrations. In addition to the Gantt chart, these programs
are capable of presenting the project or any stage in the project in other
illustrative formats, such as scheduling charts. As is the case for much of
today’s software, project management software has dozens of other help-
ful functions, too numerous to mention here.
PMs use other methods to visualize a project or to present this informa-
tion to various individuals or groups, such as the project team, investors, or
senior management. Although the Gantt chart may be familiar to members
of a project team, it can be foreign to stakeholders or individuals not familiar
with project management. How then does the PM present the visual repre-
sentation of a complex biotechnology project to an audience that is unfamil-
iar with the Gantt format and the project itself? To speak to such an audience,
the PM uses simpler formats. These are found in commonly available and
easy to use software programs. Examples of two formats, a timeline and a
PERT chart, are shown in Figures 2.4 and 2.5, respectively. Notice how these
simple formats of the information presented in the Gantt chart (Figure 2.3)
allow one to tailor formats to communicate to specific audiences.
Other direct communication tools are available to the PM, and some were
mentioned earlier in this chapter. Some are better for a particular purpose or
situation than are others, but they all serve the PM and team by communicat-
ing the project to those outside the team.
Telephones remain a frequent means of communicating one on one, and
teleconference meetings are a common audio tool used for communication
by the team. Face-to-face meetings are colocated at one site but, for many
teams, meetings in one room are infrequent, occurring quarterly or annually.
Today video conferencing is frequently used, and this technology is typically
dependable and user friendly. Electronic mail (e-mail) is an excellent means
of communication since it is near instantaneous (if the recipients read their
messages) and can accommodate a group of any size. It allows individual
or group responses. However, it is rather impersonal and is not effective
when cross-interactions must be rapid and fruitful and completed rapidly, or
when emotion is a critical component, such as resolving a long-standing dis-
agreement among team members. Also, it is more difficult to reach decisions
or clarify complex technical or scheduling issues by e-mail, and we are all
aware of misunderstandings that arise using this method of communication.
PMs employ a variety of communication methods, enhancing communica-
tion with a mixture of techniques.

64 Biotechnology Operations
Summary of Project Management in
Biotechnology Development
Chapter 4 focuses on project management, the endeavor that pulls together
the biotechnology operation, integrating functional areas into an organized
whole; this is aimed at achieving a common goal, usually the successful
development of a biopharmaceutical product. A project team, managed and
led by a PM, is necessary to achieve such lofty and expensive objectives in
our industry. A team is composed of individuals representing each tech-
nical and administrative aspect of the biotechnology operation. The PM is
the individual responsible for organizing, orchestrating, and monitoring
the various processes, tasks, or work activities of the team. The PM and his
or her authorities and responsibilities must be clearly established by upper
management and identified to the team. The PM is responsible for manag-
ing the project by ensuring proper planning, stimulating effective commu-
nication within the team and to stakeholders outside the team, guiding risk
management and problem solving, keeping metrics on progress, or lack
thereof, understanding and guiding team dynamics, tracking technical and
financial aspects of the project, coordinating with contractors, collaborators,
and consultants, and, unfortunately, providing a place for team members
and management to vent their frustrations. It is critical from the beginning
of a project to follow a written project management plan based on technical
plans previously drafted for the project. The PM also creates and manages
lists of tasks and schedules, so important to establishing, tracking, and mea-
suring a successful project. In each of these endeavors, there is reliance on
credibility, trust, knowledge, experience, and, most importantly, on project
objectives and plans from the standpoint of the team and of the PM.

65
3
Regulatory Affairs
The U.S. Food and Drug Administration: Law and
Regulations for Biopharmaceuticals
Historical Basis for FDA Regulation
Food and drug regulation evolved in the twentieth century, which was a
reflection of major changes in the way in which foods and drugs were pro-
cessed and sold. In the nineteenth century, these products were processed on
a small scale. Grain was milled locally, community butchers slaughtered ani-
mals and sold meat to neighbors, and local pharmacists and physicians formu-
lated and dispensed medications. This changed late in the nineteenth century,
notably in the food industry, as large mills and slaughterhouses became a part
of the Industrial Revolution. But abuses, like the sale of adulterated foods in
some instances, led to social revolt and the desire for governmental regula-
tory controls. Upton Sinclair’s book, The Jungle, a revealing look at practices
in the meat industry, is thought to have stimulated the U.S. Congress to pass
the Pure Food and Drugs Act in 1906 (Sinclair, 1905). For drugs, this act focused
on the need to inform the public about foods and drugs through the use of
honest labeling. A label reveals the contents of a container and cannot provide
false or misleading information in a fraudulent manner. The 1906 Act did not,
however, establish the need for review of each product by a federal agency
before it could be marketed and sold to the public.
This initiative was to come later, in 1938, when the U.S. Congress passed
the Federal Food, Drug, and Cosmetic (FD&C Act or the Act) Act of 1938. This act,
passed in the middle of the Great Depression, changed the Food and Drugs
Act of 1906 in several key ways. First, it no longer required the government to
prove fraud was committed if a drug claimed a curative or therapeutic effect.
It also required a premarket drug review of a New Drug Application (NDA),
in which the sponsor of a product, the company distributing and selling a
drug, provides written evidence that its product was safe and effective. It
authorized other government actions as well: the Federal Trade Commission
(FTC) was to review drug advertising, promotional claims, and material; the
Food and Drug Administration (FDA) would inspect drug manufacturing

66 Biotechnology Operations
facilities and enforce the law and levy fines and punishments and it prohib-
ited false therapeutic claims; and it defined classes of regulated products—
biologics, medical devices, and cosmetics—as existing under these rules. The
FD&C Act of 1938 is the foundation for today’s regulation of drugs, biologics,
and medical devices, and it was the basis for establishment of the FDA.
The FD&C Act and other food and drug laws are often responses by the
public and government to tragic and avoidable situations. The Biologics
Control Act (BCA Act) of 1902 was the result of 10 children contracting teta-
nus after taking a poorly made antitoxin. The Cutter Incident of 1955, in which
children were exposed to live polio virus from a poorly manufactured vac-
cine, was the basis for expansion of biologics regulation. Other amendments
often followed problems, abuses, or deficiencies, perceived and real, in drug,
biologics, and medical device manufacture, control, evaluation, and market-
ing. This trend has continued unabated for 50  years and, unfortunately, it
may be horror stories that lead to additional food and drug laws or amend-
ments in the future. A few of the many acts and amendments in the past
100 years are provided in Box 3.1.
Many products resulting from biotechnology are considered biologicals
or biopharmaceuticals. Two acts of Congress, the BCA Act of 1902 and the
Public Health Service Act (PHS Act), established special rules for biological
or biopharmaceuticals. Today, many biologicals result from biotechnology
endeavors. Through these acts and amendments, Congress has delegated to
the FDA the responsibility of ensuring compliance of biopharmaceuticals.
This, in turn, has profoundly impacted the means by which many biotech-
nology products are developed, manufactured, tested, distributed, and sold
in the United States.
Regulatory Organization of the FDA
The FDA is responsible for protecting the public health by assuring the safety,
efficacy, and security of human and veterinary drugs, biological products,
medical devices, our nation’s food supply, cosmetics, and products that emit
radiation. The FDA is also responsible for advancing the public health by
helping to speed innovations that make medicines and foods more effective,
safer, and more affordable, and helping the public get the accurate, science-
based information they need to use medicines and foods to improve health.
As such, the FDA is a regulatory agency responsible for many consumer
products used in the United States today, which is a huge task, especially
considering the impact these products have on our health and livelihood.
Clearly, it would be impossible for the FDA to oversee or individually inspect
each product item that is sent to consumers. The FDA also communicates
information to various interest groups, pharmaceutical, biological, and med-
ical device industries, to those who distribute or prescribe the products, such
as pharmacies and physicians, and to consumers. Each interest group has
responsibilities for reporting, to some degree. For industry oversight, FDA

67Regulatory Affairs
inspections represent but a small and selected fraction of the material that is
distributed to the user.
The FDA is an agency within the U.S. Department of Health and Human
Services (DHHS) and is composed of various organizational units or offices
and seven centers. These are shown in the current organizational chart in
Figure 3.1. The responsibilities of centers or offices are listed in Box 3.2. Specific
BOX 3.1 EXAMPLES OF FOOD AND
DRUG LAWS FROM 1906 TO 2013
Name and Year Purpose
Pure Food and Drugs Act (1906) Prohibits interstate commerce of adulterated or
mislabeled food or drugs.
Food Drug and Cosmetic Act (1938) Provides for safety testing prior to marketing,
adequate labeling, appoints FDA
responsibility.
Public Health Service Act (1944) Regulation of biological products.
Kefauver–Harris Amendment (1962) Requires drugs have proven efficacy.
Fair Packaging and Labeling Act
(1966)
Honest and informative labeling on consumer
products with FDA responsibilities.
Orphan Drug Act (1983) Encourages development of products to treat
rare diseases.
Federal Anti-Tampering Act (1983) Makes it a crime to tamper with prepackaged
consumer products.
Drug Price Competition and Patent
Term Restoration Act (Waxman–Hatch
Amendments) (1984)
Drug price and completion and patent
restoration. Generic drugs.
Prescription Drug Marketing Act
(1987)
Requires licensing of drug wholesalers, bans
diversion of drugs.
Prescription Drug User Fee Act (1992) User fees established for FDA review of
applications.
FDA Export Reform and Enhancement
Act (1996)
Controls for imported and exported products.
FDA Modernization Act (1997) Regulate advertising.
Pediatric Research Equity Act (2003) Require clinical research to include pediatrics.
Food and Drugs Administration
Amendments Act (2007)
Broaden and upgrade drug safety programs.
Biologics Price Competition and
Innovation Act of 2009 (BPCI Act)
(2010)
Created an abbreviated licensure pathway for
biological products that are biosimilar or
interchangeable with current FDA approved
reference product.
FDA Safety and Innovation Act
(FDASIA) (2012)
Promote patient access to new products.
Drug Quality and Service Act (DQSA)
(2013)
Identify and trace prescription drugs.

68 Biotechnology Operations
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69Regulatory Affairs
BOX 3.2 RESPONSIBILITIES OF SELECTED
CENTERS AND OFFICES AT THE FDA
Office of the Commissioner
Office of Orphan Products
Development (OPD)
Review applications for orphan drug
designation
Office of Combination Products (OCP) Identify primary review office for
combination products
Office of Regulatory Affairs (ORA) Ensure regulatory infrastructure and
enforcement
Center for Biologics Evaluation and Research (CBER)
Office of Biostatistics and
Epidemiology (OBE)
Statistical review and support
Office of Blood Research and Review
(OBRR)
Blood products and device review and
research
Office of Vaccines Research and Review
(OVRR)
Vaccine products review and research
Office of Compliance and Biologics
Quality (OCBQ)
Inspections of biologics facilities
Office of Cellular, Tissue and Gene
Therapies (OCTGT)
Review of genetic therapy and cell and
tissue products
Center for Drug Evaluation and Research (CDER)
Office of Compliance Surveillance, monitor, inspections
Office of Surveillance and
Epidemiology
Epidemiological review and support of new
and approved drugs
Office of Clinical Pharmacology and
Biopharmaceutics
Pharmacology review and support
Office of New Drugs Review of IND, NDA, ANDA
Office of Nonprescription Drugs Review of OTC drugs
Office of Hematology and Oncology
Drug Products
Review of drugs to treat or prevent cancers
Office of Pharmaceutical Science Drug development and testing
Office of Generic Drugs Review of ANDA applications
Office of Biotechnology Products Review of biotechnology products,
therapeutic proteins, and monoclonal
antibodies
Office of Testing and Research Analyze drugs, ensure product quality
Office of New Drug Quality
Assessment
Ensure critical pharmacological attributes,
tests
Office of Translational Sciences Statistics, clinical pharmacology
Division of Drug Information Public and professional information
Office of Medical Policy Review advertising and promotions
(Continued)

70 Biotechnology Operations
responsibilities for biologicals and biotechnology products are outlined in
Box  3.3. Amendments to the FD&C Act typically apply to biologics as well as
drugs. Some therapeutic biological products that had been reviewed and regu-
lated by Center for Biologics Evaluation and Research (CBER) in the past, such
as therapeutic monoclonal antibodies, are now reviewed by FDA’s Center for
BOX 3.2 (Continued) RESPONSIBILITIES OF
SELECTED CENTERS AND OFFICES AT THE FDA
Center for Devices and Radiological Health (CDRH)
Office of Compliance Inspections of device manufacturers
Office of Device Evaluation Review marketing applications for medical
devices
Office of In Vitro Diagnostic Device
Evaluation and Safety
Review marketing applications for in vitro
diagnostic devices
Center for Veterinary Medicine (CVM)
Office of New Animal Drug Evaluation Review marketing applications for
veterinary drugs
BOX 3.3 REVIEW OF BIOTECHNOLOGY PRODUCTS
AT THE FDA. RESPONSIBILITIES BY CENTER
Center for Biologics
Evaluation and Research
(CBER)
Center for Drug
Evaluation and Research
(CDER)
Center for Devices and
Radiological Health
(CDRH)
• Vaccines
• Plasma or serum products
• Blood products
• In vitro diagnostics for
blood
• Gene therapies
• Somatic human or animal
cells or tissues
• Pluripotent cell-derived
products
• Stem cell products
• Allergenic materials
• Antitoxins, toxoids, and
toxins
• Antivenoms
• Combination products in
which the biologic is
primary mode of action
• Monoclonal antibodies
• Therapeutic immune
therapies
• Cytokines
• Therapeutic proteins
derived by
biotechnology
• Enzymes
• Interferons
• Growth factors
• Peptides
• Small molecule drugs
• Combination product
in which the drug is
primary mode of action
• Medical devices of
biotechnology origin
• Radiation-emitting
devices
• Medical imaging agents
• Surgical and therapeutic
• Diagnostic and
Radiopharmaceutical
• In vitro diagnostics
• Combination products
in which the device is
primary mode of action

71Regulatory Affairs
Drug Evaluation and Review (CDER). Finally, certain aspects of biotechnology
product development are regulated by agencies, local, state, national, and inter-
national, other than the FDA. These are also reviewed in Chapter 4.
Food and Drug Law, Regulation, and Guidance
Laws enacted by Congress and signed by the President are the basis for FDA
functions. Regulations are established requirements, developed by an autho-
rized federal agency, to interpret the intent of laws. Regulations interpret the
law, considering the intent of Congress when the law was established, and
they apply technical, scientific, and administrative best practices to fulfill
the law. Regulations cannot be simply mandated by the FDA but must go
through a rulemaking process established by the Administrative Procedures
Act (APA Act) of 1946. The rulemaking process mandates that a regulatory
agency propose to the public every regulation and seriously consider the
comments received in response. Hence, rulemaking is a very transparent
process with significant influence by citizens and organizations. Once the
discussion period is completed, the regulation is then published and goes
into effect. Regulations are placed into the Code of Federal Regulations
(CFR), bound in numbered volumes by functional area. Food, drug, biologic,
and medical device regulations are published in Part 21 of the CFR, hence
reference to 21 CFR. Regulations have the impact of law and, if violated, are
enforceable by authorized law enforcement agencies (Chapter 4).
Regulations alone may fall short in their ability to fully interpret the law
and provide scientific or technical guidance in highly specialized areas.
Thus, regulatory agencies interpret regulations through the use of highly
technical publications referred to as guidelines. A guideline, unlike a regula-
tion, does not carry the weight of law but instead suggests to an interested
party the best technical, scientific, or administrative practices that may be
considered as a means of achieving the intent of a regulation. The FDA has
dozens of guidelines written and updated by scientists working on cutting-
edge technologies; they are available to the public. For individuals working
in biotechnology, guidelines are extremely important resources because they
facilitate targeted planning of product development by identifying scientific,
technical, and regulatory processes. This can greatly increase regulatory
compliance and prevent loss of time in development.
FDA-Regulated Products
FDA regulates a vast array of products, and these will be described as indi-
vidual classes of products along with information about the FDA center that
regulates them. An important aspect of the regulatory planning process is

72 Biotechnology Operations
to understand exactly the nature of a product and to focus regulatory efforts
to that area. Jurisdiction for product review and responsibility at the FDA
would seem obvious, but, with many biotechnology products, this may not
be the case. In  developing a regulatory plan, it is extremely important to
determine jurisdiction, and this is often based on both the type or class and
the intended use of product—information that should be available in the
targeted product profile (TPP) (Chapter 1).
Biologics
The PHS Act defines a biologic as any virus, therapeutic serum, toxin, antitoxin,
vaccine, blood, blood component or derivative, allergenic product or analogous
product…that is intended for use in the diagnosis, cure, mitigation, treatment,
or prevention of disease. For many years, this was construed to mean virtually
all biotechnology products. However, over time and with the advent of
biotechnology, biological products became very diverse. As noted earlier, some
biotechnology-derived products, such as monoclonal antibodies, enzymes,
cytokines, and simple protein therapeutics, are considered well characterized in
that their molecular nature is well known. These well-characterized products
are reviewed by CDER. In this book, the term biopharmaceutical refers to prod-
ucts under the former definition of a diverse universe composed of biologicals
or biologically-derived molecules, cells, tissues, or organisms intended for use
in the diagnosis, cure, mitigation, treatment, or prevention of disease. Since a
biopharmaceutical may be reviewed either by CBER or CDER, depending in
part on nature or indication, it is important to determine for each product the
most likely route of development.
CBER is the FDA lead regulatory office for many biologicals, notably the
blood and blood-derived products, cellular, tissue, and gene therapies and
vaccines. Three offices within CBER (Box 3.2) manage activities and regulate
these products.
Office of Blood Research and Review (OBRR) handles blood products,
most of which are derived from human whole blood or plasma. Even in a
high biotechnology world, human-derived blood and blood products, such
as red blood cells, plasma, platelets, and clotting factors, comprise a large
industry. In addition, recombinant blood products, such as clotting fac-
tors, are approved, and others are in development. Human-derived blood
products are highly regulated from their sources (blood and plasma donor
centers) to the finished product; this is partly because of the risk that such
products could contain adventitious agents. Blood establishments must be
registered, and they are routinely inspected by the FDA. Blood products are
rigorously tested for a wide variety of infectious agents. To ensure the safety
of the blood supply, materials such as blood bags and infusion lines used
in blood collection, handling, and testing are also regulated by OBRR, CBER.
Blood products used for further manufacture, such as platelets or plasma,

73Regulatory Affairs
are subjected to rigorous methods to remove and inactivate viruses such as
hepatitis or human immunodeficiency viruses.
The Office of Cellular, Tissue and Gene Therapies (OCTGT) regulates cel-
lular and gene therapies and human cell and tissue products. Most of these
products are derived from biotechnology, whereas others directly apply bio-
technology methods. Many are live microbial products, such as viral vectors
that have been engineered to carry a therapeutic gene. Others are macromol-
ecules, such as plasmid DNA, intended to have a therapeutic effect upon cell
entry.
Somatic tissues or cells, such as allogeneic skin grown in culture to replace
that of a burn patient, or bone marrow-derived cells, selected for a trait and
expanded in culture, are reviewed by OCTGT as well.
Human organs, such as kidneys, lungs, or livers, intended for whole
organ transplantation, are not regulated by the FDA. However, the agency
does regulate human cells or tissues, or products made from cells or tis-
sues, intended for infusion, implantation, transfer, or transplantation. The
agency also regulates animal whole organs such as pig skin or liver that
might be transplanted into humans. The primary reason for the regulation
of these xenogeneic products is that they are manipulated and are likely to
have a systemic effect in terms of safety. Similarly, although whether a cel-
lular product is autologous or allogeneic is important, again the level and
type of manipulation and potential distribution in the human body are of
primary importance and influence how these products are similarly regu-
lated. The safety of minimally manipulated blood and tissue products is
primarily based on registration of donor centers, careful selection of donors,
and testing of the donated cells or tissues. Current Good Tissue Practices
(CGTP) require special handling of cells or tissues, and any added materi-
als, such as supplements, that are ultimately considered a part of that prod-
uct. Human organs, such as kidneys, livers, or lungs, intended for whole
organ transplantation, are not regulated by the FDA unless this activity is
part of human clinical research.
The FDA has recently proffered guidelines for pluripotent (or stem cell-
derived) products that are now used in a number of early clinical trials. This
area of exploration is in its infancy with several pluripotent cells studies initi-
ated and several hundred studies evaluating stem cells. Stem cell technolo-
gies will continue to mature as early development pathways demonstrate
that safe and effective treatments can be developed from pluripotent cells
realizing their great potential in regenerative medicine and as therapeu-
tic products. For example, the FDA has made clear that nonclinical animal
studies are critical to understanding possible risks. Nonclinical testing must
be designed based on the source of the cell, the intended use and route of
delivery, and data from laboratory studies. The cells themselves must be
fully characterized and major issues, some of which are listed next, must be
defined in the laboratory and in animal studies.

74 Biotechnology Operations
• Mechanisms of action, physiological parameters
• Distribution and persistence in the body or migration to tissues
other than the target
• Original function in the tissue or organ of origin versus intended
therapeutic function
• Differentiation potential
• Ectopic growth potential
• Tumor, benign or malignant, formation
Interestingly, even though these issues may seem unique to stem cells, they
are in general the same questions posed by regulatory agencies to sponsors
of a wide variety of biotechnology products.
The Office of Vaccine Research and Review (OVRR) reviews biological
products that are intended to protect from or cure disease via an immuno-
logical affect. Hence, virtually all vaccines and allergenics, with the possible
exception of cancer vaccines, are under the purview of OVRR. Vaccines, like
genetic therapeutics, represent a wide variety of biotechnologies, too numer-
ous to mention here. All products reviewed by OVRR have in common an
intended mode of action: to elicit an immune response. The indication may
be therapeutic, or it might be preventative. Product types come to OVRR in
a vast array of technologies, and today most are derived from biotechnol-
ogy; only a very few candidate vaccines, such as influenza, remain as natural
products. Live bacterial or viral vectors, such as Escherichia coli or adenovi-
rus, are used both to stimulate an immune response to that organism or as
carriers intended to stimulated immune responses to other proteins geneti-
cally engineered into the host. Vaccines may be given by oral, intranasal,
intramuscular, epidermal, or other routes. Vaccines today are often part-
nered with a delivery device, making a combination product. Most biotech-
nology products engineered and intended to stimulate an immune response
goes to OVRR for review. Vaccine adjuvants, molecules intended to improve
immune response when given with an antigen, are also reviewed. Allergenic
products, often natural substances purified and then used to treat allergies
or in hypersensitivity testing, are products reviewed by OVRR as well.
CBER also reviews unique types of biological products. Exact regulatory
pathways have not been developed for every type of product that might be
conceived by the biotechnology industry. It is worth noting that one class of
biologics, those derived from plants, are also reviewed by CBER. Drugs and
biologicals may be derived from bioengineered and selectively bred plants,
and these may be reviewed by CBER or by Center for Drug Evaluation and
Research (CDER) or by both centers. One such drug is an antimalarial drug,
which was in short supply worldwide in 2011. In this situation, the product
receives review with special attention paid to the recombinant nature of the
plant from which the product, not itself a recombinant molecule, is derived.

75Regulatory Affairs
The basis for regulation and review may be based largely on the indication
and manner in which the product will be used and less on the nature, drug
or biological, of the product.
All centers and offices within the FDA and the drug regulatory agencies
of other developed countries play a significant role in providing support
for therapeutic products and vaccines used in global health, even if those
products will not be licensed in the United States. This is due in part to the
FDA’s leading international reputation for scientific product review and also
because many target countries cannot afford to have in their governments
a food and drug authority. When a biopharmaceutical firm applies to test a
product indicated for global health in the United States, it will be reviewed
by the FDA and comment will be provided. Indeed, most global health prod-
ucts are first tested in the United States or another developed country before
being fielded in developing countries, where it is necessary to conduct field
trials and it is desirable to manufacture commercial product. Hence, the
FDA, along with institutes and academic centers in the United States and
other Western countries, provides early product development support and
advice that can be transferred to target countries.
Drugs
Drugs are broadly defined as products used to mitigate, treat, or prevent
disease in man and that affect physiology or anatomy of the human body.
The industry standard for a drug is a small or large molecule that has a well-
defined chemical structure. Following market approval, drugs are recognized
as such in reference texts, such as the United States Pharmacopoeia (USP) or the
National Formulary (NF) (Chapter 7). The USP and NF are published by a gov-
ernment- charted laboratory, the United States Pharmacopeia. Pharmaceutical
is synonymous with the word drug. The historical definition of drug is of a
small molecule, or ethical, pharmaceutical, a compound that is synthesized
from nonbiological sources. Examples of drugs under this definition are acet-
aminophen or aspirin, antibiotics such as penicillin, and the anesthetic ether.
Drugs of this nature are under the purview of the FDA’s CDER (Box 3.2).
Certain molecules of biological derivation, such as monoclonal antibodies and
therapeutic proteins, are reviewed under CDER even though they do not meet
the historical definition of drug. Prescription drugs or biopharmaceuticals are
those products that must be prescribed by a licensed medical professional and
dispensed by a licensed pharmacist. Over-the-counter (OTC) drugs are those
that do not require a prescription. Most biopharmaceuticals are dispensed by
prescription.
Generic drugs are drugs that no longer have marketing exclusivity due to
patent protection but are still regulated under drug regulations. Generics
must be tested in adequate clinical studies, albeit abbreviated, and they must
receive marketing approval from the FDA under the Abbreviated New Drug

76 Biotechnology Operations
Application (ANDA). The Waxman–Hatch Act of 1984 was instrumental in
establishing regulations that guide marketing approval of generic drugs.
CDER, in addition to reviewing small molecule drugs or pharmaceuticals,
also takes the lead for review of a number of biopharmaceuticals. Examples
are shown in Box 3.3, but the list is growing as new biopharmaceuticals
enter clinical trials or receive market approval. Recombinant therapeutic
proteins are macromolecules or peptide products intended to treat disease.
Responsibility for review or co-review within CDER may also be deter-
mined by the indication or intended use. This is because CDER is also orga-
nized by disease area. Hence, a therapeutic recombinant protein intended
to treat gastrointestinal cancer might be reviewed by the oncology group,
specifically by the Division of Biologic Oncology Products (DBOP) group.
A monoclonal antibody protein directed against an infectious bacterium of
the gastrointestinal tract could receive primary review from the anti-infec-
tive group and consulting review by the gastroenterology group. Another
example of a biopharmaceutical product falling under CDER is monoclonal
antibodies. They represent a class of molecules that are produced in vitro
as a result of genetic engineering. Monoclonal antibodies are produced to
react with a variety of target proteins and to have a therapeutic effect on the
patient. Examples are antibodies directed against inflammatory cytokines;
these are engineered to stop undesirable inflammation due to autoimmune
diseases. They are produced in genetically engineered, immortalized, and
cloned cells in much the same way as other recombinant proteins. Today
we understand the molecular structure of most monoclonal antibodies.
Therapeutic monoclonal antibodies are reviewed by CDER with primary
responsibility resting in its Office of Biotechnology Products (OBP), but
may also be reviewed by experts in the disease. In the case of the mono-
clonal antibody directed against an inflammatory cytokine, rheumatolo-
gists employed in another division of CDER might work closely with OBP
on review of regulatory documents. In conclusion, the division of review
responsibility for a given biopharmaceutical may, in CDER, be more diffuse
than it is for one at CBER.
Whichever division or office within CDER or CBER has primary respon-
sibility for review of a given product, there might also be specialists from
another center involved in the review of that product as well. There are
specialists within each of the centers for functional areas such as nonclini-
cal studies and toxicology, clinical trials, manufacturing, and quality con-
trol. Hence, review of applications by any FDA center represents a team of
experts, often from a variety of offices or centers.
Medical Devices
This major class of products includes instruments, prostheses, delivery tech-
nologies, in vitro diagnostic tests, implants, apparatus, and a host of other
engineered yet nondrug and nonbiologic items. Regulatory responsibility for

77Regulatory Affairs
medical devices rests with FDA’s Center for Devices and Radiological Health
(CDRH). Examples of medical devices range from tongue depressors to cardiac
pacemakers, from dental amalgams to CAT scanners, and from devices and
software to transmit X-rays to HIV test kits. Unlike drugs and biologics, medi-
cal devices are classified, a priori, based on their risk to the user and the level of
regulatory control, review, and safety concern. Under an established hierarchy,
medical devices with the greatest risk to the user are given the highest classifi-
cation. A tongue depressor is Class I, General Controls, a test for the common
flu virus is Class II, General and Special Controls, and a heart–lung machine
is Class III, Special Controls and Premarket Clearance or Premarket Approval
(PMA). In general, the FD&C Act requires safety testing for all devices, but
does not require testing for efficacy studies of Class I and Class II medical
devices. Most Class III devices need some assurance of adequate performance.
Many Class II devices are eligible for regulatory approval under a regulation
referred to as 510(k) approval if they are substantially equivalent to a predicate
device. Manufacturers find this an attractive rapid means of seeking regulatory
approval from CDRH, but it only applies to certain devices. There are many
other differences in the regulatory requirements and review and structure of
processes leading to market application and approval for devices as compared
to drugs and biologics. Although few biotechnology products are considered
medical devices, some are a combination of a device and a drug or biologic.
Combination Products
Many biopharmaceutical products are not simply classified as biologics,
drugs, or medical devices, but instead are a mixture of these product classes.
For example, a drug aimed at a blood cancer cell, historically a drug, may be
attached to a protein, a biologic, that targets the cancerous cell. A recombi-
nant vaccine protein, a biologic, may be delivered to the skin using a novel jet
injector, a medical device. The combinations and permutations seem limit-
less, with new biotechnology combination products appearing daily.
The FDA accommodates these so-called combination products through the
Office of Combination Products (OCP) responsible for defining jurisdiction
and ensuring a coordinated review of applications. Jurisdiction is based on
the primary, or most important, mode of action for the product. It is critical to
identify and then to evaluate every potential combination product early in the
planning process so as to establish the most likely regulatory path forward.
The first step is to determine if a product is, in fact, a combination product
in the eyes of the FDA. Referring to examples cited earlier, the drug would
provide the primary mode of action because it would kill the cancer cells, and
CDER is likely the lead for review. The vaccine protein would be primary for
that example because it elicits and thus provides the desired outcome: protec-
tive immunity. Although a paradigm such as the one shown in Figure 3.2
helps a sponsor to identify the primary review office, consultation with the
FDA, through a formal Request for Designation, is recommended.

78 Biotechnology Operations
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79Regulatory Affairs
Other Classes of Biotechnology Products and Their Review at the FDA
Products for Veterinary Use
Biologicals, including biotechnology products used to treat or prevent disease in
domestic animals, are largely regulated by the U.S. Department of Agriculture
(USDA) and not by the FDA. However, the Center for Veterinary Medicine
(CVM) of the FDA is designated for animal drugs and medicated feeds. CVM
reviews marketing applications, ensuring such products are safe and effective
for their intended use. The process is similar to that applied to human drugs,
and includes the need to file an Investigational New Animal Drug (INAD)
exemption to support investigational use or a New Animal Drug Application
(NADA) to receive product-marketing approval. Animal feeds, both those that
contain medications, such as antibiotics, and those without supplements, are
under the purview of CVM, because they should not contain harmful sub-
stances and must be properly labeled. Biotechnology-derived products could
fall under both the medicated and nonmedicated classifications of animal
feeds, and these products must receive marketing approval from CVM.
Cosmetics, Food, Dietary Supplements, Homeopathic,
or Nutritional Products
Regulations do exist for cosmetics, products that are applied externally, but
have no claim of therapeutic value. Cosmetic regulations, as compared to
drug rules, are relatively simple, yet they ensure these products are safe and
not adulterated. A biotechnology-derived product could, by intended use, be
considered a cosmetic and, if so, cosmetics regulations would apply.
Products that are nutritional in nature and for which no claims are made
of a therapeutic or medicinal effect are regarded as a food by the FDA. The
FDA regulates most food products, with the exception of meat and a few
other animal-derived items that fall largely under the U.S. Department of
Agriculture’s purview. Additives for food and food colors do fall under the
purview of the FDA. Foods and additives are not considered in this chapter.
Dietary ingredients and dietary supplements are not subject to premarket
approval by the FDA, but the agency must be notified of marketing intention
nonetheless. These products are regulated by the FDA based on their nature,
but largely on the indication and the therapeutic or preventative claims
made by the sponsor. Classification of such products requires experience as
the complex guidelines are based on nutritional or medicinal claims made
by the sponsor, which themselves are often difficult to parse or comprehend.
Dietary supplements and nutritional products are now generated by bio-
technology, although the vast majority are still naturally derived or chemi-
cally synthesized. Dietary supplements, regulated largely as food and not
as drugs, are products taken by the mouth and intended to supplement the diet
and not intended to treat, diagnose, cure, or alleviate the effects of disease. Again,
the claim made on the product label is instrumental in determining the level

80 Biotechnology Operations
of product regulation and the designated regulations and review process at
the FDA. If the intended probiotic use is for therapeutic purposes, then the
associated applicable regulation under the FDA would change accordingly.
This shift in probiotic use is an example of regulatory evolution, that is how
historic regulation is applied to novel or modified products that will now
provide therapeutic benefit if given in accordance with its intended use. An
example of this, a traditional food not marketed as a food supplement with
claimed medical benefit and as a human therapeutic, is briefly described in
Box 3.4. Certain homeopathic compounds may fall under similar rules and
BOX 3.4 NONTRADITIONAL USE OF FOOD
SUPPLEMENTS AS A THERAPEUTIC
• Probiotics are microorganisms that have been traditionally
used to fortify the gut microbiota. They are regulated by the
FDA as a food supplement. As fortifiers, many probiotics have
been given Generally Recognized As Safe (GRAS) status by
the FDA.
• The potential health benefit and ability of probiotics to prevent
or cure disease have been gaining scientific traction over the
past decade; a cause–effect relationship continues to be sought
that includes clues of strengthening the immune system.
• If the intended use of a probiotic being provided to people is to
study, diagnose, cure, mitigate, treat, or prevent a disease, then
the regulation of a probiotic is as a therapeutic and falls squarely
within the purview of the medicinal branch of the FDA.
• Likewise, a study to evaluate the ability of bacterial coloniza-
tion to treat or prevent disease in patients with an immune dis-
order must be performed under an IND application submitted
to the CBER at the FDA.
• As a therapeutic, both safety and efficacy will be carefully eval-
uated by the FDA to minimize human risk and hopefully dem-
onstrate human benefit outweighs potential risks of human
exposure.
• As the increased use of probiotics continues to be investigated
for their ability to promote a positive host response to live
organisms (e.g., bacteria, fungi, or virus), the FDA issued a
draft guidance to assist with the development of these prod-
ucts intended for therapeutic purposes (Guidance for Industry:
Complementary and Alternative Medicine Products and Their
Regulation by the Food and Drug Administration, December
2006) (FDA, 2006).

81Regulatory Affairs
have their own set of definitions, depending on claims made on therapeutic
value. Certain homeopathic compounds are formulated in pharmacies for
individual patients, a process that is legal in the United States under spe-
cific circumstances. As with nutritional supplements, the indication deter-
mines the level of regulation, as pharmacies typically fall under the laws
of individual states. With homeopathic medicines, there is a point at which
the distribution or volume of sales can conflict with this definition. Then the
FDA regulations may be brought to bear, and the product may be declared a
drug, biological, or device. Few biotechnology products produced for thera-
peutic purposes would be considered homeopathic medicines or nutritional
products.
FDA Regulatory Information and Resources:
Regulatory Intelligence
This section focuses on the functions of FDA regulatory intelligence and a
biotechnology firm’s need to continually obtain information that allows an
understanding of the regulatory environment as it applies to the develop-
ment of a product. Also, a prudent biotechnology firm will have in place
processes to abstract and communicate FDA regulatory information to
operational staff and upper management. Sources of information on non-
FDA regulatory bodies are described in Chapter 4.
Regulatory intelligence is the process of finding and analyzing publically
available regulatory information. It is not necessary, nor is it possible, to
know all regulations that apply to every biotechnology product; it is more
important to understand the overall environment and possible sources of
regulatory information and to be able to locate information on particular
types of products.
Gaining regulatory intelligence is always a first step in preparing a regu-
latory plan. To find pertinent regulatory information on a particular prod-
uct or similar products, it is necessary to first understand the product and
indication, as provided in a TPP; then a structured search is initiated. The
search begins as a broad investigation to understanding the regulatory
environment using general and specific sources. This provides a general
background on the subject. It then focuses on areas of interest, moving from
one regulatory source to another until it seems that all regulatory databases
have been exhausted. Scientific databases list additional product informa-
tion, and these are examined. The result is a bibliography that provides a
regulatory history and scientific background of the product and product
class, an exact idea of the current regulatory environment, and insights into
future regulatory initiatives. Taken together, this background information

82 Biotechnology Operations
serves as a foundation for preparing the regulatory plan and a basis for
later updated information on the product and its regulation.
Today, public regulatory information, with the exception of regulatory
textbooks and some international guidelines, is available on the Internet
at no cost to the user. Hence, the key to finding the information is to use a
library of websites for searches and to apply search engines. Some searchers
use regulatory information blogs to network with colleagues, an approach
that is particularly helpful for locating very specific bits of information.
Also, before beginning a search, it is a good idea to develop a method to
catalog the information so that it is easily retrieved for review and reference.
Most regulatory libraries are electronic databases. Simple systems, such as
using office support software with desktop search capabilities, are fine for
smaller databases, although more complex and dedicated software is avail-
able for regulatory libraries consisting of thousands of references.
National regulatory agency websites, such as http://www.fda.gov, and
international or harmonization sites, such as http://www.ich.org, are a wealth
of information on FDA regulations and international standards, sometimes
providing far too many resources outside the intended scope. Compendia
methods, standards, and monographs can be readily obtained from U.S.
Pharmacopeia National Formulary (USPNF) via hard copy or is assessable
online at http://www.usp.org. Scientific literature through PubMed or a gov-
ernment and institution or university library search engine provide jour-
nal articles of a regulatory or technical nature. Professional and industry or
trade journals post articles on various regulatory topics and recent articles
offer insights into recent or hot topics.
Review and careful evaluation of brochures or dossiers of similar cur-
rently marketed products may prove to be extremely valuable in under-
standing the precedence for this type of product. Valuable product-specific
details of priority nature such as methods or manufacturing components
may be obtained from vigilant searches of the public access U.S. Patent and
Trademark Office (http://www.uspto.gov) dbase. A patent search is likely
to provide critical information regarding freedom to operate and market-
ing or potential current market exclusivity.
Regulatory intelligence does not end with an initial search but contin-
ues through the product lifecycle. Information is obtained through peri-
odic searches of websites, by using commercial regulatory intelligence
software, and through e-mail alerts, many provided by various govern-
ment agencies including the FDA, by using blogs and through diligent
personal networking, by e-mail, from professional meetings, and by
networking with other professionals. Some long-standing and helpful
U.S. sources of intelligence on biopharmaceuticals are given in Box 3.5,
although other sources, notably international guidelines, are described
in Chapter 4.

http://www.uspto.gov

http://www.usp.org

http://www.ich.org

http://www.fda.gov

83Regulatory Affairs
BOX 3.5 WEB-BASED SOURCES OF U.S.
REGULATORY INFORMATION
U.S. Government Regulatory Trade Organizations
Food and Drug
Administration
http://www.fda.gov Regulatory Affairs
Professional Society
(RAPS)
http://www.raps.org
National Institutes
of Health
http://www.nih.gov Drug Information
Association (DIA)

HTTPS

diahome.org
U.S.
Pharmacopoeia
http://www.usp.org Food and Drug Law
Institute (FDLI)

Home

Clinical Trials
Registry

HTTPS

clinicaltrials.gov
American
Association of
Pharmaceutical
Scientists (AAPS)

HTTPS

aapspharmaceutica.
org
PubMed (National
Library of
Medicine)
http://www.ncbi.nlm.
nih.gov/pubmed
Pharmaceutical
Education and
Research
Organization
(PERI)

Home

California Institute
for Regenerative
Medicine (CIRM)
http://www.cirm.ca.gov International
Conference on
Harmonization
(ICH)
http://www.ich.org
Medical Dictionary
for Regulatory
Activities
http://www.meddra.
org
World Health
Organization
(WHO)
http://www.who.int
FDA News and
Announcements

HTTPS

GXPnews.com
Regulatory Newsletters
Parenteral Drug
Association (PDA)
http://www.pda.org

HTTPS

fdcreports.com
http://www.foi.com Biotechnology
Organization (BIO)
http://www.bio.org
http://www.foi.
com
http://www.bioworld.
com
Pharmaceuticals for
Practitioners

HTTPS

pharmaportal.com

HTTPS

fdanews.com

HTTPS

fdaadvisorycommittee.
com
Association of
Clinical Research
Professionals
(ACRP)
http://www.acrpnet.
org
The Organization for
Professionals in
Regulatory Affairs
http://www.topra.
org
Applied Clinical
Trials

HTTPS

appliedclinical
trialsonline.com

http://www.appliedclinicaltrialsonline.com

http://www.appliedclinicaltrialsonline.com

http://www.topra.org

Home

http://www.pharmaportal.com

http://www.GXPnews.com

http://www.meddra.org

http://www.aapspharmaceutica.org

http://www.aapspharmaceutica.org

http://www.diahome.org

http://www.fdaadvisorycommittee.com

http://www.fdaadvisorycommittee.com

http://www.bioworld.com

http://www.fdanews.com

http://www.foi.com

http://www.fdcreports.com

http://www.ncbi.nlm.nih.gov/pubmed

http://www.clinicaltrials.gov

http://www.appliedclinicaltrialsonline.com

http://www.topra.org

Home

http://www.fdaadvisorycommittee.com

http://www.fdanews.com

http://www.pharmaportal.com

http://www.bioworld.com

http://www.foi.com

http://www.bio.org

http://www.foi.com

http://www.fdcreports.com

http://www.pda.org

http://www.GXPnews.com

http://www.who.int

http://www.meddra.org

http://www.ich.org

Home

Home

http://www.ncbi.nlm.nih.gov/pubmed

http://www.aapspharmaceutica.org

http://www.clinicaltrials.gov

Home

http://www.usp.org

http://www.diahome.org

http://www.nih.gov

http://www.raps.org

http://www.fda.gov

84 Biotechnology Operations
Regulatory Operations for FDA Applications
Regulatory Planning and the Regulatory Environment
Biotechnology endeavors are influenced in many ways by regulations and
the regulatory environment. To succeed, the biotechnology professional
must understand the regulatory landscape lying ahead before embarking on
product development. This is achieved by preparing a regulatory plan, an
early and important part of any product development strategy (PDS). Benefits
are obvious. It builds a framework for the overall operational plan and serves
as a foundation on which many aspects of nonclinical, clinical, manufactur-
ing, and control planning may be built. A regulatory plan identifies poten-
tial regulatory hurdles through inspection of product-specific regulations
and regulatory agencies. It allows a biotechnology firm to communicate two
messages to outside parties, investors, or potential partners: they will be
successful, in part, because they have identified regulatory issues specific
to their product and they intend and plan to address those matters. A well-
considered regulatory plan goes beyond reacting to the environment and
also identifies regulatory opportunities such as unmet needs or accelerated
pathways to enhance product value. Indeed, a well researched and reasoned
regulatory plan seeds new ideas into a biotechnology firm’s business plan.
Novel biotechnology products often sail into uncharted regulatory waters.
There are new diseases or unique indications for novel diseases, product
lines marketed never before, and radically new technology approaches to
unsolved problems. As noted earlier, the breadth of biotechnology products
is enormous, meaning that regulatory requirements cut a very wide and
deep swath. The regulatory plan for a given product may need to cover a
number of diverse areas and also delve into each of these areas, perhaps
setting precedent in one or more areas. Elements of a regulatory plan are
given in Box 3.6 and discussed further in Chapter 1. In biotechnology, the
regulatory planner is challenged from many directions. Even if regulatory
pathways are clear and simple with much precedent, a well-conceived regu-
latory plan adds product value by demonstrating how the product may be
a regulatory slam-dunk. The time and effort involved in preparing a regula-
tory plan are justified for any biotechnology product with reward derived
from a well-planned and efficient regulatory approval process.
Risk Versus Benefit
The FDA is chartered to protect the public health. Hence, at the heart of
regulatory judgment for any given product is the need to weigh risk versus
benefit, as it relates to individual and public health. This can be challenging
for a biotechnology firm, especially the small or virtual operation, to appre-
ciate. Nonetheless, it is critical at some point to think like the regulators and

85Regulatory Affairs
BOX 3.6 ELEMENTS OF A REGULATORY PLAN
• The product
• Characteristics
• Class
• Competitor or equivalent
• Indications: primary and secondary
• Limitations in safety or effectiveness, stability, shipping/
handling, scaling, price competitive, and population
• Special safety considerations
• Product blemishes
• The regulatory environment
• Scope of applicable regulations: Global, national, state, and
local
• Purposes for regulations: Medical, agricultural, environ-
mental, and safety
• Regulatory agencies or guidelines: FDA, USDA, ICH,
European, Japanese, and rest of world
• Laws, regulations, and guidelines
• Precedence for product class or competitor products
• Global strategy
• Global market targets
• Sequence or timing of applications
• Exclusivity by market
• Application and approval methods by market or country,
ex-U.S.
• Influences on regulatory environment
• Political: U.S. and ex-U.S.
• Social: U.S. and ex-U.S.
• Advisory committees
• Local or state authorities
• Special interest groups
• Regulatory communication
• Formal applications: IND, NDA, BLA
• Written communication: Letters, amendments
(Continued)

86 Biotechnology Operations
consider the risk-to-benefit balance that a product must bring to the market.
Any sponsor has surely considered the benefit that his or her product might
provide to mankind. In fact, the benefits of any biotechnology product have
most probably been touted to the world, in an effort to garner financial and
public support, during research in early development phases. But it is not
typical, certainly not in the early stages, of biotechnology development, to
admit that a product also carries risks; it is counterintuitive to the entrepre-
neurial environment to delve deeply into the possibility of product-associ-
ated risk. Traditionally it is not good business practice to highlight potential
product risks to the public. However, the FDA must, by law, consider the
possibility that any product can cause harm, even that the risks might out-
weigh the benefits. Regulatory agencies look at any product as possessing
possible risk as well as benefit and they have an obligation, as part of their
mission to public health, to evaluate and, sometimes, to publicize product
risks. This is a reason some biotechnology firms are asked by the FDA to
place a black box warning onto the labels of a marketed product. These dif-
ferent ways of thinking create a tension between biotechnology firms and
regulatory agencies.
In developing a regulatory plan throughout the development cycle, it falls
on regulatory and quality professionals to carefully weigh product benefits
against risks and pass the results of this analysis along to members of the
product development team. This is done so that risks can be addressed before
they become an issue with regulatory authorities or, worse, cause harm to
public health. Regulatory risk identification and management begins early
BOX 3.6 (Continued) ELEMENTS OF A REGULATORY PLAN
• Meetings or teleconferences
• Public conferences
• Timing of each major communication
• Options for special designations and pathways
• Accelerated approval
• Emergency use and treatment
• Orphan product designation
• New drug product exclusivity
• Fast track designation
• Breakthrough therapy
• Priority review
• Rolling review
• Regulatory risks

87Regulatory Affairs
in the development cycle, as a formal and broader process of product and
project risk analysis and management, as discussed in Chapters 1 and 5.
Here, we point out the simple fact that regulatory agencies perceive poten-
tial risks early in a product’s life cycle. If risks are not recognized as such by
the sponsor, then the FDA may demand that they be both recognized and
mitigated. This process may go on through the entire cycle of product devel-
opment. Failure to heed warnings by the FDA on risk can lead to regulatory
action by the FDA. The regulatory professional is often the biotechnology
team member to elicit support of product development team members to
identify and manage technical risks so that they do not later become prod-
uct or regulatory risks.
If after a careful evaluation, it is determined that a product’s risks out-
weigh benefits and these risks cannot be effectively mitigated, then a biotech
firm is encouraged to withdraw or fail the product early in the development
process to ultimately minimize losses and avoid potential harm to people.
Applications Seeking FDA Investigational Use or Marketing Approval
The development life cycle for a biopharmaceutical is heavily influenced
by regulatory requirements, hurdles some might say. These requirements
are considered met only after a biotechnology firm has communicated
scientific plans or results to the FDA. Indeed, the regulatory process is a
dialogue between the agency and the sponsor. The sponsor, a legal entity
responsible for the product, may be an individual, but, in most cases, it
is a corporation or an institute. A sponsor such as a biotechnology firm
has one individual responsible for signing regulatory documents, and this
person is the designated sponsor’s representative. During the develop-
ment life cycle, a dialog between the FDA and the sponsor’s representative
is carried on through a series of meetings, teleconferences, and written
documents. These steps in communication with the FDA can and should
represent important milestones in meeting objectives in the overall prod-
uct development cycle. The overall sponsor–FDA communication process,
shown in Figure 3.3, is much the same for drugs and biologics even though
the nomenclature differs in some respects. Certain aspects of medical
device development, including major documents, can differ significantly
from drugs or biologics.
The FDA regulatory process begins with a designation by the sponsor that
a candidate product will be used in humans for the treatment or prevention
of a disease or disorder. The process of developing a TPP (Chapter 1) is criti-
cally important in part because the sponsor clearly identifies the intended
use, indication, and nature of the product. Consideration as a medical device,
a drug, a biologic, or combination product commits to a regulatory pathway
that has been established by precedent and regulation or guidelines. Thus,
the TPP process is a critical first step in the development of a PDS and for
regulatory planning.

88 Biotechnology Operations
Biotechnology research
laboratory:
Potential product
Intended for
human use?
Drug or biologic?
Nature of product
and intended use (indication)
Veterinary or
laboratory use
Target product profile and
product development
strategy
Briefing for pre-IND
meeting
Phase 1 studies
Phase 2 studies
Pre-IND meeting with
FDA
Phase 3 studies
Yes
No
Complete technical
requirements,
prepare IND
Pre-phase 2 meeting
with FDA (optional)
Pre-phase 3 meeting
with FDA
Prepare
BLA or NDA
File BLA or NDA
Pre-BLA meeting with
FDA
Correspondence and
meetings with FDA
BLA or NDA information
filings and reports
File IND
Phase 1 study report,
annual reports
Phase 2 study report and
annual reports
Reports and amendments
Phase 4 studies,
promotional review
and reports
REMS
Correspondence and
meetings with FDA
FDA concurrence
Marketing approval
FIGURE 3.3
Regulatory activities and milestones in biopharmaceutical development.

89Regulatory Affairs
Investigational Use Applications. The Investigational
New Drug Application
The Investigational New Drug (IND) application is a request to the FDA to
perform human safety and effectiveness studies on a biopharmaceutical or
drug. The IND application often represents the first legally binding docu-
ment that is submitted by a sponsor to the FDA. However, the IND filing
at the FDA is often preceded by less formal means of communication, such
as a pre-IND meeting or teleconference. A new drug is a construct or mol-
ecule that has an ingredient or combination of ingredients that have not
yet received marketing approval. A product is considered investigational,
although being evaluated for safety and efficacy under an IND. Indeed,
even a compound with a slight molecular variation from an approved com-
pound may necessitate filing an IND with the FDA to investigate safety
and efficacy. Products approved for an indication are also considered a new
drug if they are to be used (investigated) outside the approved label claim
or used for another indication. The investigational use of a new drug is
the use of that new drug in a human clinical study or studies by or on
behalf of a sponsor (Chapter 9). The IND process focuses on submission,
by the sponsor to the FDA, of a clinical protocol, a written plan to test that
drug in the first clinical study ( first time in man) and the scientific data and
background information related to that drug. The format for an IND can
be found at http://www.fda.gov. Many other countries have an investiga-
tional process and associated applications, but the process and format vary
across borders.
In the Unites States, the sponsor and FDA are both responsible to the
public for ensuring that a new drug will not present unnecessary risks to
those receiving it and that any risks are balanced with potential benefits.
The contents of an IND, outlined in Box 3.7, are standard for any IND,
and the sponsor is responsible for generating this application and sub-
mitting it to the FDA. In doing so, the sponsor must attest to the validity
of data and background information provided to the FDA by completing
and signing a form (FDA Form 1571). The information provided in an IND
is critically important to the FDA, as this represents the basis for their
review and determination of risk versus benefit assessment. As shown in
the IND format (Box 3.7), the information comes from a variety of sources,
which are described in Chapters 1,4 through 6,8, and 9. Of considerable
importance is Section 6 of the IND, providing the Clinical Protocol and
other clinical investigation documents, which are described in Chapter 9.
The nonclinical information (described in Chapter 8) and any laboratory
studies related to laboratory studies, such as biodistribution, are provided
in Section 8 of the IND. Previous human use of this or closely related
products are communicated in Section 9 of the IND. Section 7 of the IND
is reserved for chemistry, manufacturing, and control of the product
(described in Chapters 6 and 7).

http://www.fda.gov

90 Biotechnology Operations
BOX 3.7 CONTENTS OF AN IND
• Section A(1)*: Cover page is Form FDA 1571
• The sponsor’s statement to certain questions about the
product and responsible individuals is placed onto a stan-
dard form provided by the FDA. Signature sponsor’s repre-
sentative. Delegation of responsibilities.
• Section B(2): Table of contents.
• Section C(3): Introductory statement and general investiga-
tional plan.
• A review of the background, perhaps noting the research
basis for the product, information on the disease or condi-
tion, and an explanation of why the product was chosen to
treat or prevent the indication.
• Section (4): Reserved for FDA requests.
• Section D(5): Investigator’s Brochure (IB).
• IB is a summary of critical information found in the IND,
and is written by the sponsor to fully inform the clinical
investigators and staff. Not required for single center spon-
sor–investigator IND.
• Section E(6): Clinical protocols
• The protocol describes in detail the nature of the study
and all procedures that will be taken during the clinical
trial. Supportive documents include the Form FDA 1572, a
signed statement of the principal investigator, clinical pro-
tocol synopsis, CVs of clinical staff, sample consent form,
and other documents related to the clinical investigation.
• Section F(7): Chemistry, manufacturing, and control information
• Here the sponsor describes the manufacture of the product
and provides detailed information on the raw materials,
process, and facilities. Quality control assays are identified
and certificates of analysis presented. An environmental
statement is required.
• Section G(8): Pharmacology and toxicology information
• All nonclinical studies are described with full study reports.
All nonclinical or preclinical studies that are related to
(Continued)
* The FDA has used two systems, one numbering (1–10) and the other lettering (A–I), to
identify the elements of an IND.

91Regulatory Affairs
Common Technical Document
The IND format continues to be replaced by a more universally accepted
investigational application format, the Common Technical Document (CTD),
developed by the International Conference on Harmonization (ICH). The
goal of this harmonized format is to reduce or avoid duplicative testing
during the drug development process and eliminate unnecessary delays in
the global development of regulated products. The contents of the IND and
CTD are the same, but the reporting format or sectional structure differs, as
presented in Figure 3.4; additional details and more specific information on
CTD can be found at http://www.fda.gov. Rather than the previous nonstan-
dard format of 10 sections that make up an IND application or 20 sections of
a market application, the CTD format has five modules, irrespective of the
type of application. Of the five modules, only four of them (Modules 2–4) are
actually universal to all regions and recognized as part of the CTD. Module 1
is region specific, intended for administrative purposes. For example, a CTD
submitted to the FDA includes in Section 1 a cover letter, Form FDA 1571,
and a Table of Contents required by the FDA. Together with four subsequent
modules—quality, safety, efficacy, and multidisciplinary—the modules
facilitate ease of navigation and accommodate for regional differences in
BOX 3.7 (Continued) CONTENTS OF AN IND
safety or toxicology and key studies that demonstrate
potential efficacy, such as pharmacokinetic and pharmaco-
dynamic studies, are included.
• Section H(9): Previous human experience with the investiga-
tional drug.
• Descriptions and references to all previous uses of this or
closely related products with special attention to safety
issues in humans.
• Section I(10): Additional information.
• Certification of compliance (Form FDA 3674), summary of
FDA communications, letters granting permission to refer-
ence Master Files or other INDs
• References
• A list of scientific publications cited in the text
• Appendices
• Large documents, such as nonclinical toxicology reports
or pertinent scientific publications, may be included if they
have been abstracted or cited in an earlier section.

http://www.fda.gov

92 Biotechnology Operations
submission requirements for several geographic areas to include the content
and format of technical data to be accepted by the United States, Canada,
Europe, and Japan. Again, the CTD format applies to applications for all
investigational stages as well as those to support approval and marketing.
Electronic Submission of a CTD
The CTD format provides a rigid structure or platform to accommodate an
electronic submission, as the electronic CTD (eCTD) format. The content of
Module 1
Regional administrative
information
(Not part of the CDT)
Module 3
Quality
Module 4
Nonclinical
study reports
Module 5
Clinical study
reports
Module 2
Common technical
document summaries
2.1 CTD table of contents
2.2 CTD introduction
2.3 Quality overall summary
2.4 Nonclinical overview
2.5 Clinical overview
2.6 Nonclinical written and tabulated summary
Pharmacology
Pharmacokinetics
Toxicology
2.7 Clinical summary
Biopharmaceutics and associated analytical methods
Clinical pharmacology studies
Clinical efficacy
Clinical safety
Synopsis of individual studies
Administrative information and prescribing information
1.1 Table of contents of the submission including module 1
1.2 Documents specific to each region
(e.g., application forms, prescribing information)
3.1 Module 3 table of contents
3.2 Body of data
3.3 Literature references
4.1 Module 4 table of contents
4.2 Study reports
4.3 Literature references
5.1 Module 5 table of contents
5.2 Tabular listing of all clinical studies
5.3 Clinical study reports
5.4 Literature references
FIGURE 3.4
Common technical document (CTD) structure.

93Regulatory Affairs
an eCTD submission is designated into specific modules, sections, and docu-
ments creating a backbone structure. This structure then results in each doc-
ument to be contained in a subfolder of a specified section, and each section in
the folder of its specified module. The eCTD format is the only acceptable elec-
tronic submission format of regulatory documents to the FDA. Furthermore,
for some device submissions, the FDA offers a software system (eSubmitter)
for use in the electronic submission of certain types of device applications.
Overall it is easy to appreciate the level of standardization and presumed
simplicity that electronic submissions offer large corporations. Unfortunately,
complying with the electronic submission requirements has created great
challenges for small biotechnology companies and academic institutions.
There are meticulous requirements in formatting and naming conventions
often requiring expensive software packages, regulatory expertise to ren-
der electronic documents required to generate an acceptable electronic sub-
mission, and only those submissions that are demonstrated to be error-free
are accepted into the electronic submission system. Document rendering to
ensure an acceptable format for FDA electronic software acceptance takes
countless hours and typically requires a small group of dedicated staff for
each eCTD submission requiring additional resources, which is challenging
for a biotech company that may generate only a few submissions over the
course of a 1-year period. One option to acquire the ability to compile and
submit eCTD documents to the FDA would be to use template documents.
This may be the most cost effective option but does require a significant
investment in time and effort with a steep learning curve. A second option
is to purchase an e-Submission software package to include publishing and
validation capabilities. Although more straightforward, this option is more
costly than the piecemeal approach previously described using template doc-
uments and rendering them via trial and error. A final strategy to consider
may be to hire a consultant or to outsource data management for a submis-
sion. This may turn out to be the most expensive option but requires the least
amount of training and is expedient.
The IND is a legal document, and it is important for the sponsor to provide
only information that is true and accurate, and to disclose any and all infor-
mation relevant to the product, notably safety data. On receipt, the FDA has
30 calendar days to review the IND and decide whether or not to allow the
sponsor to proceed with the proposed clinical study. If during this 30-day
review period, the FDA decides there is insufficient information to evalu-
ate safety based on the information provided in the IND application, then
additional information will be requested of the sponsor. If the 30-day review
period is reached and the FDA review team identifies issues regarding prod-
uct safety or potential undue risk to human subjects, then the application
will likely be placed on Clinical Hold until these issues are resolved. A formal
clinical hold is a mechanism used to ensure that the study will not proceed
until the FDA is satisfied with the sponsor’s ability to address all safety con-
cerns identified as a result of the review process.

94 Biotechnology Operations
This formal iterative process is dictated by documented communications
usually in the form of letters and amendments to the IND application. A
sponsor has the right to appeal a clinical hold but, in doing so, must pro-
vide significant data to refute or better explain the safety concerns brought
forth by the FDA. Release from a disputed clinical hold or appeal typically
involves a process of scientific negotiation and may include the role of an
FDA appointed ombudsman but this process takes time, and thus a clinical
hold or appeal process is an undesirable process for any biopharmaceutical
development program.
To better ensure the chances of a successful IND, a sponsor often com-
municates and discuses with the FDA his or her intentions to submit an
IND, well before the IND is written. This is a formal process with several
steps. The FDA provides guidelines for meetings with sponsors, classify-
ing each meeting type as either A, B, or C and offering guidance on the
associated timing and how to plan a successful meeting at various stages
of development and for different purposes. Once a sponsor has some sci-
entific information about a product and clinical trial design, the time is
right to request a pre-IND meeting. To benefit the most from this meeting,
it is advisable to prepare well in advance by crafting specific questions for
the FDA to comment and provide input either agreeing with the develop-
ment plan, clinical approach, and regulatory strategy or not. This dialogue
with the FDA is an opportunity to evaluate sensitive areas or potential
areas of specific safety concerns of the FDA review team. In scheduling
a pre-IND meeting, the sponsor submits a formal written request to the
FDA. This request includes an information packet or premeeting package,
outlining information and data relevant to the product, and summaries
of the clinical protocol, nonclinical safety data, and objective of the meet-
ing. Also included in this information are potential meeting topics and
associated questions, a list of meeting participants and their role in prod-
uct development along with proposed meeting dates. This information is
submitted to the FDA in advance, typically 30  days or more, prior to the
proposed meeting dates. This is accompanied with a request that the FDA
meet either in person or by teleconference with the sponsor to discuss any
issues. The sponsor also queries FDA staff’s opinion on specific items of
concern. For example, the sponsor may ask whether a particular nonclini-
cal study was necessary, or if a certain manufacturing method or the con-
cept clinical protocol design was acceptable. The FDA will usually provide
written feedback after review of the information packet just prior to the
scheduled meeting date. In the interest of being sensitive to the many time
commitments of FDA review staff, the FDA will provide a statement in its
written response. For example, the FDA might respond to the request: If you
find that our written responses and advice are sufficiently clear and complete to
obviate the need for further discussion, please inform us as soon as possible so that
the meeting time may be cleared. Alternatively, the FDA may recommend the
meeting as proposed.

95Regulatory Affairs
Timing of the request for a pre-IND meeting is crucial because the sponsor
must have acquired enough information about a product to allow the FDA
to make an informed response to the specific questions being asked. At the
same time, it is not appropriate for a sponsor to approach the FDA requesting
pre-IND feedback shortly before filing an IND. As previously mentioned,
a formal process with associated deadlines and responses is required for
FDA interactions to occur. The result of a well-defined, -timed, and -executed
meeting with the FDA is thoughtful recommendations from the agency to
the sponsor which are based on questions provided to the FDA in advance of
the meeting. Pre-IND communication greatly reduces the chances of a clini-
cal hold on the future IND application if, in fact, the sponsor addresses the
issues raised by the FDA in their pre-IND responses.
Responsibilities for the sponsor and FDA to communicate do not end
with filing an IND application and receiving clearance to begin a clinical
study. Once the product is considered an Investigational Drug, the pro-
cess of submissions, responses, meetings, and teleconferences has only just
begun. Meetings and communications between the sponsor and FDA con-
tinue throughout the life of an IND and the development cycle it supports.
These are shown in Figure 3.3. A few commonly used meeting venues, in
addition to pre-IND, are noted here. A meeting is sometimes held prior
to initiating Phase 2 clinical studies (pre-Phase 2), and one is always held
prior to performing a pivotal clinical study, Phase 3 (pre-Phase 3 meeting),
and prior to submitting the marketing application, a NDA or Biologics
License Application (BLA) (pre-NDA or pre-BLA meeting). These meet-
ings allow the FDA and the sponsor to agree to clinical study designs that,
if successful, support the next step of the approval process (e.g., market-
ing application). Sponsors also request, during the course of development,
a meeting with the FDA to discuss a special issue, such as the need to
do an additional nonclinical study or the design of a new manufactur-
ing plant. Although these subject matter-specific agendas may be com-
bined with a clinical or milestone meeting, they typically require input of
experts outside the clinical arena, and thus result in individual meetings
or teleconferences.
Throughout the life of the IND, note that is not unusual for an IND to
be active for more than 10 years, the sponsor must file written reports and
data with the FDA. Annual reports outline progress on the program and
changes in technology, summarize product manufacturing and disposition,
and summarize clinical and nonclinical studies, and are provided on the
anniversary date of the IND. In addition, and at anytime during the year,
significant changes made or findings related to safety of the investigational
product, notably toxicology or adverse reactions seen in clinical or nonclini-
cal studies, must be immediately communicated to the FDA in writing. There
is also a need to keep the FDA abreast, by written correspondence, of matters
arising in development. In any given year, a sponsor is likely to submit sev-
eral letters, many containing updates or modifications to protocols or data, to

96 Biotechnology Operations
the IND. Each letter is considered an amendment under that IND and there-
fore represents a legally binding statement. For example, the 10th letter or
amendment to an IND would be numbered by the FDA as BB-IND32401-010,
where BB stands for a biological, IND32401 is an FDA-assigned number
given to that original IND (on first receipt), and each future amendment to
the IND is labeled sequentially. So sequential number 10 (0010) represents
the 10th amendment to the original IND submission. Ensuring the accuracy
and completeness of each submission, managing meetings and premeeting
information, and maintaining all submissions to an IND are important tasks
and require professional regulatory support.
If undue risk is noted by the FDA, the agency can, at any time, impose a
clinical hold and halt ongoing or prevent planned clinical studies. Clearly,
there is a need for the sponsor to be diligent and to have a means, typically
through a formal regulatory process and professional staff, to monitor all
aspects of product development and to report findings and changes to the
FDA in a timely manner.
Marketing Applications: BLA and NDA
A marketing application (NDA or BLA) from a sponsor is a request from the
sponsor to the FDA for approval to enter a product into interstate commerce
and to make claims of safety and efficacy about that product in the labeling.
Hence, a marketing application is composed of all the information that is rel-
evant to that product, all clinical and nonclinical study designs and results,
and everything else known or discovered about the product, its research,
manufacture, and control. NDAs and BLAs, submitted to CDER and CBER,
respectively, are massive documents, containing narrative summaries, tabu-
lated information, raw data, and explanations of how the data were gener-
ated, analyzed, interpreted, and submitted. The sponsor also proposes the
product labeling and provides critical documents stating the indication and
making claims for the product and the product label, which is the written
information affixed to the container of product. The proposed labeling must
provide warnings, contraindications, directions for use, and other informa-
tion. An outline of the most important (and required) product labeling, the
package insert, is shown in Box 3.8. The Physician’s Desk Reference and
other publications provide a collection of product labeling for FDA-approved
drugs and biologicals, bound into one volume. Product labeling may also be
found at the website of the FDA and those of product sponsors or manufac-
turers. Biotechnology firms seldom make draft product labeling available to
the public until a product has been approved.
The proposed labeling drives the application review process at the FDA.
Each of the product claims in the proposed labeling must be supported by
the data submitted to the FDA in the NDA or BLA. For example, if the spon-
sor claims in the product labeling that a monoclonal antibody would stop the
growth of prostate tumors for a period of 2 years (an endpoint) and prolong

97Regulatory Affairs
BOX 3.8 ELEMENTS OF PRODUCT LABELING
(PACKAGE INSERT)
Trade name and chemical name.
• Description: Describes the drug’s nature and classification.
Identifies how it is supplied in the final container and lists
any excipients.
• Clinical Pharmacology: This gives the mechanism of action,
pharmacodynamics, pharmacokinetics, and known drug
interactions.
• Clinical Studies: All pivotal clinical studies and important
smaller studies are described, to include endpoints and
outcomes. Data are often summarized in graphic or tabu-
lar format and all important results, efficacy and safety, are
identified.
• Indications and Usage: The exact indication is given exactly
in a very few brief sentences.
• Contraindications: Here are listed any situations in which
the product should not be used.
• Warnings: Messages to the prescriber and user are noted,
sometimes in bold and capitalized text to stress special
safety issues. Warnings may be listed by organ system (e.g.,
cardiopulmonary events) or by disease (e.g., malignancies).
These may be in bold and surrounded by a black box.
• Precautions: These are items that the user or physician
should watch for, issues less common or important than
those given in warnings. They are general, written for phy-
sician and user, or they are information for patients, specifi-
cally written for the user. Instructions for special situations
are also placed here, and specific items are highlighted as
paragraph headings. Recommendations may be given to
stop using a product, for example, if a disease progresses
or if certain symptoms are noted. Drug interactions, use
in nursing mothers or in pregnancy, pediatric and geriat-
ric use, or use in other special populations are generally
included in this section.
• Adverse Reactions: General adverse reactions are first
described, which are coming from reactions seen in the clin-
ical studies. Here again key results of clinical trials are often
presented in graphic or tabular format. A table presenting
(Continued)

98 Biotechnology Operations
the life expectancy of the patient for 4  years (a second endpoint), then the
design of the pivotal clinical trials must be focused on acquiring data to con-
firm those endpoints. For example, if product labeling submitted to the FDA
claims a monoclonal antibody is 98.7% pure, then the manufacturing and
quality control data provided in the BLA must show both a specification for
this purity level and provide data, from testing multiple lots of product, that
it does in fact reach that purity level. Then BLA must further demonstrate
that the monoclonal antibody will be manufactured and tested in such a
way that there is high probability that future lots of product will achieve that
same 98.7% purity. If the product labeling claims this monoclonal antibody
did not result in autoimmune disease in nonhuman primates when it had
been given more than 4 years, then that claim must be evident from the non-
clinical data submitted under the BLA.
Further, for each NDA or BLA, it is typical for the FDA to request addi-
tional data from the sponsor, sometimes asking that an additional study
be performed, and inspections of clinical, nonclinical, and manufacturing
facilities are routine during the examination product. These inspections
are referred to as Pre-approval Inspections (PAI), biomonitoring (BIMO), or
post-approval. Exact wording on labeling claims may be open to negotiation
BOX 3.8 (Continued) ELEMENTS OF PRODUCT
LABELING (PACKAGE INSERT)
the most common adverse events is usually provided here.
The most common adverse events are then described in a
narrative paragraph. Finally, adverse events are listed by
body system, cardiovascular through urogenital.
• Overdosage: This describes what is known if a patient should
take more than the prescribed amount.
• Dosage and Administration: Information on how product is
provided to a patient in final or dosage format is provided.
This repeats and provides details on what has been given
under description.
• How Supplied: If multiple formats exist (e.g., liquid or a tab-
let), then each is described. The NDC number is given for
each.
• References: A few key scientific publications are cited.
• Administrative: Manufacturers’ name, address, and license
number with dates are given.
• Patient Information: Instructions that are for the patient,
especially concerning proper handling, storage, or use,
may be given on a separate but attached pamphlet.

99Regulatory Affairs
between the sponsor and FDA, but the claims must always be supported by
data. Inevitably, the FDA asks a sponsor to make changes to their proposed
labeling; discussions can be contentious but negotiations inevitably result in
fair and balanced wording to labeling, and the FDA always makes the final
decision. Note the cyclic nature of the product labeling in a well-conceived
biopharmaceutical development project. In Chapter 1, we described the need
for TPP (draft labeling) to drive the development process and now, at the
NDA or BLA stage, we finalize that labeling. All the effort of the biopharma-
ceutical development team went into generating data that would support the
claims made in the draft labeling, presumably the same claims seen in the
proposed and final labeling. With product labeling, the development process
is truly a cycle, beginning with a visualization of what will appear in label-
ing and ending with proposed and approved labeling, which is supported
by many years of effort and investment in between!
On submission of the NDA or BLA, the sponsor must pay a significant sum
of money, referred to as an FDA user fee. This fee supplements FDA resources
for product review and is not refundable if the application is denied by the
FDA. Additional fees must be paid for each manufacturing facility at appli-
cation and periodically for the marketing life of the product. Review by the
FDA takes months since each claim must be carefully examined in light of
the data presented in the application. No wonder that a prudent biotechnol-
ogy operation carefully prepares an NDA or BLA and submits it to the FDA
only once it is supported by data. Failure is expensive for a sponsor in many
respects!
The format for an NDA and BLA has changed to harmonize with the inter-
national community. As previously mentioned in this chapter, most coun-
tries have adopted the CTD format, and the electronic or eCTD is encouraged
over the historic hard-copy paper submission.
Medical Device Applications. 510(k) and PMA
Sponsors of new medical devices, regulated through FDA’s CDRH, are faced
with a variety of pathways to market approval. For any given device, the
pathway is determined largely on the risk posed to the user by that class
of device and, to a lesser extent, on the nature of the device and previous
experience with that device. As noted earlier, medical devices are in one of
three classes based on the risk posed by the device. Class I, generally low risk
devices, must be developed under general controls, processes that include the
quality systems regulation (QSR) for manufacturing and record keeping.
Very few Class I devices require premarket notification, or 510(k) process.
Class II devices, of moderate risk, are subject to both general controls and spe-
cial controls and most are subject to premarket review and clearance under

100 Biotechnology Operations
510(k) premarket notification process, a process described in Subsection
“Medical Devices”. Class III devices carry potentially more risk and include
many life-sustaining or -supporting implantable devices. These are subjected
to the most rigorous controls. In addition to general and special controls,
they are subject to PMA. Some Class III devices may be marketed under
the 510(k) rules, but most must undergo the PMA process. The PMA pro-
cess demands proof that the device is both safe and effective, which usually
means clinical studies are required. For human studies of medical devices, an
Investigational Device Exemption (IDE) is required of devices presenting the
highest risk to the user. No matter which route is taken to market, medical
device labeling is required in a device marketing application, and it cannot
be false or misleading.
Few biotechnology products are considered medical devices alone, but
some are combination products and so the sponsor of a combination device
must consider medical device regulations in their development scheme.
However, the inclusion of a medical device into a combination product does
not lessen the regulatory hurdles, rather it likely increases them due to the
added product complexity. As noted earlier, the final determination of which
center at the FDA will be designated as the lead review for a biopharmaceuti-
cal combination product is based on which component, device or biological,
primarily produces the major effect. More often than not, it is the biological
that is designated the primary mode of action and, in such cases, the product
will follow the IND to BLA (or IND to NDA for a drug–device combination)
route to approval with concurrent review by CDRH.
A device submitted to the FDA for clearance under 510(k) must demon-
strate substantial equivalence to a legally marketed predicate device. The
device must have performance standards, it must have an indication for
use, the proposed labels must be accurate, and it must be fully described
to CDRH in the 510(k) application. There must be evidence that it will be
legally marketed and information on the device’s safety and efficacy profile
must be provided. The PMA and its contents are equivalent to those of the
BLA or NDA, because they demand a significant amount of nonclinical and
clinical study data to support label claims and because the manufacturing
controls are quite stringent. Clearly, most device manufacturers would pre-
fer to register their devices under 510(k) approval process.
In vitro diagnostics (IVDs) are a class of medical devices that have a
few special rules. Since many biotechnology products are used as key
reagents with IVDs, their regulation is of great interest to biotechnology
firms. IVDs encompass a host of products, from complex instruments to
test kits used to diagnose life-threatening diseases to simple diagnostic
laboratory reagents. Many IVD biotechnology products are marketed fol-
lowing 510(k) premarket notification, and a few must enter the market
through PMA route; either pathway for IVD is under review by CDRH.
Also under the review of CDRH are biotechnology products, reagents,
and instruments designated for Research Use Only, Investigational Use

101Regulatory Affairs
Only, Analyate-Specific Reagents, and Laboratory Developed Tests.
Biotechnology firms may find a ready market for their products, some-
times originally developed as laboratory reagents, with IVD manufac-
turers who purchase reagents to include in their test kits. Before making
a decision to enter the IVD field, firms should understand the possible
impact of entering a regulated arena. Considerable diligence, research,
and regulatory planning are advised.
Special Documents, Pathways, or Exemptions
• Master Files: The FDA provides a means to file confidential informa-
tion for any type of product; the submission is called a Master File
(MF), and the contents are maintained similar to other regulatory
filings. There are five recognized types of MFs designated: Type I, II,
III, IV, and V, with the most common MF used by the biotechnology
industry to be Type I for manufacturing site, facilities, operating pro-
cedures, and personnel, Type II for drug substance or drug product,
intermediate product, and materials used in their preparation, and
Type III for packaging material. The advantage of filing an MF is to
allow the FDA to review important information without divulging
confidential information to other industry partners on manufactur-
ing processes or testing methods for example. The MF mechanism
allows for the use of information that is already on file at the FDA
to be accessed for the new application that should minimize review
time. This mechanism of cross-referencing information may also be
used to avoid duplicating costly and lengthy pharmacology or toxi-
cology studies. The MF may be referenced by the FDA in support of
an IND, NDA, or BLA, with a letter of permission from the sponsor
of the MF to FDA. The FDA reviews the MF only as an informa-
tional source and in no case does the FDA constitute the contents of
the MF as an application for investigational use or market approval.
For  example, a biotechnology Firm, A, produces a synthetic mole-
cule that is sold to another biotechnology Firm, B for use in Firm B’s
in vitro diagnostic. Firm A prepares an MF that describes in detail
specific proprietary information, for example the manufacture and
control methods used to make and test this molecule and Firm A
then submits or files it with the FDA. Firm A also prepares a letter of
cross reference or letter of authorization (LOA) for Firm B, and in it
they give the FDA permission to review their MF as part of Firm B’s
IND 510(k) filing. Firm B provides Firm A’s letter of cross reference
in their application. In effect, Firm B does not have access to the tech-
nical details of Firm A’s proprietary information, yet the FDA can
review this information in great detail and on behalf of Firm B to
ensure that it is pure and potent and suitable for use in Firm B’s IVD.
MFs are commonly used in a number of ways to support both the

102 Biotechnology Operations
regulatory and business interests of companies. MFs are required to
be kept current through the standard annual reporting mechanism.
• Animal Rule: This regulation applies to biopharmaceuticals for
which there is no possible or ethical way to test efficacy in humans.
Examples are reasonably well-understood diseases caused by bio-
logical weapons of terrorism or mass destruction. It allows the
sponsor to test the efficacy of the product, a countermeasure, in a
well-developed and surrogate (to man) animal model. The product’s
safety is then studied in human clinical studies under IND. The IND
or PMA process is used throughout development with the only dif-
ference being that efficacy studies are typically conducted in at least
two animal species.
• Accelerated Approval: Drugs or biologicals indicated for the preven-
tion or cure of serious or life-threatening diseases are eligible for a
program known as Accelerated Approval. Under this regulation, the
FDA may approve such products on the basis of a surrogate or clini-
cal endpoints if these are likely to predict clinical benefit. Clinical
studies are still required, but they may not require the stringent and
long clinical processes for other drugs. There are restrictions on the
approval, however, and sponsors carefully choose this route in spe-
cial projects.
• Emergency Use and Treatment IND: An FDA regulation allows inves-
tigational drugs to be used outside the standard clinical protocol
in serious or life-threatening situations and with FDA concurrence.
This regulation may speed a drug to a patient who might other-
wise be ineligible to enroll in the study, such as someone living far
from the study site. There are caveats to this as the sponsor must
supply product at no cost, and there is risk that the drug may be
misused or result in harm to the patient. Biotechnology firms devel-
oping life-saving products agree to Emergency Use with caution,
but consider it important as a good faith effort to speed a product to
needy patients. Treatment IND is a slightly different approach for
investigational biopharmaceuticals. There must be preliminary evi-
dence of efficacy and an indication for a serious or life-threatening
disease or it may be used if there is no alternative drug available
and if death is expected from the disease within months. Examples
are advanced cases of AIDS and cancers. The product is typically
in Phase 3 clinical studies, and the information on its use must be
reported to the FDA.
• Orphan Product Designation: This leverage option is commonly used
by biotechnology firms, because many biopharmaceuticals are
developed for the diagnosis or treatment of rare diseases or condi-
tions. By definition, a rare disease or condition affects fewer than
200,000 people in the United States per year. It provides incentives

103Regulatory Affairs
for firms to develop biopharmaceuticals that are used less frequently
and therefore have a smaller market value. This program has been
quite successful for more than 30  years and is coordinated by the
Office of Orphan Products Development (OPD), FDA. A common
European and FDA application is available. The sponsor benefits by
receiving both 7  years of FDA-administered market Orphan Drug
Exclusivity (ODE), exclusive market rights for the indication and a
tax credit of up to 50% of R&D costs, waived FDA fees, clinical trial
tax incentives, and protocol assistance, and reduced statistical bur-
den such as numbers of subjects required in a Phase III human clini-
cal study to support approval. Also, the FDA awards development
grants to sponsors of orphan products.
• New Drug Product Exclusivity: This is protection from competition,
for 3 or 5 years after marketing approval, for the holder of an NDA
or BLA when the drug is a new chemical entity that is a product
unique both in nature and the marketplace. This exclusivity, in addi-
tion to that provided by a patent, encourages biotechnology firms to
develop novel products.
• Fast Track Designation: Another program to expedite products to
patients with serious or life-threatening diseases or conditions,
and where there are clinical data to demonstrate the potential to
address an unmet medical need, is called Fast Track Designation.
The sponsor must request Fast Track Designation from the FDA, and
certain criteria must be met. However, a sponsor with Fast Track
Designation receives special consideration from the FDA, such as
additional meetings in which guidance may be provided, Priority
Review of market applications, and even a program to review cer-
tain sections of the NDA or BLA incrementally, thus saving time in
the review process.
• Breakthrough Therapy: A breakthrough therapy designation is
granted to products that are intended to treat serious condition
and have preliminary clinical evidence demonstrating substantial
improvement over the current standard of care therapies. Request
for consideration of this product designation occurs during clini-
cal development under an active IND. Benefits of this designation
include intensive development guidance, organizational com-
mitment to involve senior managers and experienced reviewers,
Rolling Review, and other actions that would expedite the review
process. If the FDA determines the product does not meet the break-
through criteria, they will provide a nondesignation letter to  the
sponsor stating this status was not granted and explain the reasons
for the decision.
• Priority Review: This expedited review process is intended for a
product developed to treat a serious condition or a labeling change

104 Biotechnology Operations
to a pediatric study. A request for Priority Review is to be submitted
with the marketing application, and if successfully granted, it will
provide a shorter clock for marketing application review to 6 months
from the standard 10-month review period. The FDA will acknowl-
edge, in writing, if a Priority Review has been granted.
• Rolling Review: Product approval for a Rolling Review entitles the
sponsor of a product to submit sections of an application for FDA
review prior to the assembly of a complete marketing application
submission. The intent is to avoid delays associated with comple-
tion and assembly of a complete application and instead allow the
FDA to review individual sections of an application such as chem-
istry, manufacturing and controls, toxicology, or clinical sections.
The most common and beneficial review to take place as a Rolling
Review is preliminary evaluation of clinical data that demonstrate
effectiveness.
Generic Drugs and Biosimiliar or Follow-on Biologics
Generic drugs have the same active ingredient as brand name marketed
drugs and represent look-alikes that enter the market following expira-
tion of patent protection. They are often made by manufactures other than
the company that originally made the brand name drug, and they must be
tested in small, head-to-head studies—laboratory and clinical pharmaco-
kinetics and pharmacodynamics—to demonstrate chemical identity and
pharmacokinetic and pharmacodynamic similarity. The approval process
for a generic drug at the FDA follows the sponsor filing an ANDA, and
indeed it is just that.
It is unlikely that generic drugs will be derived from biotechnology.
However, their equivalent, biosimilars, also referred to as follow-on biolog-
ics, biogenerics, or generic biologics, are biologic look- and perform-alikes.
The FDA approved the first biosimilar product Zarxio (filgrastim-sndz)
manufactured by Sandoz, Inc., a Novartis company (Princeton, NJ) in March
2015. Zarxio is biosimilar to Neupogen (filgrastim), which is marketed by
Amgen, Inc., (Thousand Oaks, CA) originally licensed in 1991. Zarxio is
approved for the same clinical indications as Neupogen and can now be pre-
scribed by physicians as a substitute for Neupogen. Congress has met stiff
resistance on two fronts: first, from biotechnology firms that currently hold
the market in the absence of patent protection and second because there are
serious questions regarding whether or not biological molecules can, in fact,
be reproduced to mimic exactly the purity, potency, efficacy, and safety of
the predecessor molecule. Clearly, any biosimilar product will need to be
thoroughly tested in adequate and well-controlled preclinical and clinical
studies. The extent of testing continues to be debated, but the biogenerics
have now reached the market.

105Regulatory Affairs
Other Regulatory Activities
Many additional regulatory activities must be completed during the product
life cycle, some before and others after product registration. Several examples
are as follows:
• Establishment registration: Establishments manufacturing any drug or
biopharmaceutical or medical device, whether U.S. or foreign, must
be registered with the FDA.
• Licensing issues: Divided, shared, or contract manufacturing. To
accommodate the complex and sometimes specialized manufac-
turing schemes required for biopharmaceuticals, the FDA allows
manufacturing of one product at two or more sites. For example, a
biopharmaceutical might be produced by fermentation at one site,
then shipped to a second site for purification, formulated at a third
site, and filled and labeled at a fourth site. Such divided or shared
manufacturing, generally done largely by contractors for the spon-
sor, is allowed if it is carefully controlled and defined and if each site
is a registered establishment.
• Proprietary name: A sponsor wishes to have a unique name, apart
from the often long and confusing chemical name, for their bio-
pharmaceutical. To avoid duplication for confusion in labeling, the
FDA is responsible for approving the proprietary name for each
biopharmaceutical.
• National drug code: In addition to the unique name, the FDA issues
with marketing approval a unique drug number, a National Drug
Code (NDC), and this is clearly marked on all labeling.
Public Meetings and Advisory Committees
A cornerstone of good government is the right to speak in public for or against
an issue, especially if that issue arises from a government or government-
regulated activity. Biotechnology products and the marketing approval of
biopharmaceuticals and medical devices are no strangers to the public arena,
and their use and release into the environment have been a matter of debate
ever since recombinant Pseudomonas syringae was sprayed on strawberry
fields of California in 1983 to prevent frost damage. The photograph of sci-
entists dressed in protective gear and spraying the bacterium elicited much
debate worldwide. We noted earlier that a key part of the formal process to
make or to change a regulation is the public rulemaking process in which
the public has an opportunity to review and comment on proposed regula-
tions before rule publication and codification. In addition, regulatory agency

106 Biotechnology Operations
processes may be influenced by public petition: requests made to produce,
remove, or change a regulation. Other rules allow the public to demand eco-
nomic or environmental impacts for a regulation, and these processes also
mandate the public be informed and allowed to influence the government’s
decision. All of this applies to the FDA and other regulatory agencies. States
have similar rules that result in public hearings or meetings. Just as it was in
1983, public debate is a very important aspect of rulemaking.
The FDA also uses advisory committees to its and the public’s advantage
by asking expert panels to review data on safety and efficacy for products
near completion of review and recommend approval. The FDA has estab-
lished these committees for every class of drugs, biologicals, and medical
devices under its purview. Members meet at established intervals to make
recommendations on a variety of subjects. At the top of their list are product-
specific recommendations, notably whether or not a product should receive
market approval. This is typically done after the FDA has completed the
review. For this, the committee is asked to answer a series of questions, such
as, Does the committee view this product to be safe for its intended use?
Panel members vote, but the recommendation is not binding to the FDA,
and the agency will sometimes decide in a manner not consistent with the
panel’s majority recommendation. Clearly, such meetings and the commit-
tee’s voting record and recommendation are extremely important to the bio-
technology firm sponsoring the product put before a committee. Another
function of advisory committees is to make recommendations on groups or
classes of products. For example, if a class of monoclonal antibody, repre-
sented by several similar products, appears to cause an unexpectedly high
number of allergic reactions, the FDA might ask a committee to meet and
discuss the situation and perhaps make a recommendation, such as posting
a warning on the label. Advisory committees also perform more mundane
tasks such as reviewing research laboratories at the FDA. It is very important
that members of these committees be experts yet have no conflict of interest
such as working in a commercial environment with the products on which
they make recommendations or receive money from the sponsor. Indeed,
a member should have no strong personal bias for or against a technology.
As one might imagine, it can be a challenge for the FDA to find the right
experts to serve on advisory committees. Committee meetings are open and
announced to the public, comments are solicited, and minutes and votes are
a matter of the public record.
Having read this chapter, one might ask, What is there about a biopharma-
ceutical operation that is not made public and that can be kept confidential
and proprietary? The answer is quite a lot; much, perhaps most, of the tech-
nical information that the firm considers proprietary, and all of the financial
facts are kept from public view. The FDA does not delve into a firm’s finances
or the public or private nature of a company, which is a territory for the
Securities and Exchange Commission or the Internal Revenue Service. The
FDA does not consider marketing other than whether or not promotion is

107Regulatory Affairs
in line with approved labeling and in a few other areas related to market
approval. Proprietary information that must be disclosed to the FDA by the
biotechnology firm in applications or correspondences is maintained as con-
fidential and remains hidden from public view. Regulatory agencies, unlike
many organizations in political capitals, do not leak confidential information
to the public or to the media.
Although a limited amount of information is accessible to the public through
Freedom of Information Act (FOIA), this information is nonproprietary or
reportable observations from inspections and provided in warning letters or
Form FDA 483. Indeed, proprietary information is redacted before public dis-
closure through FOIA.
Postmarketing Requirements and Activities
Interaction with regulatory authorities does not stop once market approval
is given. The FDA can withdraw marketing approval any time after it has
been granted. This has been demonstrated in cases where the Agency inves-
tigates a proposed noncompliance issue which results in the ability to prove
cause (i.e., demonstrating noncompliance with regulation). This happens
when a sponsor (holder of the market approval letter) fails to meet report-
ing requirements or if a product proves unsafe. What are postmarketing
requirements of the sponsor? Some of them are as follows:
• Post-approval maintenance of the approved NDA or BLA: Sponsors must
file annual reports with the FDA as long as they are marketing an
approved drug. Elements of an annual report include but are not lim-
ited to labeling, chemistry, manufacturing and control, nonclinical
testing, and clinical data. Reporting of changes from the original mar-
ket approval is especially important to regulatory agencies. It may be
necessary for the sponsor to report significant changes immediately
and not wait for the annual report. At the time of market approval,
it is normal for the FDA and sponsor to agree to certain postmarket-
ing clinical studies, such as Phase IV or monitoring of special patient
populations. These commitments by the sponsor also include adver-
tising and labeling changes, product complaint reporting schemes, or
events that trigger product recalls. The FDA and the public take these
commitments quite seriously.
• Reports of adverse drug events (experiences): ADEs are explained in
Chapter 9. Briefly they involve reactions in patients using a biophar-
maceutical, and they must be reviewed by the sponsor if they fall
under certain guidelines for severity or frequency and might be
related to the product. Physicians and users may report these expe-
riences to the sponsor and sometimes directly to the FDA. Direct
communication is referred to as MedWatch for many medications,

108 Biotechnology Operations
whereas specific products such as vaccines have a unique report-
ing system. Certain types of products, such as vaccines, have unique
reporting requirements. The sponsor, in turn, must report serious
experience situations to the FDA in an Alert Report within 15 days.
The rules are not complex, but they are considered extremely impor-
tant to maintaining public health and a safe source of biopharma-
ceuticals, as well as keeping a positive image for the firm and the
biopharmaceutical industry as a whole.
• Risk evaluation and mitigation strategy: The FDA has instituted this
program, referred to as REMS, to improve postmarket approval
safety of medical products. REMS includes guides for medications,
patient-friendly labeling, and improved communication from the
FDA or sponsor with health care providers to better ensure proper
use of products. The document is a plan that is submitted by the
sponsor to FDA for approval as part of the marketing application.
• Dear doctor letters: The FDA believes that an effective means of
communicating new information, especially risks, for prescrip-
tion biopharmaceuticals is by ensuring well-informed prescribing
physicians. Letters to doctors often fill that objective, along with
announcements in medical journals, and through the public media.
• FDA letters to manufacturers: Letters to manufacturers are another
matter, since these are targeted directly to the sponsor and are often
issued in response to compliance issues. Communication between
sponsors and the FDA are therefore discussed in Chapter 4.
Advertising and Promotion
Drugs, biologics, and medical devices are heavily marketed to various target
populations—physicians, nurses, pharmacists and patients, or end users—
and we accept this in our society. As compared to many other countries,
biopharmaceutical marketing and promotion are lightly regulated in the
United States. As discussed elsewhere, marketing and advertising activities
are regulated through claims made on the label. A biopharmaceutical label is
defined as, A display of written, printed, or graphic material on the immedi-
ate container of a drug. Labeling, as defined by the FDA and used as a noun
is “any written, printed or graphic material on the drug, on any of its con-
tainers or wrappers, or on any material accompanying it” (Federal Register,
2006). Hence, the package insert, that lengthy document that contains pre-
scribing, safety, and dosage information, and is stuffed into boxes of OTC
or prescription medications, is labeling. Promotional labeling is any labeling
used in advertising or marketing activities. Promotional labeling is at the
heart of biopharmaceutical sales, and so it becomes a point of contention
whenever it does not reflect the approved labeling. For example, one of the
most egregious violations of FDA marketing rules is to promote the use of a

109Regulatory Affairs
biopharmaceutical for an indication or use that is not given in the approved
label. This is known as promotion for off-label or unapproved use. Although
the FDA does not restrict licensed medical practitioners from prescribing
medications for or advising patients to take medications outside the labeled
information, the FDA does not allow sponsors in anyway to promote this
practice. Hence, it is only legal to promote biopharmaceuticals, or other drugs
and biologicals, in accordance with the approved label. Advertisements must
be balanced and complete, again as driven by information included in the
FDA-reviewed and -approved label.
A product’s label will almost certainly change during the postmarketing
period, and this results in refined definitions for what can and cannot be
included in promotional materials. To ensure that biopharmaceutical pro-
motional information is in line with current labeling, the FDA insists that it
be provided for review by the agency at certain times during product devel-
opment. Investigational products may never be promoted. Most or all pro-
motional materials for approved products must be submitted to the FDA as
they will be used immediately after market approval. This first advertising
campaign is referred to as a launch. Also, most other promotional materials
generated postlaunch must also be submitted for review. Dissemination of
scientific and medical information is also closely monitored by the FDA and
direct-to-consumer advertisements, such as television and newspaper ads,
is controlled. An important subject of ongoing debate is whether or not the
consumer or the health practitioner is given adequate information about
safety issues that are known to be or could even possibly be related to a
product.
Summary of Regulatory Affair Activities
in Biotechnology Operations
This discussion completes this chapter on regulatory affair operational
activities, and it reiterates themes that were introduced in Chapter 1. Having
a historical perspective of FDA regulation and understanding organization
of the FDA provides a foundation to better understand the regulatory con-
siderations, regulation, and guidelines throughout the product development
process. Early in this chapter, the different types of regulated medicinal
products were highlighted under the broad categories of drugs, biologics,
devices, and combination products. Further mention is made of biotechnol-
ogy products that are considered cosmetics, dietary supplements, or veter-
inary products but are not regulated as medical products. The regulation
of medical devices is complex since they include a wide variety of product
types and today involve many combination products. Developing regula-
tory strategies to enable efficient biopharmaceutical development based on

110 Biotechnology Operations
understanding how each biopharmaceutical product is regulated by the
FDA is a process collectively termed regulatory intelligence. Use of regula-
tory intelligence allows us to strategize and plan for biotechnology opera-
tional adjustments at every stage of product development.
It is also important to continuously assess the risk-to-benefit balance
during product development with an eye toward understanding how the
regulator will interpret our analysis of biopharmaceutical development
information. In most cases, the benefits must outweigh the associated
or anticipated potential risks. The IND is a formal application to and for
review by the FDA, and the process used to obtain their permission to use
an investigational product in a human clinical study. Another application,
the BLA or NDA, is used to request FDA authorization for approval to mar-
ket a medical product, biologic or drug, respectively, in the United States.
Each application has specific format requirements with different formats for
paper versus electronic submissions. It is important to recognize that user
fees may be associated with the submission of a regulatory application. An
important and essential part of the successful development of a biotechnol-
ogy product is managing an active relationship with the FDA and being
responsive to any FDA request.
An introduction and various examples of special documents, pathways, or
exemptions established specifically or expediting product development for
particular underserved clinical indications have been provided. Also pre-
sented in this chapter are a brief discussion on generic drugs or biosimilars
that have now reached the market, a summary of the approval mechanism,
and the testing requirements that continue to cause much debate. Adequate
testing of generic or biosimilars remains controversial since demonstration
and testing of comparability are usually required only for the active ingre-
dient, and overall the product must pass the brand product specifications
with regard to potency and percent impurities. It is well documented that
the impurity profile of medical products is the primary contributor of safety
issues.
The FDA has many regulatory responsibilities and oversight that are briefly
mentioned in this chapter which include review of establishment registra-
tion information and of priority naming conventions, licensing issues, and
issuing product codes. As a public service, the FDA organizes public forums
inviting a subset of experts but is open to the public and often attended by
special interest groups—patients, lobbyists, or other stakeholders who are
interested to hear firsthand on the potential issues and current thinking on
specific topics. It is also very common for the FDA to rely on outside expert
opinion that is solicited by establishing advisory committees and meetings
on specific topics.
Information has been provided in this chapter on the various types of
formal FDA applications including those for investigational product use,
labeling, and commercialization. Also, recall that compliance with detailed
postmarketing requirements is essential to lawfully market medical products

111Regulatory Affairs
throughout the product lifecycle. These marketing requirements include
advertisements, promotions, and medical claims.
Regulatory planning starts early and involves successful product develop-
ment through the very end. Planning must consider all regulatory aspects of
the product and its development; each step in the cycle. The product labeling
is central to the development lifecycle, beginning with a draft labeling or
TPP and ending with approved labeling for the marketed product.
References
FDA. 2006. Guidance for Industry: Complementary and Alternative Medicine Products
and Their Regulation by the Food and Drug Administration. US Food and Drug
Administration. http://www.fda.gov/downloads/regulatoryinformation/
guidances/ucm145405 (accessed May 31, 2016).
Federal Register. 2006. Department of Health and Human Services. US Food and
Drug Administration. http://www.fda.gov/OHRMS/DOCKETS/98fr/06-545.
pdf (accessed May 31, 2016).
Sinclair U. 1905. The Jungle: The Uncensored Original Edition. Sharp Press, Tucson, AZ.

http://www.fda.gov/OHRMS/DOCKETS/98fr/06-545

http://www.fda.gov/downloads/regulatoryinformation/guidances/ucm145405

http://www.fda.gov/OHRMS/DOCKETS/98fr/06-545

http://www.fda.gov/downloads/regulatoryinformation/guidances/ucm145405

http://taylorandfrancis.com

113
4
Regulatory Compliance
Regulatory Compliance
Information in this chapter builds upon an understanding of regulatory
operations (Chapter 3) by examining the broad world of regulatory com-
pliance, and discussing the FDA requirements to integrate quality into
all aspects of biopharmaceutical development programs and by review-
ing many regulations outside of the FDA that impact most biotechnology
operations.
Quality Systems to Meet Regulatory Compliance
Compliance and Quality Systems
The Oxford English Dictionary (1997) defines compliance as “the act or instance
of complying; obedience to a request or command.” Further defined for bio-
technology product development, compliance is the act of meeting a plethora
of rules, regulations, and directives. Compliance impacts each biotechnology
development function every day. It involves constant vigilance to identify and
understand each applicable regulation and, most importantly, it drives the bio-
technology firm to institute and integrate programs that ensure obedience to
these regulations and directives. Compliance is achieved largely by ensuring
quality in all aspects of development and at every step in the development
cycle. This is best done by instituting quality systems, described in Chapter 5.
Indeed, results of FDA inspections repeatedly demonstrate that firms with
mature and effective quality systems consistently have, in the eyes of regu-
latory agencies, fewer deficiencies than do operations with deficient quality
systems. With this in mind, it is easy to consider the need to integrate, into a
biotechnology operation, scientific and technical skills, regulatory guidance,
and quality systems.

114 Biotechnology Operations
Quality systems are composed of quality hallmarks, features of a well-
established, compliant, and smooth operation (Chapter 5). In this chapter, we
discuss the intersection of compliance with quality system. Three examples
of quality systems—current Good Manufacturing Practices (cGMP), cur-
rent Good Laboratory Practices (cGLP), and current Good Clinical Practices
(cGCP)—are outlined in the following and each will be further described
in Chapters 6 and 7 (manufacture; quality control), in Chapter 8 (nonclini-
cal), and in Chapter 9 (clinical), respectively. These are excellent examples of
systems applied by most nations to protect public health by ensuring safety
and efficacy of biopharmaceuticals. They are presented here as U.S. FDA
regulations, but similar good practices of many nations are currently being
harmonized into international compliance guidelines and so these elements
now reach worldwide.
Current Good Manufacturing Practices for Manufacture
and Quality Control
cGMPs were established to prevent drug and medical device manufacturers
from producing and selling adulterated products to the public. In brief, adul-
terated products are those that contain harmful ingredients, misrepresent
strength, are mixed with other substances than are approved, do not con-
form to performance standards, packaging, labeling, and storage conditions,
in which strength/quality/purity do not match the label, or lack adequate
manufacturing controls. This, in turn, was desired by the public because
adulteration had occurred in medical product manufacture, and the practice
was not tolerated by the public. cGMP is an established quality system that
has been shown to have a positive effect on the quality of biopharmaceuti-
cals and hence cGMPs have been adopted as the manufacturing standard,
worldwide.
In the United States, biopharmaceutical manufacturing compliance is
based upon regulatory requirements for manufacturing processes and utili-
ties, codified for human and animal drugs and in 21  CFR 210 and 211 as
well as in other sections of FDA regulations. Important elements of cGMP
are listed in Box 4.1. cGMPs are now quite well harmonized worldwide and
any differences in cGMPs largely reflect the nature of the product or differ-
ences in its usage. It is repeatedly stated that “…quality cannot be tested into
a product but that the sum total of what constitutes that product must be
of the highest quality” (Federal Register Online, 1996). Biopharmaceutical
manufacture and control are, under cGMP, based upon the idea that a prod-
uct, the process to make it, and the laboratory control tests must be designed
in a manner that meets the intended use. cGMPs strive to meet that stan-
dard. These quality systems concepts are discussed further in Chapter 5 and
examples or application of cGMP to actual manufacturing and control pro-
cesses are demonstrated in Chapters 1 through 4, 6 and 9.

115Regulatory Compliance
BOX 4.1 HIGHLIGHTS OF CURRENT GOOD
MANUFACTURING PRACTICES (cGMP)
• 21 CFR 210—Current Good Manufacturing Practice in manu-
facturing, processing, packaging, or holding of drugs
Status, applicability, and definitions
• 21 CFR 211—Current Good Manufacturing Practice for fin-
ished pharmaceuticals
A. General provisions
B. Organization and personnel
– Responsibilities of quality control (assurance) unit, per-
sonnel qualifications and responsibilities
C. Buildings and facilities
– Design, construction, lighting, ventilation, plumbing,
sewage, washing and toilet, and sanitation
D. Equipment
– Equipment design size, location, construction, cleaning,
calibration, maintenance; automatic, mechanical and
electronic equipment, filters
E. Control of components and drug product containers and
closures
– Receipt and storage, quarantine and release, testing and
use of components, containers and closures; retesting;
rejection; drug product containers and closures, waste
removal and flow
F. Production and process controls
– Written procedures, vendor qualification, deviations;
yield; equipment identification; sampling and testing
of in-process materials and drug products; time limita-
tions; control of microbial contamination; reprocessing.
G. Packaging and labeling control
– Materials examination; issuance of labels; tamper-
evident packaging; inspection; expiration
H. Holding and distribution. Warehousing and distribution.
(Continued)

116 Biotechnology Operations
BOX 4.1 (Continued) HIGHLIGHTS OF CURRENT
GOOD MANUFACTURING PRACTICES (cGMP)
I. Laboratory controls
– Testing and release for distribution; stability testing;
special testing; sampling plans, reserve samples; labo-
ratory animals
J. Records and reports
– Cleaning and use logs, equipment calibration records,
component, container, closure and labeling records; master
production and control, laboratory, distribution and com-
plaint records and review;
K. Returned and salvaged drug product
• 21 CFR 600—Biological products
A. General provisions
B. Establishment standards
– Personnel; establishment, equipment, animals; records,
retention samples, product deviations; temperatures
during shipment
C. Establishment inspection
D. Reporting adverse experiences
• 21 CFR 610—General biological products standards
A. Release requirements
B. General provisions
– Methods and processes: General safety, inactivation,
sterility, purity, identity, constituent materials, combi-
nations, cultures
F. Dating period limitations
G. Labeling standards
– Container and package labels, name of product, manu-
facturer and distributor, export
• 21  CFR 630  and 640—Standards for human blood and blood
products
• 21  CFR 660—Standards for diagnostic substances for labora-
tory tests (blood products)
• 21  CFR 680—Additional standards for miscellaneous (biological)
products
(Continued)

117Regulatory Compliance
Current Good Laboratory Practices for Nonclinical Laboratory Studies
cGLP regulations were established because certain individuals were perform-
ing nonclinical laboratory studies in an unscientific or uncontrolled manner
and the results of these studies could not be trusted. Important toxicology
data were found to be questionable. In response, a regulation, 21 CFR 58, was
established in 1979 with the purpose of ensuring the quality of nonclinical
safety studies for medical substances. The key elements of FDA cGLP regula-
tions are outlined in Box 4.2. Taken together, cGLPs ensure that testing has
been performed in a sound scientific manner and with an established qual-
ity system. Notable are requirements for study protocols, appropriate data
capture, accurate reports, internal quality audits, and acceptance of results by
both the scientists and a quality assurance professional.
Current Good Clinical Practices for Clinical Studies
cGCPs represent a quality system that ensures the highest quality science
and ethical treatment of human subjects for clinical studies of all types and
at all phases of development. With cGCP the burden for quality is shared
between the principal parties conducting a clinical trial: sponsor, investiga-
tor, and, if one is used, contract research organization (CRO). Unlike cGLPs
and cGMPs, which are to be found in one or a few sections of 21 CFR, cGCPs
are codified in a number of chapters and sections of the regulations. This
is due in large part to the broad scope of clinical trials overall, the fact that
they involve FDA functions, and generally recognized and codified rules
for the conduct of research that involves human subjects no matter what
the reason for their enrollment. The key components of cGCP are outlined
in Box 4.3. In the biopharmaceutical industry, an important foundation of
cGCP is that the regulation both protects users of biopharmaceutical prod-
ucts and also safeguards the well-being of human subjects, those individu-
als taking personal risk by volunteering to test new products. Indeed, the
protection of human subjects is paramount in cGCPs, as it should be.
BOX 4.1 (Continued) HIGHLIGHTS OF CURRENT
GOOD MANUFACTURING PRACTICES (cGMP)
• 21 CFR 820—Quality system regulation (for design and manu-
facture of medical devices)
• 21 CFR 1270—Good Tissue Practices
• 21 CFR Part 11—Electronic records; Electronic signatures
• Controls for closed and open systems, signature manifesta-
tions and record-linking, electronic signature components
and controls, identification and passwords.

118 Biotechnology Operations
BOX 4.2 ELEMENTS OF CURRENT GOOD
LABORATORY PRACTICES (cGLP)
• 21 CFR 58—Current Good Laboratory Practices for nonclinical
laboratory studies
A. General provisions
– Definitions, applicability, and inspections
B. Organization and personnel
– Personnel, management, study director, quality assur-
ance unit
C. Facilities
– Animal care and supply; handling test and control arti-
cles, laboratory areas, specimen and data storage
D. Equipment
– Equipment design, maintenance, and calibration
E. Testing facilities operations
– Standard operating procedures, reagents and solutions,
animal care
F. Test and control articles
– Test and control article characterization, handling and
mixtures
G. Protocol for and conduct of a nonclinical laboratory study
J. Records and reports
– Reporting study results, storage of records and data,
retention of records
K. Disqualification of testing facilities
– Grounds for disqualification, notices, final orders,
actions, public disclosure, and suspension
• 21 CFR Part 11—Electronic records; Electronic signatures
• Controls or closed and open systems, signature manifesta-
tions and record-linking, electronic signature components
and controls, identification and passwords.

119Regulatory Compliance
Compliance for Biopharmaceuticals: Other Regulations
of Importance
Compliance for Import of Biopharmaceuticals into the United States
Importation of biopharmaceuticals is regulated by a number of agencies in every
country of the world. For the United States, Center for Biologics Evaluation and
Research or Center for Drug Evaluation and Research oversee importation and
BOX 4.3 REGULATIONS FOR CURRENT GOOD
CLINICAL PRACTICE (cGCP) AND CLINICAL TRIALS
• 21 CFR Part 11—Electronic records; Electronic signatures
• Controls or closed and open systems, signature manifesta-
tions and record-linking, electronic signature components
and controls, identification and passwords
• 21 CFR 50—Protection of human subjects. Informed consent
• General requirements, elements and exception for informed
consent, additional safeguards for children
• 21 CFR Part 54—Financial disclosure by clinical investigators
• 21 CFR Part 56—Institutional review boards
• Organization, personnel, functions, operations records and
reports, administrative action for noncompliance
• 21 CFR 312—Investigational new drug application and foreign
clinical trials
• Responsibilities of sponsors and investigators: responsibili-
ties of sponsors, transfer of obligations to a contract research
organization, selection of investigators and monitors,
informing investigators, review of investigations, record-
keeping and retention, inspection of records and reports,
disposition of investigational drug, assurance of IRB review,
disqualification of clinical investigator.
• Drugs intended to treat life-threatening and severely-
debilitating illnesses, emergency use.
• Foreign clinical studies not conducted under an IND, pub-
lic disclosure of data and information.
• 21  CFR 314—Applications for FDA approval to market a new
drug
• 21 CFR 320—Bioavailability and bioequivalence requirements.

120 Biotechnology Operations
exportation of biologics or drugs, respectively, to ensure they comply with all
U.S. laws and regulations. The FDA works closely with Customs and Border
Protection (CBP). Inbound shipments in violation are detained by CBP on
behalf of FDA or the United States Department of Agriculture (USDA). FDA
must be advised if a final biological or drug product is manufactured overseas.
A foreign manufacturer must have a U.S. FDA license to manufacture and dis-
tribute that product. This means the product must have an approved marketing
application and, before this is granted, the foreign manufacturer must usually
pass FDA inspection. A product approved and manufactured in the United
States may, however, be exported from the United States to another country
without additional FDA authorization to export. In such cases, FDA provides,
on behalf of a biopharmaceutical sponsor and to a foreign regulatory agency,
a Certificate to Foreign Government to substantiate marketing approval in the
United States. Investigational biopharmaceuticals are another matter. The man-
ufacturer need not have a U.S. FDA license but they must declare a valid and
active IND by number and name. CBP screens such shipments carefully, notify-
ing FDA and USDA if paperwork is in any way out of order or incomplete.
Compliance for Medical Devices
There are aspects of medical device compliance that differ from other FDA
products, and the biotechnology firm developing a combination product
with device components is well advised to understand these nuances. As
noted earlier, registration and listing of U.S. establishments developing or
manufacturing devices is critical to understanding medical device compli-
ance. Also, devices are classified according to the level of risk to the user.
Quality systems regulations (QSRs) and guidelines demand a strict quality
system for development and production of medical devices. While some
aspects of device QSR, identified in Chapter 5, now apply to drugs and bio-
logics, they are quite detailed for devices. Certain reporting requirements are
also unique to medical devices, as identified under the FDA’s medical device
reporting (MDR) regulations. Additionally, there are stringent rules on track-
ing of certain medical devices. Medical device import and export compliance
has many similarities to drugs and biologicals, but some processes do differ
and are important to firms engaged in international transport, manufacture
or marketing of medical devices or combination products.
Inspection and Enforcement
We are certainly all aware that in any society it is necessary to enforce
laws and regulations. Yet, skirting or blatantly disobeying regulations just
seems to come naturally to certain individuals, typically those motivated by

121Regulatory Compliance
personal gain, and so societies have established means of ensuring, or try-
ing to ensure, compliance by everyone. These are (1) enforcements, a means
of imposing on individuals the observance of law and (2) inspection, the
official and careful examination of an item or an activity. Biological, drug,
and medical device activities have, in the past, been found to be deficient and
in some cases there has been proved a serious intent to produce adulterated
product, to falsify nonclinical or clinical study data or to avoid providing
human subjects with their legal rights. Such behavior does, unfortunately,
exist. In an effort to ensure that all biopharmaceutical products are both
safe and effective and to increase public confidence in the biotechnology
industry, the FDA inspects virtually all aspects of regulated development
and enforces regulations intending to keep biopharmaceutical products
safe and effective.
Inspections
Inspections provide one means of ensuring compliance and most coun-
tries have enacted laws to allow regulatory inspectors to review facili-
ties, records, and operations that produce or distribute investigational or
approved products. U.S. FDA inspections are typically conducted for the
following reasons:
• Periodic review of an operation to ensure continuing compliance
• Supplier of products to the government
• Directed review due to issues related to a product
• Revisit, following finding of deficiencies on an earlier inspection
• Following a recall or complaint
• Preapproval visit based upon a market application or amendment or,
more rarely (but in the case of a new technology, an investigational
new drug [IND] Application).
The Food, Drug, and Cosmetic Act of 1938 gives the FDA broad author-
ity in what may be inspected as long as the items—facilities, records, even
vehicles—bear on whether a product (e.g., a biopharmaceutical or active
ingredient) or service (e.g., a nonclinical or clinical study) is in compliance
with the Act. Personal, financial, or business information is not a target of
inspections and technical information is kept confidential for inspectional
reports. Individuals are not required to sign affidavits but information they
disclose may be used in a case against the firm.
Let us examine the FDA inspection process as it might happen at a bio-
pharmaceutical firm. Primary reason or types of FDA inspections include
preapproval, routine, and directed or for-cause. An inspection typically begins
in the morning of a weekday as FDA inspectors present their credentials
and state the reason for their visit. If a firm refuses entry, then the FDA

122 Biotechnology Operations
will seek an administrative inspection warrant or, if serious breaches of
the law are suspected, a criminal search warrant. The inspection itself
involves a review of the plant, facilities, and records. FDA inspectors are
highly trained and inspection teams, varying in size from one to a dozen
FDA employees, include individuals with various expertise. For example,
the team sent to a biomanufacturing plant might include individuals expe-
rienced in record review, others with expertise in technologies used at the
plant, and specialists with a deep understanding of general manufactur-
ing processes and regulations. Inspections may be brief, lasting less than a
day and conducted by one individual, or they may take weeks and involve
teams of inspectors, visiting continuously or sporadically. Inspectors care-
fully research the history of a product and the facility before they visit, and
they are guided by FDA’s Inspection Operations Manual. Further, inspec-
tors now use a systems approach when visiting an operation. There are
compliance trends that lead to investigational emphasis, and these issues
should be evident to the biopharmaceutical community through meetings
and from press releases by the FDA. Today, for example, this is corrective
and preventive action (CAPA), sources of active pharmaceutical ingredi-
ents (APIs), and production and process or facilities and equipment con-
trols, but in the future it may be other topics that the FDA and the public
believe require immediate attention to ensure a supply of safe and effica-
cious products. A thorough investigation begins at the top, looking at man-
agement responsibility and involvement, moving to design control and
always touching on the hot topics. During an FDA inspection, typically
at the end of each day, a debriefing session takes place to ensure aware-
ness and status of the inspection and also provides an opportunity for any
clarification or misunderstanding. Upon completion of the inspection, the
inspector conducts an exit interview with management and provides a list
of any notable observations.
A variety of documents may be prepared by the FDA as the result of an
inspection.
• Form FDA 483, Inspectional Results, lists notable observations made
by the inspectors. It is issued to the firm before the inspector leaves
on the final day.
• Upon returning to their FDA office, inspectors file details of their
findings and present evidence or exhibits of deficiencies, uncovered
in the Establishment Inspection Report (EIR).
Inspections result in one of three courses of action, as recommended by the
FDA. For the biopharmaceutical firm, the preferred outcome is no action indi-
cated (NAI); a clean bill of compliance health, if you will. Another possible
outcome is voluntary action indicated (VAI) and the third is official action
indicated (OAI). A prudent biotechnology firm will take any inspectional
findings of VAI or OAI very seriously. The report on their firm becomes a

123Regulatory Compliance
matter of public record, and competitors and customers may file a Freedom of
Information Act (FOIA) request to obtain Form FDA 483 or the EIR, redacted
of confidential information. Management and quality staff of the firm is
involved in all reviews of and responses to inspectional findings. In the ideal
situation, a team of supervisors carefully examines the inspectional findings
and compares them to regulations cited and to company records or proce-
dures identified and described. Indeed, a systems approach is applied and
the firm typically generates a voluntary plan to correct each deficiency cited
by inspectors; this plan is submitted by the sponsor to the FDA for review.
Negotiations between the agency and management of the firm may follow
and there is usually a final resolution and agreement, satisfactory to regula-
tory authorities. Such is the outcome for most VAI situations. Enforcement
action is, however, indicated for OAI determinations. Time and again it has
been shown that FDA has the upper hand in these matters and rarely does a
firm avoid the need to admit to and correct OAI deficiencies found on an FDA
inspection.
Overseas biopharmaceutical manufacturing facilities produce prod-
ucts that are imported into the United States by national and international
firms. There are strong economic drivers: lower cost of goods and operating
expenses, ease of sourcing materials, and relatively low wages to prepare
bulk substances and even final product. This shift in international manufac-
turing has increased the demand for the U.S. FDA inspections of overseas
operations. Bulk API or final product cannot be imported into the United
States without an appropriate FDA approval, to include approval of the
overseas manufacturing facility and hence the products made in that facil-
ity. FDA inspectors perform routine inspections of overseas biopharmaceu-
tical manufacturing and quality control operations and, in larger countries,
FDA employees may even reside in that nation. Further, the FDA performs
100% screening of API and product that enters into the United States, to
include surveillance inspections of imported goods to ensure compliance
with the U.S. requirements. Ultimately, the U.S. firms, or the U.S. represen-
tative of a foreign firm, the importer, is responsible and held accountable
for safety and efficacy of foreign-manufactured ingredients used in their
finished product.
Enforcement Actions
The Food, Drug, and Cosmetic Act of 1938 went beyond inspectional author-
ity and action and delegated certain authority for enforcement to the FDA.
This gave the agency authority for seizure, injunction, civil penalties, and
criminal prosecution, or import and export restrictions for certain prod-
ucts. FDA enforcement actions may, however, only be applied to certain
acts, with the most common being production or delivery of adulterated
or misbranded product into interstate commerce or of adulterating or
misbranding the product once it is in commerce, refusing to permit an

124 Biotechnology Operations
inspection, failing to register a manufacturing facility, and adulterating or
removing labeling. Adulteration and misbranding require further defini-
tion as these acts apply to biopharmaceuticals, but not every nuance can be
listed here. Adjectives used in specific definitions of adulterated include:
putrid, filthy or decomposed, lacking indicated strength, quality or purity,
out of cGMP compliance, or having a deficient container. Phrases used to
define misbranded labeling are: false or misleading, failure to list essential
elements such as name of drug or manufacturer and directions for use, and
directions that result in a dangerous situation when followed. The point is
clear and the public agrees that products meeting definitions of adulter-
ated or misbranded should be pulled from the market. Consider now how
this discussion on FDA inspection and enforcement directly relates to dis-
cussions regarding regulatory operations, quality systems, manufacturing
and control, and nonclinical or clinical studies.
The FDA may only bring to bear enforcement actions if the product is
introduced into interstate commerce. Courts have interpreted the terms
adulterated, misbranded, and interstate commerce quite broadly and it is
virtually impossible for a firm or even a university or institution to avoid
compliance with FDA regulations. So, what might the FDA do if the sponsor
fails to correct deficiencies uncovered by the FDA? There are many possi-
bilities but those most commonly used are an enforcement letter or warning
letter to the sponsor, forced recall of the product, and judicial enforcement.
Debarment or disqualification from participating in the FDA regulated
activities is an option when responsible individuals are identified. It is not
unusual for the FDA to take two or more of these actions before resolving
a case.
• Debarment: It is imposed when action is sought against individuals.
For example, an officer of a biotechnology firm may be debarred
from working in the regulated industry for a period of time. The
investigator of a clinical study or the director of a nonclinical may be
debarred from conducting further studies to support the develop-
ment of a regulated product after proven egregious behavior.
• Letters: For OAIs or when VAIs are not resolved to the satisfaction
of the FDA, the sponsor is sent a strongly worded warning letter in
which the FDA states the case against the biopharmaceutical firm.
The letter is addressed to an individual, usually an executive, at
the firm. The agency then posts this letter at http://www.fda.gov
for public access. Letters also list additional enforcement action,
including possible criminal action that could be taken against
the firm or high-level individuals at that firm unless the matter is
resolved to the satisfaction of the FDA. Not surprisingly, several
issues are resolved to FDA’s satisfaction shortly after a warning
letter is issued, the ultimate step in ensuring compliance through
administrative means.

http://www.fda.gov

125Regulatory Compliance
The FDA always has at its disposal judicial actions. The FDA, like many other
regulatory agencies, is not alone authorized to bring enforcement action but
must use judicial tools in conjunction with the Department of Justice. Judicial
Enforcement is reserved for situations that cannot be resolved or those in
which public health or safety of individuals is at risk. FDA may apply the
following judicial actions:
• Seizures or Recalls: The FDA may send federal marshals to a plant
with instructions to seize all product and the FDA may then order
the company to announce and to pay for a complete recall of all
product: sold, on the shelf or in distribution.
• Injunctions: Injunctions—temporary, preliminary, or permanent—are
legal tools used to keep a party from doing something or to proactively
make them do something. For example, an injunction on a manufactur-
ing facility may prevent, by law and legal enforcement any employee
and/or management to manufacture, process, pack, label, hold, or dis-
tribute product in a facility that produces product that has been called
into question by the FDA authorities. Unlike an administrative action,
injunctions carry the force of criminal penalties. Consent decrees of
permanent injunctions may result and they can remain in effect forever
or they can expire on a particular date.
• Criminal prosecution: It really happens, firms and individuals will
go so far as to face criminal prosecution over a disagreement with
the FDA. Others flee the country before they can be prosecuted. The
Department of Justice is always involved and there is coordination
with the FDA’s Office of Criminal Investigations. The capstone to this
process is that criminal prosecution involves strict liability, which
means that a corporate officer need not commit the act or even know
that a specific act was committed. Prosecution can rest upon failure
of a responsible individual to seek out and remedy when situations
occur or have occurred. In most cases of criminal prosecution, the
FDA involves officers of a firm and may or may not involve technical
operators or supervisors. Food for thought.
Product Liability
The biopharmaceutical firm must also be concerned about another legal issue
involved in possible adulteration or misbranding issues. Product liability, or
other civil actions related to poorly designed products, incorrect manufac-
ture or control, or inadequate or misleading clinical or nonclinical studies
that result in harm to a private party, such as the user of a biopharmaceutical,
can result in civil actions. Civil suits are commonly pursued in the United
States and it does not require FDA action for a biotechnology firm or for the
officers of that firm to end up in a court of law accused of selling bad product
or of putting a human at risk.

126 Biotechnology Operations
Compliance with Non-FDA Regulations:
International, National, State, and Local
When we think of compliance in biotechnology operations, most people have
a mental image of meeting regulations of the Food and Drug Administration.
While U.S. FDA compliance is important to most biotechnology firms, some
will never need to consider 21 CFR. Yet every company will face non-FDA
compliance issues. These issues can arrive with little warning and they can
have a tremendous and, unfortunately, negative impact on operations. For
example, virtually every biotechnology firm ships biologicals and chemi-
cals across state lines and international borders. Shipping such materials is
highly regulated by several agencies at the national level and perhaps also
at the state level. Another example is disposal of waste generated in labora-
tories and during nonclinical and clinical studies.
This chapter provides an overview of regulatory compliance situations
that are frequently encountered by biotechnology firms in the United States
and yet do not fall under the purview of U.S. FDA. The subject matter has
been organized under headings related to a particular activity, but the
reader will find that a single activity may be regulated by two or more agen-
cies at the local, state, and federal levels.
International and Foreign National Regulatory Authorities
for Medical Biotechnology Products
National interests and international political differences can be major hurdles
to multinational regulatory approval of biotechnology products. Attempts
are underway by both regulatory agencies and biopharmaceutical firms to
eliminate these differences through transnational harmonization and, for
much of the world, by strengthening national regulatory authorities (NRAs)
in some countries. While these efforts may not bring every national agency
into agreement, they are making a difference in many international biotech-
nology markets, notably for countries consuming the greatest amounts of
biopharmaceuticals. Despite the lack of movement by some nations, NRAs
are generally moving in the direction of harmonization. Today, it is pru-
dent to assume the fastest route to multinational approval for biotechnology
products in a multinational marketplace is through the early application of
harmonized documents.
Organizations encourage and provide guidelines for harmonization. For
example, the International Federation of Pharmaceutical Manufacturers and
Associations (IFPMA; http://www.ifpma.org), a trade organization, and
several NRAs are working to harmonize international regulations through
the International Conference on Harmonization (ICH). ICH, a nonprofit
group mentioned throughout this book, promulgates harmonized guidelines on

Home

127Regulatory Compliance
various subjects applied to biopharmaceutical development (http://www.
ich.org). ICH topics are divided into four major categories and ICH topic
codes are assigned according to these categories: (1) Q, or quality topics, are
those relating to chemical and pharmaceutical quality assurance (e.g., qual-
ity control test validation and stability testing); (2)  S, or safety topics, are
those relating to in vitro and in vivo preclinical studies (e.g., carcinogenicity
testing); (3) E, efficacy topics, are those relating to clinical studies in human
subject (e.g., dose response studies, Good Clinical Practices); and (4) M,
multidisciplinary topics, are cross-cutting topics, which do not fit uniquely
into one of the aforementioned categories (e.g., medical terminology, or elec-
tronic standards, and the common technical document, or CTD). A partial
list of ICH documents relevant to biotechnology product development is
in Box 4.4. Most ICH guidelines are accepted by the FDA and so they are a
particularly helpful guidance for ensuring safe and effective biopharmaceu-
ticals enter the U.S. marketplace.
The FDA also provides at its website hundreds of additional guidance
documents for virtually every aspect of biotechnology product develop-
ment (http://www.fda.gov/regulatoryinformation/guidances/default.htm).
Examples of FDA guidance documents are listed in Box 4.5. It is worth not-
ing that the U.S. Federal government regulations, those for all agencies,
can be searched online by keyword or numeric citation at the Government
Printing Office’s electronic Code of Federal Regulations website, http://
www.ecfr.gov/.
The WHO also certifies the quality of products in international com-
merce and provides international standards for nonclinical and clini-
cal testing of drugs and biologicals (http://www.who.int). However, the
WHO website is complex and it can be difficult to identify specific guide-
lines. As a general rule, WHO guidelines and standards are neither as
detailed nor as stringent as those of developed countries. The WHO guid-
ance on pharmaceutical development is an example (http://www.who.
int/topics/pharmaceutical_products/en/). WHO also provides guidelines
for clinical trials, including an international registry of clinical research
and templates for study documents, and biological product development,
including those for nonclinical safety testing, formulation, distribution,
and purchase.
European and Japanese regulations continue to have, in these countries and
much of the rest-of-the-world, a direct impact on development of biopharma-
ceuticals. While each member of the European Economic Community (EEC)
has a national regulatory authority and national regulations, harmoniza-
tion within the EEC is being led by the European Medicines Agency (EMA),
responsible for scientific evaluation of medicines for use in the EEC, and the
Committee for Human Medical  Products (CHMP), responsible for prepar-
ing opinions on questions concerning medicines for human use and cen-
tralized marketing assessment and authorizations. Additional information

http://www.who.int/topics/pharmaceutical_products/en/

http://www.ecfr.gov/

http://www.ich.org

http://www.who.int/topics/pharmaceutical_products/en/

http://www.who.int

http://www.ecfr.gov/

http://www.fda.gov/regulatoryinformation/guidances/default.htm

http://www.ich.org

128 Biotechnology Operations
BOX 4.4 EXAMPLES OF ICH GUIDELINES USED
IN BIOTECHNOLOGY DEVELOPMENT
• Quality
• Q1A—Stability testing of new drug substances and products
• Q2(R1)—Validation of analytical procedures
• Q3A(R2)—Impurities in new drug substances
• Q5A(R)— Viral safety of biotechnological products derived
from cell lines of human and animal origin
• Q5D— Derivation and characterization of cell substrates used
for production of biotechnological/biological products
• Q6B— Specifications: Test procedures and acceptance crite-
ria for biotechnological/biological products
• Q7— Good manufacturing practice guide for active phar-
maceutical ingredients
• Q8(R2)—Pharmaceutical development
• Q9—Quality risk management
• Q10—Pharmaceutical quality system
• Safety
• S1B—Testing for carcinogenicity of pharmaceuticals
• S2—Guidance on genotoxicity testing and data interpretation
• S4—Duration of chronic toxicity testing in animals
• S6— Preclinical safety evaluation of biotechnology-derived
pharmaceuticals
• S9—Nonclinical evaluation for anticancer pharmaceuticals
• Efficacy
• E2A—Clinical safety data management
• E3—Structure and content of clinical study reports
• E6(R1)—Good clinical practice
• E8—General considerations for clinical trials
• E9—Statistical principles for clinical trials
• Multidisciplinary
• M1—Medical dictionary for regulatory activities (MedDRA)
• M2—Electronic standards for the transfer of regulatory
information
• M3—Nonclinical safety studies
• M4—Common technical document

129Regulatory Compliance
BOX 4.5 EXAMPLES OF U.S. FDA GUIDELINES
FOR DEVELOPMENT OF BIOPHARMACEUTICAL PRODUCTS
• Chemistry, Manufacturing, and Controls (CMC)
• Points to consider in the characterization of cell lines used
to produce biologics. FDA. August 1993.
• Points to consider in the manufacture and testing of
monoclonal antibody products for human use. February
1997.
• Content and format of chemistry, manufacturing, and
controls information and establishment description infor-
mation for a vaccine or related product. FDA. January
1999.
• Drugs, biologics, and medical devices derived from bio-
engineered plants for use in humans and animals. FDA.
September 2002.
• INDs for Phase 3 and Phase 3 studies—Chemistry, manu-
facturing, and controls information. FDA. May 2003.
• Comparability protocols—Protein drug products and bio-
logical products—Chemistry, manufacturing, and controls
information. FDA. September 2003.
• Sterile drug products produced by aseptic processing—
Current Good Manufacturing Practice. FDA. September
2004.
• CGMP for Phase 1 investigational drugs. FDA. July 2008.
• Process validation. General principles and practices.
Guidance to industry. November 2008.
• Assay development for immunogenicity testing of thera-
peutic proteins. December 2009.
• Characterization and qualification of cell substrates and
other biological materials used in the production of viral
vaccines for infectious disease indications. FDA. February
2010.
• Analytical procedures and methods validation for drugs
and biologics. FDA. July 2015.
• Recommendations for microbial vectors used for gene ther-
apy. FDA. October 2015.
(Continued)

130 Biotechnology Operations
BOX 4.5 (Continued) EXAMPLES OF U.S. FDA GUIDELINES
FOR DEVELOPMENT OF BIOPHARMACEUTICAL PRODUCTS
• Quality
• Medical device quality systems manual. December 1996.
• Labeling for human prescription drug and biological
products—Implementing the new content and format
requirements. January 2006.
• Quality systems approach to pharmaceutical CGMP regu-
lations. FDA. September 2006.
• Q10—Pharmaceutical quality system. April 2009.*
• Request for quality metrics. FDA. July 2015.
• Clinical
• Structure and content of clinical study reports. July 1996.*
• Protocol development guideline for clinical effectiveness
and target safety trials. July 2001.
• Guidance for clinical investigators, sponsors, and IRBs:
Adverse event reporting to IRBs—Improving human sub-
ject protection. FDA. January 2009.
• Adaptive design clinical trials for drugs and biologics.
FDA. February 2010.
• Early clinical trials with live biotherapeutic products:
Chemistry, manufacturing, and control information. FDA.
September 2010.
• Guidance for clinical investigators, sponsors, and
IRBs: Investigational new drug applications (INDs)—
Determining whether human research studies can be con-
ducted without an IND. FDA. September 2013.
• Guidance for clinical investigators, industry, and FDA
staff: Financial disclosure by clinical investigators. FDA.
February 2013.
• Oversight of clinical investigations—A risk-based approach
to monitoring. FDA. August 2013.
• Considerations for the design of early phase clinical trials
of cellular and gene therapy products. FDA. June 2015.
(Continued)

131Regulatory Compliance
regarding exact responsibilities of various agencies within the EEC is given
at the EMA website at http://www.emea.europa.eu. The Japanese Ministry
of Health, Labor and Welfare, website (http://www.mhlw.go.jp/english/;
Pharmaceuticals and Medical Devices tab), provides regulations and guide-
lines for biopharmaceutical products marketed in Japan and this regulatory
guidance also influences regulatory agencies of countries of East  Asia and
the Pacific region. Australia (Therapeutic Goods Administration, http://
www.tga.gov.au) and Canada (Health Canada, http://www.hc-sc.gc.ca) have
strong regulatory infrastructure for biomedical products, as well. In addi-
tion, many trade organizations, such as IFPMA, the Biotechnology Industry
Organization, BIO, (http://www.bio.org), and the Pharmaceutical Research
and Manufacturers of America (PhRMA), (http://www.phrma.org) actively
support national regulatory authorities, ICH and the process of harmoniza-
tion, and international guidance, since it is good business and good regula-
tory practice for member firms.
Finally, marketing approval is important if biotechnology products are to
be used in developing countries, many burdened with much of the world’s
infectious diseases. This is especially important for drugs and biologicals
BOX 4.5 (Continued) EXAMPLES OF U.S. FDA GUIDELINES
FOR DEVELOPMENT OF BIOPHARMACEUTICAL PRODUCTS
• Preclinical
• Formal meetings between the FDA and sponsors or appli-
cants. FDA. May 2009.
• Nonclinical safety studies for the conduct of human clini-
cal trials and marketing authorization for pharmaceuti-
cals. FDA. January 2010.*
• Process validation: General principles and practices. FDA.
January 2011.
• Rare diseases: Common issues in drug development. FDA.
August 2015.
• Product development under the animal rule. FDA. October
2015.
* FDA Guidelines based upon ICH Guidelines.

http://www.tga.gov.au

http://www.phrma.org

http://www.bio.org

http://www.hc-sc.gc.ca

http://www.tga.gov.au

http://www.mhlw.go.jp/english/

http://www.emea.europa.eu

132 Biotechnology Operations
developed to treat diseases such as AIDS and malaria. As noted earlier,
EMEA, FDA, and the drug regulatory agencies of other developed coun-
tries are influential in the development and regulation of products for
global health. Also, the WHO and several nongovernmental organizations/
agencies (NGOs), are actively involved in searching for means to gain regu-
latory approval in countries that do not currently have national regulatory
authorities or lack a science-based review system for biopharmaceuticals.
Transporting Infectious or Otherwise Hazardous Materials
Successful transportation, national and international, of materials and
products is important to any biotechnology firm. The transportation com-
munity, such as national and international shippers, and transportation
regulatory agencies, like the U.S. Department of Transportation (DOT)
are diligent about the materials acceptable for shipment (http://www.dot.
gov). Proper shipping procedures for hazardous and infectious materials
are enforced because they protect employees of the shipping firms, ensure
public health, and allow all compliant firms to transport a variety of mate-
rials, some considered hazardous. While most biologicals and chemicals
can be shipped, many require special precautions in packaging, labeling,
and handling.
The shipper bears virtually all responsibility for ensuring safe shipping of
infectious or otherwise hazardous materials. Therefore, the shipper must be
aware of the various and sometimes complex regulations and then properly
classify, identify, package, mark, label, and document the substance being
shipped. References to common shipping regulations or guidelines for the
United States are listed in Box 4.6. The International Air Transportation
Association (IATA) is a clearinghouse for international air transport and for-
eign, national regulations (http://www.iata.org). By regulation, the shipper
of dangerous goods must be a trained person and they must comply with
regulations and also certify that materials will arrive at their destination in
good condition and not present any hazards to humans and animals dur-
ing shipment. Commercial carriers refuse to accept any package that fails
to comply with international, national, and the shipper’s regulations or
guidelines. Failure to comply with shipping regulations often means that a
material does not reach its destination and, for spills or human exposure to
infectious, chemical, or radiological substances, can result in substantial fines
for noncompliance.
Biological materials are especially important to the biotechnology indus-
try. They are considered as (1) infectious (etiologic) agents; (2) diagnostic
(clinical) specimens; or (3) biological products. Infectious substances are
those known or reasonably expected to contain pathogens. Pathogens are
microorganisms (including bacteria, viruses, rickettsia, parasites, and fungi)
or recombinant microorganisms (hybrid or mutant) that cause infectious
disease in humans or animals. This includes (1) all cultures containing or

http://www.dot.gov

http://www.iata.org

http://www.dot.gov

133Regulatory Compliance
suspected of containing an agent which may cause infection; (2) human or
animal samples that contain such an agent in quantities sufficient to cause
infection should an exposure to them occur due to a transport mishap; (3)
sample from a patient with a serious disease of unknown cause; and (4) other
specimens not included earlier but designated as infectious by a qualified
person, for example, a physician.
Diagnostic specimens are any human or animal material including, but not
limited to, excreta, secreta, blood and its components, tissue or tissue fluid,
being transported for diagnostic or investigational purposes, but excluding
live infected animals. Diagnostic specimens resulting from medical prac-
tice and research are not considered a threat to public health. An  example
is a serum sample not suspected of containing an infectious agent that is
shipped to a laboratory for routine testing.
BOX 4.6 SOURCES OF INFORMATION ON
TRANSPORTATION OF BIOTECHNOLOGY
MATERIALS OR PRODUCTS
• Transportation within the United States
• Hazardous materials regulations, 49  CFR Parts 171–178, U.S.
department of transportation
• Interstate shipment of etiologic agents, toxins, radiologic agents,
42  CFR Part 70–75, U.S. public health service, centers for
disease control and prevention (CDC)
• Occupational exposure to bloodborne pathogens, 29  CFR Part
1910.1030, The department of labor, occupational safety,
and health administration
• Visit the website of the intended shipping firm, FEDEX, USPS,
or UPS, for guidance.
• International transportation
– Recommendations of infectious substances and diagnostic speci-
mens, United Nations. http://www.who.int/csr/emc97_3m.
pdf
– Technical instructions for the safe transport of dangerous
goods by air, International civil aviation organization
(ICAO)
– International air transportation association (IATA). http://
www.iata.org

http://www.who.int/csr/emc97_3m

http://www.iata.org

http://www.iata.org

http://www.who.int/csr/emc97_3m

134 Biotechnology Operations
Biological products may have special licensing requirements. These speci-
mens are further defined as those products derived from living organisms
that are manufactured and distributed in accordance with the requirements
of national governmental authorities. They are used either for prevention,
treatment, or diagnosis of disease in humans or animals, or for development,
experimental or investigational purposes and include, but are not limited to,
finished or unfinished products such as vaccines and diagnostic products.
This general definition would include many biotechnology products. Hence,
diligence and thorough research of any and all guidelines is required by
any biologics manufacturer prior to shipment. As noted earlier, import or
export of infectious agents is highly controlled. For infectious materials and
vectors imported from foreign countries, there are requirements for importa-
tion permits and shipping labels issued by the U.S. Public Health Service and
posted on the website of Centers for Disease Control and Prevention (CDC),
U.S. Department of Health and Human Services (http://www.cdc.gov).
Biotechnology firms also ship a variety of chemical substances and dan-
gerous goods, defined as a substance capable of posing an unreasonable risk
to health, safety, or property when transported by commercial carrier or by
air, or identified as explosive, corrosive, flammable liquid, oxidizer, or com-
pressed gas. Indeed, one of the most often overlooked chemical dangerous
substances transported by biotechnology firms is solid carbon dioxide, or dry
ice. Several steps must be taken before a chemical substance is transported.
First, the shipper must determine if the good can be shipped by commer-
cial carrier. If so, then written guidelines are followed for packaging, mark-
ing, and declaring the good. A Dangerous Goods Declaration is an essential
part of every shipment and it appears outside, on the carton, along with clear
labeling as to contents, risk and response in case of spill or carton damage.
Importing, Possessing, or Transferring Controlled
Biotechnology Materials
The Animal and Plant Health Inspection Service (APHIS), USDA, regulates
the importation, possession, and transfer of many controlled plant and ani-
mal materials (http://www.aphis.usda.gov). Specifically, under the regula-
tion defining the scope of regulated materials (7 CFR 340), the Biotechnology
Regulatory Services (BRS) division of APHIS is responsible for importation,
interstate movement, and field release of genetically-engineered plants. The
BRS website (http://www.aphis.usda.gov/biotechnology) is the most com-
plete and up-to-date source of information for those engaged in agricultural
biotechnology. While many agricultural pests, actual or potential, are obvi-
ous, we must also consider that even if a material is not to be used for agricul-
ture it could, in the eyes of USDA, be considered a pest to agriculture. Hence,
through APHIS and BRS, importation is controlled for virtually all animal-
and plant-origin materials and animal products and byproducts as well as
biological materials that contain or have been in contact with materials of

http://www.aphis.usda.gov/biotechnology

http://www.aphis.usda.gov

http://www.cdc.gov

135Regulatory Compliance
animal origin. The USDA also controls selected microbial agents that could
pose a risk to animals or plants in this country. This includes in vitro mate-
rials, such as cell lines. Requests for permits authorizing the importation of
such controlled materials must be submitted to APHIS and importation can-
not commence until the application is approved.
APHIS regulates genetically engineered plants by administering the
Federal Plant Protection Act and the Lacey Act. This legislation authorizes
APHIS to control interstate movement, imports to the United States, and
release (for field testing) of “organisms and products altered or produced
through genetic engineering, which are plant pests or for which there is rea-
son to believe are plant pests” (CFR, 2011). A plant pest is a risk to other plants
and ecosystems. The term is generally applied to weeds, insects, diseases, or
untested genetically modified organisms (GMOs). GMO release in the United
States is discussed later. Applying the term plant pest to a genetically engi-
neered plant means only that the nonpest nature of the plant has yet to be
demonstrated. APHIS requires a permit and concurrence of individual state
departments of agriculture for movements across state lines. For field test-
ing of a new plant, referred to as environmental release, a permit may also
be required from APHIS. For selected plants, one of two other processes, the
notification process or the petition process, may be used in place of a permit.
Today, many firms are developing genetically engineered plants that pro-
duce drugs or biological compounds intended for medical or veterinary
treatments. The FDA has responsibility for regulating the active ingredients
produced by these plants. APHIS ensures engineered plants do not pose a
significant plant pest risk, a risk to threatened and endangered species, or
a risk to people working with them. An APHIS permit is required to take
such plants to the field so as to reduce the risk of harm to other organisms,
to evaluate any special considerations for containment, and to prevent such
plants from entering the food supply.
APHIS is also responsible for protecting animals important to agriculture.
This agency facilitates international trade, monitors health of animals before
they enter the United States, and regulates the import and export of animals,
animal products, and biologics. It is in the import and export area that the
National Center for Import and Export (NCIE) has the greatest interaction
with the biotechnology industry. Generally, a USDA veterinary permit is
needed for import of nonhuman materials derived from animals or exposed
to animal-source materials. A wide range of materials, for example, animal
tissues, RNA/DNA extracts, hormones, anti-sera, and monoclonal antibodies
for in vivo use, are regulated.
USDA also regulates the care and use of laboratory animals (http://www.
usda.gov). Any biotechnology firm that does business with a research animal
breeder or vendor or itself houses or uses animals is familiar with this exten-
sive set of regulations.
The U.S. Fish and Wildlife Service, part of the U.S. Department of Interior,
enforces possession or transfer of certain species (e.g., endangered birds or

http://www.usda.gov

http://www.usda.gov

136 Biotechnology Operations
primates) or any part of those species (e.g., feathers, eggs, blood, or tissue)
under the Endangered Species Program for the Endangered Species Act
(http://fws.gov/endangered). Permits are required to transport or hold spec-
imens and the law is enforced at U.S. borders by CBP, part of the Department
of Homeland Security.
The Bureau of Industry and Security (BIS), of the U.S. Department of
Commerce, formerly the Bureau of Export Administration, oversees U.S.
exports of dual-use commodities, technology, and software (http://www.bis.
doc.gov). The Bureau has the lead role in both export licensing process and
enforcement operations. Their mission, based upon national security, is to
control exports of sensitive products to entities that could misuse U.S. technol-
ogies and products. BIS licenses exporters of certain products, including bio-
technology-related products such as fermentation equipment. In doing so, it
requires exporters to notify other parties of the sale and the conditions of sale
and to obtain written acknowledgment from the end-user of the intended use.
Licensing conditions are sometimes necessary to make certain that approved
items are in the correct location and being used in an appropriate manner.
This Bureau does not regulate all goods, services, and technologies but it does
control the export of certain microorganisms, toxins, and equipment used to
make these items. The items are provided in the Commerce Controlled List
(see  the Regulations tab at http://www.bis .gov). To further complicate
matters, other U.S. government agencies regulate more specialized exports.
For example, the U.S. Department of State has authority over defense articles
and defense services. A list of other agencies involved in export controls can
be found on the website for BIS.
The Public Health Security and Bioterrorism Preparedness
and Response Act of 2002
This law has certainly complicated movement or use of many biotechnology
products within the United States and across its borders but it has also enhanced
security while allowing legitimate biotechnology efforts to continue. It includes
sections on “Enhancing Controls on Dangerous Biological Agents and Toxins,”
providing for the regulation of biologicals specified by the Department of
Health and Human Services and the Department of Agriculture. It recom-
mends interagency coordination between the two departments regarding
control of overlapping agents and toxins and provides for criminal penalties
regarding certain biological agents and toxins. The CDC has primary responsi-
bility for implementing the provisions of this Act (http://www.cdc.gov). APHIS
is the agency fulfilling roles designated to USDA.
The USDA Regulations are within 7 CFR and 9 CFR while the CDC regula-
tions are 42 CFR 73. In general, the regulations are aimed at animal and plant
agricultural and human health threats, respectively, but there is some over-
lap. The regulations establish and enforce safety procedures for listed agents
and toxins, including

http://www.bis .gov

http://www.cdc.gov

http://www.bis .gov

http://www.bis .gov

http://fws.gov/endangered

137Regulatory Compliance
• Measures to ensure proper training and appropriate skills to handle
agents and toxins, and proper laboratory facilities to contain and
dispose of agents and toxins
• Safeguards and security measures to prevent access to listed agents
and toxins for use in domestic or international terrorism or for any
other criminal purpose
• Procedures to protect animal and plant health, and animal and plant
products, in the event of a transfer or potential transfer of a listed
agent or toxin in violation of the safety procedures, as well as safe-
guards and security measures
• Appropriate availability of biological agents and toxins for research,
education, and other legitimate purposes
The regulations themselves cover requirements for registration, security
safety and emergency response plans, training, transfer, record keeping,
inspections, and notifications. They regulate molecular parts of organisms,
since particular genes or proteins from these organisms might also con-
stitute a risk to public health. Even a small amount of nucleic acid from a
select agent may be regulated. A permit system allows a research investiga-
tor or biopharmaceutical product developer to import, keep, transfer, or test
(e.g., field test a genetically engineered plant) an agent. Since the penalties
for improper or illegal possession, use, or transfer of the agents are severe,
those biotechnology firms using even seemingly safe and innocuous agents
or molecules should become familiar with the select agent list and the regu-
lations well before they consider transferring the material to their labora-
tory. Noteworthy is the fact that this and other Acts often contain unusual
or unexpected and sometimes confusing or conflicting clauses. Examples of
issues from the Act of 2002 are provided in Box 4.7.
Importation or Exportation of Biotechnology Products for
the Purpose of Treatment of Diseases in Humans
FDA regulations, as discussed in the section “Compliance for Import
of Biopharmaceuticals into the United States” define requirements for
import or export of any “virus, therapeutic serum, toxin, antitoxin, or
analogous product” for the “prevention, treatment, or cure of diseases
or injuries of man” (CFR, 2015). It is important to note the roles customs
and other Federal agencies play in the importation and exportation of
these products, since the scope of regulations encompasses many biotech-
nology products. Labeling requirements are absolute since they inform
customs and other government officials. If  a product is intended for
human use, then it must be labeled and may be inspected and sampled
by CBP (http://www.cbp.gov). FDA may be contacted and may even
inspect the shipment. If there is no evidence that the product is licensed

http://www.cbp.gov

138 Biotechnology Operations
BOX 4.7 A DISCUSSION ON THE SELECT AGENTS
AND PUBLIC HEALTH SECURITY AND BIOTERRORISM
ACT OF 2002 (PUBLIC LAW PL107-188) AND THEIR
IMPACT ON BIOTECHNOLOGY OPERATIONS
• In 2002, Congress passed the public health security and bioter-
rorism act, identifying the need to keep a list of select agents and
toxins. Select agents (biological agents or toxins) were specifi-
cally identified, or declared by USDA and Department of Health
and Human Services. The U.S. Center for Disease Control and
Prevention maintains and changes the list, approves laborato-
ries qualified to transfer, use, or control select agents and regu-
lates, to a certain extent, practices in those laboratories. A full
and current list of select agents is at http://www. selectagents.
gov. Some examples are botulinum neurotoxins, SARS-
associated coronavirus, certain hemorrhagic fever viruses,
Shiga toxin, Rift Valley fever virus, Xylella, a bacterial pathogen
of citrus crops, swine flu virus, and avian influenza virus. Most
would agree that we do not want just anyone, and certainly not
our residential neighbors, holding these agents.
• Congress also mandated that certain individuals, including
among others those adjudicated as mentally defective or (hav-
ing) been committed to any mental institution, and/or those hav-
ing been discharged from the Armed Forces of the United States
under dishonorable conditions, and/or those determined to be
unlawful users of the Controlled Substance Act (21 USC 802). In
the United States, there are many citizens who have at one time
been in a mental institution, discharged dishonorably or used a
controlled substance, to include LSD, codeine, cocaine or mari-
juana, as defined by the Department of Justice, Drug Enforcement
Administration, Officer of Diversion Control. Current informa-
tion can be identified at http://www.deadiversion.usdoj.gov
• The purpose of this discussion is not to argue or in any way
malign the Act of 2002; it was well-meant and passed by
knowledgeable legislators and the president. The purpose is to
stress how very important it is for a professional working in
biotechnology to be familiar with a host of detailed public laws
and regulations before embarking upon a laboratory or devel-
opment project. Further, the professional should consult with
regulatory authorities or legal counsel whenever perceived
issues or conflicts arise.
(Continued)

http://www.selectagents.gov

http://www.deadiversion.usdoj.gov

http://www.selectagents.gov

139Regulatory Compliance
by FDA, then it is held by CBP. If the product is of animal origin (e.g., a
horse antiserum against snake venom), it may require a USDA permit,
as well. If any part of the product is from an endangered species, it will
also need a permit from Fish and Wildlife Service under the Endangered
Species Program. In effect, CBP serves as a gatekeeper at the U.S. borders,
acting on behalf of several federal agencies. In summary, importation and
exportation of all biotechnology materials and products must be carefully
researched by the shipper with the expectation that the regulations of
multiple federal agencies could complicate, delay, or stop the movement
of these goods.
BOX 4.7 (Continued) A DISCUSSION ON THE SELECT
AGENTS AND PUBLIC HEALTH SECURITY AND
BIOTERRORISM ACT OF 2002 (PUBLIC LAW PL107-188) AND
THEIR IMPACT ON BIOTECHNOLOGY OPERATIONS
• Now, in light of terms of the Act of 2002 identified earlier,
consider in three examples, provided in the following, YOUR
responsibility as a manager or supervisor at a biotechnol-
ogy firm when a select agent is handled by your employees.
1. My operation handles, under proper conditions, recom-
binant Corona virus to infect animals for testing Corona
virus vaccines. I know some employees are veterans of the
armed forces; must I determine if each veteran was honorably
discharged?
2. We will use small amounts of botulinum toxin in a mouse
model to develop QC assays to test our monoclonal antibody,
intended to neutralize the toxin. Although Botox® is currently
a product licensed for medical use and widely available in doc-
tor’s offices, must I query each employee to ensure that no per-
son was at any time committed to a hospital mental ward, if
even for one day and decades ago?
3. Our laboratory is studying protein expression of Xyella in an
effort to develop antibiotics and combat this disease of citrus
crops. Must I query all employees to ensure that no one has ever
used marijuana or possessed or taken a cough remedy or another
medicine containing codeine?

140 Biotechnology Operations
Occupational Health and Safety
A biotechnology firm must have effective health and safety policies and
practices for one simple reason: it protects employees, their most valuable
asset. We all know that work can affect our health and, when queried, people
state that good health is a leading factor in quality of life. If a workplace is
safe, people enjoy their jobs and are more interested and involved in their
employment.
The biotechnology laboratory work environment includes hazards but it
need not be unsafe. We work with harsh chemicals, acids, corrosives, radio-
chemicals, and biological agents such as viruses and toxins. In research labo-
ratories, individuals are often in close contact with these materials and, in
biopharmaceutical manufacturing, there may be large volumes of potentially
hazardous materials in the workplace. The work environment may also have
carcinogens, flammable gases or liquids, steam and hot fluids.
As with just about everything in biotechnology, the key to providing a safe
and comfortable work environment is good planning. Every biotechnology
firm should have a health and safety policy and plan and procedural docu-
ments, all receiving the full support of upper management and line supervi-
sors. Good policies emphasize prevention rather than reaction to incidents
or accidents. Standards for health and safety are based upon risk assess-
ments and regulatory requirements. Health and safety plans state objectives
or goals and standards or specifications. The results, based upon measur-
able outcomes, are compared at regular intervals against health and safety
objectives.
Biotechnology firms should have a visible organization or structure to sup-
port a health and safety plan. An environmental health and safety specialist
is the individual responsible for developing, implementing, and monitor-
ing industrial safety programs within the biotechnology company. While a
smaller firm may not require a full-time health and environmental safety offi-
cer, a consultant, such as an occupational safety specialist, is an important
member of the corporate team. These professionals inspect laboratories and
product development, manufacturing and testing areas to ensure compliance
with federal Occupational Safety and Health Administration, state, and local
regulations and corporate policies. They evaluate new equipment and raw
materials for safety, and monitor employee exposure to chemicals and other
toxic substances. A safety specialist also conducts training programs in haz-
ardous waste collection, disposal, and radiation safety.
Finally, management must make every effort to encourage a safe and
healthy culture within the biotechnology firm. Communication is an
important part of the process, with periodic seminars and, most impor-
tantly, an effective means for employees to express their health and safety
concerns to management. A safety training program gives employees an
opportunity to learn more about safety as it relates to their particular job
assignments.

141Regulatory Compliance
Local, state, and federal agencies, notably OSHA of the Department of
Labor, regulate health and safety in the workplace (http://www.osha.gov).
As early as 1985, OSHA began to examine the health and safety issues related
to biotechnology. OSHA originally felt that no additional regulations were
needed for such workplaces since other standards, such as those for general
laboratory safety, provided an adequate basis for protection and safety. These
OSHA regulations and standards are at 29 CFR 17. In addition, blood-borne
pathogen guidelines, which apply to all occupational exposure to blood
or other potentially infectious materials, and exposure to other infectious
organisms are responsibilities of both OSHA (http://www.osha.gov/SLTC/
biologicalagents/index.html) and the National Institute for Occupational
Safety and Health (NIOSH), Centers for Disease Control (http://www.cdc.
gov/niosh/homepage.html).
In summary, one of the most important aspects of safety, from the point
of view of the employee in a biotechnology firm, is to have a clear under-
standing of hazards in their workplace. Simple acts, such as participation
in training, wearing safety glasses, proper disposal of waste, and review of
material data safety information are operational keys to health and safety in
the biotechnology work environment. A health and safety plan and effective
training can go a long way to reach these objectives.
Environmental Regulations in Biotechnology
There are many environmental hazards in biotechnology operations and
there also are numerous federal, state, and local regulations as well as agen-
cies to enforce them. Failure to heed environmental guidelines by a firm
puts both the company and community at possible risk. These issues, or
even perceived problems, are often highly publicized within a state or com-
munity and, when a violation becomes known, it creates a negative image
of the biotechnology industry as a whole. To make matters worse, there
already are concerns, worldwide, about the release of genetically modified
molecules or organisms into the environment. And there are the less publi-
cized, but still very real issues, related to environmental release of materials
from biotechnology laboratories or operations.
A relevant example is the receipt, handling, and disposal of radioiso-
topes, functions regulated by the Nuclear Regulatory Commission (NRC),
Department of Energy (http://www.nrc.gov/) and by state and sometimes
local agencies or other federal agencies. For example, OSH handles certain
aspects of exposure to ionizing radiation (29 CFR 1910.1096). NRC manages
radioactive materials by controlling the production, shipment, use, and dis-
posal of these materials. It does so through licensing responsible entities,
such as universities or biotechnology firms. NRC also allows individual
states to regulate certain activities through the Agreement State Program.
A biotechnology firm wishing to purchase, receive, use or dispose of a radio-
isotope must apply for and receive a license and agree to keep careful records,

http://www.cdc.gov/niosh/homepage.html

http://www.osha.gov/SLTC/biologicalagents/index.html

http://www.nrc.gov/

http://www.cdc.gov/niosh/homepage.html

http://www.osha.gov/SLTC/biologicalagents/index.html

http://www.osha.gov

142 Biotechnology Operations
train employees, follow detailed rules, accept unannounced inspections, and
pay fines for noncompliance.
Sponsors of INDs, BLAs, or NDAs are required to file an environmental
impact statement or seek categorical exclusion for each product and under
each regulatory filing with FDA.
Biotechnology firms also face complex regulations, many local, deal-
ing with the disposal of chemicals and biological substances. With a few
exceptions, it is not difficult to dispose of small amounts of nonhazardous
chemicals or biological materials. However, in biotechnology operations,
notably manufacturing, larger amounts of biological and chemical materials
may need to be released into the environment. Examples include disposal
of a large mass of recombinant bacteria following fermentation or of large
volumes of organic solvents following molecule purification. In most cases,
these cannot simply be sent to the local landfill or flushed down the com-
munity sewer. Regulations for waste disposal often fall under the purview of
the Environmental Protection Agency (EPA) (http://www.epa.gov) but one
can expect to find complex and extensive state and local regulations, as well.
Indeed, local officials often understand all regulations—federal, state, and
local—that apply to their community.
Major regulatory applications, such as IND, NDA, or BLA, must consider
environmental issues related to the manufacture and use of each product.
National environmental law and policy, notably the National Environmental
Policy Act of 1969, drive this requirement. Each of these documents must
contain either an Environmental Assessment or the sponsor must state
and show that the actions described in the document are categorically
excluded from an Assessment. Environmental Assessments can be large
and complex documents and they are not required for most biopharma-
ceuticals, at least not in early development. However, with some products
in the IND stage and with many products in large-scale manufacture, the
sponsor must complete an assessment prior to submitting a regulatory
application to FDA. The agency provides guidelines for those wishing to
choose between claiming exclusion or preparing an assessment.
Genetically Modified Organisms or Molecules
Several federal agencies are responsible for regulating the release of GMOs
or chemicals into the environment. The role of USDA has been mentioned.
The EPA is also involved because of the Microbial Products of Biotechnology
section of the Toxic Substances Control Act, or TSCA. TSCA authorizes EPA
to, among other things, review new chemicals before they are introduced
into commerce (http://www.epa.gov/opptintr/biotech and www2.epa.
gov/regulation-biotechnology-under-tsca-and-fifra). FIFRA, the Federal
Insecticide, Fungicide and Rodenticide Act mandates EPA registration of
all pesticides, including those derived from biotechnology (e.g., genetically-
modified microbial pesticides and herbicide-tolerant crops). Intergeneric

http://www2.epa.gov/regulation-biotechnology-under-tsca-and-fifra

http://www2.epa.gov/regulation-biotechnology-under-tsca-and-fifra

http://www.epa.gov/opptintr/biotech

http://www.epa.gov

143Regulatory Compliance
microorganisms, that is, microorganisms created to contain genetic mate-
rial from organisms in more than one taxonomic genus, are considered new
chemicals under TSCA and therefore EPA reviews and regulates the use of
intergeneric microorganisms in commerce or for commercial research.
Plants and domestic farm animals are genetically engineered in the
United States and such endeavors, especially their field testing and com-
mercialization, are highly regulated. The USDA is the lead regulatory
agency for genetically engineered crop, providing regulatory guidance
and licensing of crops, for planting, commercial and investigational, in the
United States. BRS at APHIS takes the lead on such matters at USDA and
the primary regulation is 7  CFR 340. Environmental regulations, such as
those handled by EPA, may also apply. And if a food or a biopharmaceuti-
cal is derived, in part or in whole, from a genetically engineered crop, then
FDA also provides guidance and may need to give final approval prior
to commercialization. Regulations for genetically modified domestic farm
(i.e., nonlaboratory) animals, including those used for food or production
of biopharmaceuticals, are complex and any given situation must be care-
fully researched with special considerations given to guidelines provided
by USDA, FDA, and EPA.
The Office of Biotechnology Activities (OBA), Office of the Director,
National Institutes of Health (NIH) is involved in activities that affect many
biotechnology firms (http://www4.od.nih.gov/oba). These activities include
biomedical technology assessment, biosafety, and biosecurity. Examples in
genetic research and biotechnology are that OBA: (1) monitors human gene
transfer and aspects of stem cell development; (2) manages several review
committees for novel technologies; (3) advises other government agencies
or departments; (4) develops policies and procedures, reviews established
Institutional Biosafety Committees (IBC); (5)  provides information to the
public; and (6) develops registries of activities.
Although OBA is not chartered by Congress as a regulatory agency, as is
the FDA, it has significant influence in several areas of biotechnology and
can have an immediate and significant impact on fields of biotechnology,
notably emerging technologies, and on biotechnology firms themselves.
OBA is influential largely by providing guidelines to the public. A typical
action is a decision as to whether an institution, such as a university, may
receive NIH funding. For example, if OBA decided that a type of human
gene cloning experiment was not appropriate, then any university or bio-
technology firm that did such experimentation could lose all NIH funding
for any research purpose. Since many biomedical entities, such as insti-
tutes, universities, and even some biotechnology firms in the United States,
receive NIH funding, OBA guidelines have significant impact. Furthermore,
unlike many FDA activities in which the information is held in confidence,
OBA activities are largely public and both the press and interest groups
often monitor and publicize issues and the biotechnology firms that are
involved with OBA.

http://www4.od.nih.gov/oba

144 Biotechnology Operations
OBA promulgates the NIH Guidelines for Research Involving
Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines), which
is the premier guidance document for nucleic acid research. It specifies all
aspects of genetic engineering and production of GMOs, outlining respon-
sible research practices in basic, animal and clinical nucleic acid research.
OBA sponsors the Recombinant Advisory Committee (RAC), experts
appointed to monitor scientific progress in basic and clinical research
involving recombinant nucleic acid and human gene transfer. RAC rec-
ommends changes to the NIH Guidelines and its members review human
gene therapy protocols.
OBA also manages compliance with the requirements for Institutional
Biosafety Committees or IBCs. An IBC must be established by any entity that
receives NIH funding and performs genetic engineering. Many biotechnol-
ogy firms that do not receive NIH funding also use an IBC for the purpose
of reviewing experiments involving genetic engineering or transfer of GMOs
to the environment. The primary role of an IBC is to ensure that all recom-
binant DNA research conducted at or sponsored by that institution is con-
ducted in compliance with the NIH Guidelines, but the roles of IBC have been
expanded at many institutions to include other aspects of laboratory research
with genetically modified materials or organisms.
OBA maintains databases on technologies under their purview and thus
fosters transparency on novel technologies that may be of concern to scien-
tists and to the general public.
Somewhat apart from these government agencies, but important to certain
biotechnology firms, is the National Science Advisory Board for Biosecurity
(NSABB), now hosted by the National Security Agency. NSABB advises U.S.
federal agencies on security issues related to life sciences, notably on ways
to minimize the possibility that knowledge and technologies emanating
from vitally important biological research will be misused in a manner that
threatens public health or national security.
Taken together, these advisory committees, while not themselves regu-
latory agencies, make recommendations that are very influential to gov-
ernmental regulatory agencies and to the biotechnology industry overall.
Their impact is particularly aimed at cutting-edge technologies. The pru-
dent biotechnology operation, public or privately held, will carefully moni-
tor the activities of each committee and be constantly and fully aware of
recommendations or guidelines they produce.
International Diligence in Biotechnology Operations
Biotechnology is an international endeavor. Most biotechnology firms
expect to sell their products or services overseas as well as in the United
States. Sometimes biotechnology research can be completed in a national

145Regulatory Compliance
environment, heeding only U.S. requirements but this is a rare situation and
most biotechnology firms or endeavors are or will become transnational or
intercontinental businesses. A  biotechnology firm must diligently plan to
incorporate their operation and, of course, their services and products, into
international environments and markets. International awareness and com-
pliance is therefore especially important to the success of biotechnology firms.
International regulations and guidelines are far too numerous to cover
in this chapter. However, a single example, genetic engineering of plants
from which food is derived, is given to emphasize the value of understand-
ing international, as well as country-specific, information prior to embark-
ing on biotechnology product development. Two entities, the Food and
Agricultural Organization (FAO) of the United Nations (http://fao.org) and
the Biotechnology Organization, BIO (http://BIO.org) are actively involved in
efforts to harmonize international guidelines for genetically modified foods
or GMOs that provide foods. The biotechnology firm proposing to export
recombinant organisms, food produced by recombinant plants or animals,
or equipment, supplies and raw materials or services for the production of
such products, should consider the guidance provided by and experiences of
these organizations or their members.
An example of an international guideline intended to harmonize the
movement of genetically engineered foods is the Codex Alimentarius
(http://www.codexalimentarius.org/), a collection of internationally adopted
food standards presented in a uniform manner. Codex standards are
meant to ensure that consumers receive products that meet internation-
ally accepted and minimally acceptable quality levels, are safe, and do not
present a health hazard in accordance with FAO guidelines. The Codex is
written by an international Committee and is approved by a WHO body,
the FAO. The priority of the Codex Commission is to protect the health of
consumers and ensure fair practices in food trade. One would therefore
assume that by following the Codex, a biotechnology firm could trade their
product or service worldwide. Unfortunately, this is not the case, since
national laws and regulations for genetically modified foods still differ
considerably on many points. As with most international guidelines, the
Codex guidelines are binding, in a national sense, only when fully rati-
fied by all parties. However, most international guidelines dealing with
biotechnology products and services are not yet, and some may never be,
accepted by all nations.
There are many regulations impacting biotechnology operations and their
reach is worldwide. Indeed, regulations outside FDA likely dominate the
FDA aspects. It  is impossible to imagine a product that is not touched by
one or more of the regulatory bodies mentioned in this chapter. A sample
problem is provided in Box 4.8 as a means of testing the reader’s knowledge
in this area.

http://www.codexalimentarius.org/

http://BIO.org

http://fao.org

146 Biotechnology Operations
Summary of Regulatory Compliance
Biotechnology activities of all types are highly regulated. FDA regula-
tions apply to all aspects of biopharmaceutical research, development, and
commercialization. Regulatory compliance, which simply means meet-
ing FDA regulations, is a necessary and important aspect to developing a
BOX 4.8 APPLICATION OF NON-FDA
REGULATIONS. AN EXERCISE
• This brief exercise is offered to provide an appreciation for the
complex analysis that must be performed in an effort to deter-
mine which non-FDA regulations and appropriate regulatory
bodies, state, local, national, and international, apply to your
biotechnology operation.
• Suppose your biotechnology firm wishes to ship from London
to Maryland a live, attenuated, recombinant bacterium, derived
from an infectious agent. You would then grow, in small
amounts, that bacterium in the laboratory and pilot manufac-
turing area. This would then be studied as a candidate vaccine
in the laboratory, in animals, and then, perhaps, in a Phase 1
study in humans.
• A few situations you must consider are listed below. Please
add to the list, to include federal, state, and local governments,
expand on the regulatory role of each agency, and cite the regu-
lations themselves.
• Export from the United Kingdom: Agencies in the United
Kingdom
• Transport within the United Kingdom: Agencies in the United
Kingdom
• International transport by common carrier
• Import and entry into the United States
• Transportation within the United States
• Classification as an agent within the United States
• Research in the laboratory in the United States
• Development in the biomanufacturing facility, the animal test-
ing facility and the clinical facility in the United States
• Environmental concerns and disposal in the United States

147Regulatory Compliance
biopharmaceutical throughout the entire product development pathway.
Regulatory compliance is achieved by operating within systems that are
established and followed to ensure product quality and adherence to appli-
cable regulations. Quality systems include implementation of best practices
such as GMP for manufacturing activities, GLP for preclinical safety evalu-
ation, and GCP for clinical activities. FDA takes a systems approach when
evaluating an organizational level of compliance, beginning with one sys-
tem then expanding to other systems, thereby assessing the overall com-
prehensiveness of the quality system. In this way, FDA develops a level of
confidence that systems instituted by a sponsor are sufficient to support
product safety. Inspections are utilized to evaluate staff, training, documen-
tation, material flow, manufacturing and testing procedures, data integrity
and interpretation, each of which contributes to achieving and maintaining
regulatory compliance. Similar compliance requirements exist for medical
devices. There is an increased level of regulatory control and review for
those devices posing greater potential risk to the end user. The FDAs’ QSR
for medical devices establishes a rigorous system, a powerful tool used to
achieve and maintain regulatory compliance. QSR is now generally accepted
and applied to the regulation of drugs and biologics. A noteworthy and pow-
erful component of regulatory compliance is inspection and enforcement.
FDA directed inspections are usually triggered for one of three reasons:
(1) required preapproval inspection, (2) routine inspection of a regulated
facility, or (3) for cause (FDA, 2016) precipitated by a serious adverse event
report, a complaint or follow- up to a previous inspectional deficiency. At the
conclusion of an inspection, if a serious noncompliance issue is identified by
the FDA, then a list of inspectional observations, commonly referred to as a
FDA 483, is issued. An unacceptable response to a 483 can result in enforce-
ment action by FDA, such as issuance of a warning letter. Noncompliance or
suspected noncompliance weighs heavily on a biotechnology company plac-
ing the company at great risk for product liability concerns, negative indus-
try perception, and decreased product sales.
The biotechnology is highly regulated industry; therefore any entity devel-
oping biopharmaceuticals must be compliant with FDA regulations. This is
essential to the viability and success of any biotechnology firm. The most
successful approach is to demonstrate quality compliance through a compre-
hensive quality systems approach; this is resource intense, and requires sig-
nificant resources, regulatory expertise, and extensive checks and balances.
Summary of Non-FDA Compliance
In any given country, several federal agencies, in addition to a food and
drug agency, may regulate a single activity or function performed by a bio-
technology firm. Hence, international, national, state, and local regulations
must be considered, and there can be considerable overlap in regulatory
authority. Due diligence and careful planning regarding all regulatory

148 Biotechnology Operations
compliance is essential to the success of any biotechnology firm. Congress
continues to change the laws regarding various aspects of biotechnology
operations and at the same time executive agencies change or add regula-
tions, while courts interpret application of laws. Adding to the complex-
ity of regulatory compliance, any given government agency may change its
mission or focus or become overwhelmed with regulatory submissions. As
biotechnology becomes more global, the U.S. biotechnology firm must con-
sider international guidelines and the regulations of countries other than
the United States. To be successful, the biotechnology operation must be
aware and diligent of all U.S. and global compliance issues as well as FDA
regulations and guidelines.
References
CFR. 2011. Introduction of Organisms and Products Altered or Produced Through Genetic
Engineering Which are plant pests or which there is reason to believe are plant pests.
CFR Part-340. National Archives and Records Administration.
CFR. 2015. Food and Drugs Chapter I- Food and Drug Administration Department of Health
and Human services. CFR Part-600. US Food and Drug Administration.
Federal Register Online. 1996. Medical Devices; Current Good Manufacturing
Practice (CGMP) Final Rule; Quality System Regulation. 61(195): 52601–62.
http://www.wais.access.gpo.gov (accessed May 31, 2016).
Oxford English Dictionary. Oxford University Press. 1997. University of Oxford,
Oxford, UK.

http://www.wais.access.gpo.gov

149
5
Quality Systems
Overview of Quality in Biotechnology
Quality impacts every aspect of a biotechnology operation. While this
might seem like a bold statement, those involved in biotechnology would
certainly agree it is true. The requirement for quality in biopharmaceuti-
cal development is backed by a host of regulations (Chapters 3 and 4). As
applied to biotechnology operations, a state of quality is necessary in all
endeavors and quality increases the value of services and products. We
refer to quality systems, quality by design (QbD), quality control, and the
roles of quality in compliance to name but a few quality terms. Indeed, the
word quality has various meanings to different individuals and for each
situation in which the word is used, so it is perhaps best defined in the
context of each usage. This book tries to do just that. Yet, we need to begin
this chapter on quality systems by establishing a basic definition for qual-
ity. The reader may take their choice from any one of five definitions given
below.
• “Quality. The degree of excellence of a thing; general excellence.”
(Oxford English Dictionary, 1997)
• “Quality is the totality of features and characteristics of a product
or service that bear on its ability to satisfy stated or implied needs.”
(British Standards Institute, 1991)
• “…specified requirements for a product can be stated in terms of an
established design…and [where]confidence in product conformance
can be attained by demonstration of…capabilities in production.”
(International Standardization Organization, 1994)
• “High quality is freedom from defects.” (source unknown)
• “Continuous improvement and waste reduction.” (Henry Ford)

150 Biotechnology Operations
Practical man that Henry Ford he goes; right to the point of saving money to
produce a quality automobile at low cost.
Even though 100 people might provide 100 different definitions for qual-
ity, a common theme in each definition would be that a product is fit or
fit for use, that it performs as intended and, hence, the user is satisfied.
But, how do quality and these definitions affect the biotechnology indus-
try? First, everyone in the business of biotechnology is either producing
a product, such as a patent, a vial of therapeutic medicine or a recombi-
nant crop plant, or they are producing a service, such as testing products
in animals or humans, manufacturing active ingredients or testing those
ingredients. Everyone engaged in biotechnology wants their product to be
fit for the intended use and thus to have satisfied customers, it is as simple
as that.
In this chapter, the reader is introduced to the concepts and practices of
quality, and the terminology associated with this field. A brief history of
quality is used by way of introduction. This is followed by a review of the
hallmarks of quality, that is, criteria that are common to most of the quality
systems used in biotechnology. To finish, we review modern ways of incor-
porating quality into biotechnology endeavors, identify how quality systems
are generally applied in our industry and provide guidelines for establishing
and managing a quality system in a biotechnology firm.
History: Evolution of Quality Concepts and Practices
Although it is not obvious, quality has a long background, outlined in
Figure  5.1. Quality is rooted in the ancient history, beginning when indi-
viduals made goods for themselves or, through barter, for their neighbors.
Since buyer and seller usually lived in the same village, the act of provid-
ing a bad product led to a bad reputation for both product and producer,
or seller. As it does today, a negative image resulted in loss of business for
the producer and it also led to social and economic pressures to make only
quality products. With the industrial revolution, manufacturers were at a
distance to the buyer and thus there was little face-to-face contact between
the parties. New forces ensured quality under this rapidly evolving system.
Oral, then written, warrantees were developed. For example, manufacturers
began to list themselves as the source of a product and, in some cases such
as processed foods, they also provided consumers with the ingredients. If
the product was of high quality, this served as a form of advertisement.
However, poor quality products led to a negative image and even to con-
flicts between the buyer and seller. This led to various quality initiatives

151Quality Systems
and, over the past 100  years, a number of ideas for ensuring quality of
products and services have been adopted to ease those conflicts. Some of
the most familiar are
• Standards for measuring instruments, for example, kilogram weight
standards
• Marks or seals, for example, UL Seal of Approval on electrical
appliances
Quality
Outside groups certify
quality
Late twentieth century
Code of Federal
Regulations 21
Part 820
Quality System
Regulation
Work groups focus on
quality
Today
Quality Assurance
Guaranteed to customer
Today
Tradesmen compete on
quality of their products
Prior to nineteenth century
Industrial revolution demands group
efforts toward quality products
twentieth century
Quality systems identified
in written documents
Today
Standard International
Quality Systems
Today
ISO 9001 Certified
Quality Management System
FIGURE 5.1
Evolution of quality systems.

152 Biotechnology Operations
• Supervisory responsibility for quality production on the assembly
line, for example, Henry Ford ordering his foremen to be responsible
for quality in each of their assembly line areas
• Regulatory quality requirements were codified, such as the Food
and Drug Act, to mandate quality in drugs and medical device
development
• Worker responsibility for quality production on the assembly line,
for example, as touted by automobile manufacturers in the late 1980s
• Statistical trend analysis, for example, medical chart reviews by
health insurance firms to ensure that patients received the best
health care for the price
• Fresh ideas, such as ISO 9001, Total Quality Management and Six
Sigma, as introduced over the last three decades
Therefore, when and how did the concepts and practices of quality come to
be applied to biotechnology? Quality was applied to virtually all aspects of
biotechnology operations very early, as soon as biopharmaceutical develop-
ment began in the 1980s. For what reasons was quality applied to biotech-
nology? For business reasons—good quality results in an excellent product
or service and this, in turn, leads to healthy sales. For compliance reasons—
various regulatory agencies said quality assurance was necessary for most
biotechnology products, certainly for those with a potential impact on the
health or safety of the user or on public health, in general.
Long before biotechnology endeavors moved from laboratory benches and
into development, the pharmaceutical industry had, for these reasons, applied
quality concepts to all aspects of drug development. The medical device field was
not far behind, adopting slightly different but no less stringent quality systems.
Since many biotechnology firms must compete within those industry segments
and because they are often regulated by the likes of the U.S. Food and Drug
Administration (U.S. FDA) or the U.S. Department of Agriculture, the evolving
biotechnology industry had no choice but to be compliant with modern quality
standards. Does this mean that most, if not all, biotechnology product devel-
opment or services must consider quality? Yes, today quality is a must. How
then can a biotechnology firm begin to develop a product or service without first
instituting a quality system and how could they continue to develop products or
services with a weak quality system? They cannot. Pretty blunt language but it is
a fact, you cannot provide services or develop products in biotechnology unless
you follow quality systems that are accepted by the consumer and regulatory
agencies for that product or service. Most biopharmaceutical firms must follow
several quality systems. In summary, compliance with regulations is a major rea-
son for using quality systems in biotechnology operations.
Another compelling reason is that quality constitutes good business prac-
tice. When a biotechnology firm provides a quality product or a service, cus-
tomer satisfaction increases and with it increases sale of the product or service.

153Quality Systems
Under a quality system, the number of complaints is significantly reduced.
Within a firm, employee pride and satisfaction are enhanced in a quality envi-
ronment and significant cost savings are realized from increased productivity
and reduced waste of materials. Quality also means speeding products to the
market and keeping them on the market with result as excellent profit.
Quality Systems Approach to Product Development
Biotechnology firms develop and market products. To reach the market place
firms adapt or invent various development systems—manufacturing, qual-
ity control, or nonclinical studies to name a few—for the purpose of product
development. A system that cannot be neglected is a quality system, an orga-
nized body of immaterial things, if you will, aimed at ensuring the utmost qual-
ity of the product or service. A quality system then takes into consideration the
many facets or hallmarks of quality that have been adopted by our industry.
Hallmarks of quality are listed in Box 5.1 and discussed further in a later section
BOX 5.1 HALLMARKS OF QUALITY SYSTEMS
Management responsibility
Defined quality system(s)
Quality by design and design control
Contractor, vendor, and consultant control
Product identification and traceability
Process control
Environmental control
Inspection and testing (quality control)
Control and release of material, services, or product
Change control and corrective or preventive actions
Packaging and labeling
Preservation, storage, and handling
Servicing
Customer concerns and adverse event reports
Risk and risk management
Documentation
Training
Auditing

154 Biotechnology Operations
of this chapter. Hallmarks are immaterial things, tools really, that are applied to
ensure a quality product and, hence, user satisfaction.
Several quality systems have been defined, for example, ISO 9001 and
current Good Manufacturing Practices (cGMP). Each falls under a guiding
authority (e.g., U.S. FDA), each has a specific objective, and each is applied to
a particular functional area. For example, cGMP is a quality system applied
to the manufacture of biopharmaceutical products while current Good
Laboratory Practices (cGLP) are applied to nonclinical safety testing of prod-
ucts. Specific quality systems are described throughout this book. Quality
systems are sometimes defined by regulatory bodies while others represent
consensus within an industry. In the United States, cGMP and cGLP are rec-
ognized and enforced by the U.S. FDA while elsewhere international com-
mittees (ICH, International Conference on Harmonization) often define or
redefine quality systems.
One or more of the following six quality systems is often incorporated into
biotechnology operations. Each is briefly described in the following and they
will be mentioned in greater detail elsewhere in this book.
• Current Good Manufacturing Practices: cGMPs are regulations used
worldwide to ensure the quality manufacture and control of drugs
(Chapters 6 and  7), biopharmaceuticals and medical devices, world-
wide. Despite attempts to harmonize cGMPs between nations, there
are differences in national cGMPs and an international version as well.
In addition, cGMPs also have unique guidelines pertaining to manu-
facture of special classes or even of unique types of products. For exam-
ple, the cGMPs for medical devices considers engineered products
and, in some cases, the software to operate those products. Each class
of biotechnology product may also have unique aspects of cGMPs; for
example, vaccines have several unique manufacturing guidelines and
monoclonal antibodies also have special quality features.
• Current Good Laboratory Practices: cGLPs are applied worldwide for
evaluating the safety of medical products in nonclinical (in vitro or
animal) studies. As is the case with cGMPs, cGLPs are not fully har-
monized across countries and there are differences in how they are
applied to various product classes. Additional information on cGLPs
is provided in Chapters 4 and 8.
• Current Good Clinical Practices (cGCP): cGCPs are regulations used
worldwide for evaluating the safety of medical products in clini-
cal (human) studies at all phases of development. As compared to
cGMPs and cGLPs, cGCP regulations are harmonized across coun-
tries, although there are differences in some aspects of this quality
system due to various types of products and cultural or political
features of national regulations. Additional information on cGCPs is
given in Chapters 4 and 9.

155Quality Systems
• ISO 9001: ISO, an acronym for International Standards Organization,
is an internationally recognized standard for quality of virtually
any manufactured product and many classes of services. While it
is not often applied for the development of biopharmaceutical prod-
ucts, it is a standard quality system for medical device development,
manufacture, and control in much of the world. Additionally, many
service providers to the biotechnology industry are ISO 9001 certi-
fied. ISO 9001 emphasizes quality processes, making the case that a
desired result is achieved more efficiently if activities and resources
are managed as a process, rather than as isolated events. ISO 9001,
like other quality systems, is based upon the hallmarks of quality.
Indeed, the hallmarks of quality were originally promulgated by the
ISO organization. A firm seeking ISO 9001 certification institutes a
quality system under ISO 9001 guidelines and then requests a pre-
certification inspection. If they pass, certification is used to demon-
strate the quality nature of their products or services. ISO 9001 is
used throughout the biotechnology industry but is less commonly
applied to biopharmaceutical development. ISO provides many
other quality systems, each designated by a unique ISO number and
each designed for a specific operation. For example, ISO 17025 certi-
fication is for testing and calibration laboratories.
• Quality system regulation (QSR): QSR was developed specifically for
the regulation of medical devices but, because QSR is also highly
effective at maintaining quality in operations, it has been more
broadly adopted by other centers of the FDA as a quality standard
for a variety of medicinal products. QSR, defined in 21 CFR 820, sim-
ply provides a framework, considerations, and general processes to
ensure establishment of the essential elements of a quality system.
• Pharmaceutical quality system (ICH Q10): The International Conference
on Harmonization provides the pharmaceutical quality system,
a comprehensive system based upon quality concepts. These con-
cepts include cGMP, pharmaceutical development, and quality risk
management, applicable throughout the product lifecycle (e.g., prod-
uct development, technology transfer, commercial manufacture, and
product discontinuation). Implementation of this quality system
is intended to foster innovation and continuous improvement and
strengthen the connection between pharmaceutical development
and downstream manufacturing activities.
Each of these distinct quality systems serves a purpose and has a unique
scope. It  should not be surprising that the institution of any quality sys-
tem by a biotechnology firm should be preceded by a conscious decision to
institute the system and careful planning to ensure that it is implemented
correctly.

156 Biotechnology Operations
Planning a Quality System
Defining Objectives and Ensuring Management Support
Like any endeavor in the biotechnology industry, quality is planned before
a system is instituted. To initiate quality planning there are decisions on the
exact nature, scope, and objectives of the quality system to be instituted.
Most firms begin with a single quality system and then grow into additional
systems as they are required. If product is to be manufactured in-house, the
first quality system would be cGMP. Alternatively, if cGMP manufacture and
control were delegated to a qualified contractor then the firm’s first qual-
ity system might be cGLP or cGCP. A biotechnology company that does not
have FDA-regulated products, such as an agricultural biotechnology firm,
might wish to become ISO 9001 certified or it could elect to first establish ISO
17025 for laboratory functions. And a firm producing medical devices would
likely consider the quality systems regulation (21 CFR 820).
Once a quality system has been chosen, then formal, written documents,
the quality manual and the quality plan, are considered as early elements
of quality development. To begin, management empowers an individual or
group of individuals to prepare a quality manual and begin to write the
quality plan. These might be drafted by consultants but more commonly
a quality professional is employed by the firm at this juncture. The result
is that everyone at the firm understands the purpose and objectives of the
proposed quality system and what will be done to incorporate it into daily
operations. But, most importantly, everyone is now aware that management
currently supports and will continue to support quality initiatives. This
ensures that employees, contractors, and consultants of the biotechnology
firm buy into the development of a quality system.
Experience suggests that these first steps are the most critical to success, and
yet they are often the most difficult to achieve in a biotechnology firm. This
is because managers and executives, especially entrepreneurs, are typically
unaware of the importance of quality systems as good business practice and
as a means to achieve regulatory compliance. Far too often, a firm will hastily
develop a quality system without management support and absent either a clear
objective or a quality plan. In such cases, there is little planning and the quality
system is, more often than not, inappropriate for the firm’s business objectives,
hastily constructed and wasteful of resources. Getting started on a quality sys-
tem therefore involves significant thought, management support, and planning.
The Quality Manual
Once management has made the decision to establish a quality system at a bio-
technology firm, the next step is to create a quality manual. A quality profes-
sional is appointed the champion and lead author of this document. The manual

157Quality Systems
is a short, usually under 10 pages, document that provides elements, listed in
Box  5.2, of the intended quality system. Each section must be tailored to the
particular biotechnology operation. Once written, the quality manual is signed
by the senior management. It states, for all employees, contractors, and consul-
tants of the biotechnology firm, the corporate expectations with regards to the
quality system and employee performance under that system. Just as a corpo-
rate employee manual spells out human resource policies, the quality manual
provides quality policies as guidance to all employees, explaining to each why
quality is considered important to them and to their customers.
For example, if the chosen quality system is cGMP, then the manual would
discuss the firm’s commitment to manufacture quality product, choose
competent subcontractors, perform internal audits, apply specifications,
and follow other hallmarks of a cGMP system. The first chapter of a quality
manual, the statement by management, is critical and must be signed by the
highest levels of management. The second chapter spells out responsibili-
ties for everyone in the firm, providing an organization chart and authority
and responsibilities for quality assurance and other functional departments
(e.g., manufacturing). It clearly defines for everyone the areas involved in the
planned quality system. The remainder of the manual spells out quality sys-
tem functions in a general sense, leaving details for the quality plan.
The Quality Plan
Having stated objectives and elements of the quality system in the qual-
ity manual, the firm now identifies a route to best implement the chosen
quality system. An initial step is to review the business plan, the targeted
product profile (TPP) and product development plan (PDP) (Chapter 1) and
then, under the objectives stated in the quality manual, write a supporting
BOX 5.2 ELEMENTS OF A QUALITY MANUAL TO SUPPORT
A QUALITY SYSTEM
Chapter 1: Introduction and statement of management
Chapter 2: Responsibilities and organization
Chapter 3: Overview of studies performed under the quality system
Chapter 4: Type of quality system(s)
Chapter 5: Employee management
Chapter 6: Safety
Chapter 7: Facilities, equipment, and reagents
Chapter 8: Special operations
Chapter 9: Records and documents
Chapter 10: Other elements of the quality system

158 Biotechnology Operations
quality plan. This means the quality system is appropriate for and supports
the intended operations and biotechnology products or services that will be
developed by the firm. This may sound easy, but far too often previous and
inappropriate experiences become the sole basis for preparing the new qual-
ity plan. Each quality plan is unique and must focus on the new biotechnol-
ogy product and development objectives. In other words, the quality shoe
must fit the business and operational foot.
To begin, the quality planner studies the objectives and processes of all
functional and operational areas, current and intended, for that firm. For
example, biotechnology firm A may be focused upon genetically engineered
plants, agricultural products, and with the intention of doing all product
development in-house. Firm B may develop medical biopharmaceutical prod-
ucts but, as a virtual company, intend to have most manufacturing, nonclini-
cal and clinical efforts completed by contractors. In contrast, firm C may plan
to produce recombinant biological molecules for use in research laboratories
and in the manufacture of in vitro diagnostic medical devices. Each of these
three examples requires a unique quality plan since each firm faces a differ-
ent set of regulatory requirements, operational processes, and business objec-
tives. Operational research is critical to establishing the correct quality plan.
It is also very helpful to appoint a quality steering committee and to work
with project management committees. Members of these committees instruct
the quality planner on the technology and resources in their departments
and, at the same time, work closely with quality professionals to establish the
corporate quality plan. Typically, there is a bit of attitude adjustment required of
any product development team embarking on the quality planning process.
This is especially true at a firm that has no past experienced working with
a quality system. A quality committee with regular meetings ensures that
cooperation between the functional area managers and the quality planners
begins at the outset and continues through the process of quality planning.
Project committee meetings are another means of disseminating quality
planning information and for communicating objectives and quality proce-
dures between quality professionals and other functional area supervisors.
Why is there a great need to sell the concept of quality to staff? Managers
of other departments may view quality as a threat, since it represents someone
from outside their department reviewing critical functions and even suggest-
ing operational changes. The journey of establishing a quality system reaches a
first milestone the moment managers and supervisors from throughout the firm
visibly and fiscally support the draft quality plan. A positive attitude from man-
agers goes far because it instills a team spirit and positively influences everyone
on the product development team. Using tact and convincing arguments, qual-
ity professionals strive to instill this attitude on fellow team members.
Support of peers and managers cannot stop with the quality plan, and qual-
ity support must continue beyond establishment of the quality system. This
includes continual sharing of information, full integration of the quality staff
into product development teams, provision for additional quality resources

159Quality Systems
as the scope and complexity of efforts expand, and continual dialogue
between quality and functional departments. As will be discussed in later
chapters, such processes are not unique to the new biotechnology firm but
apply equally to the established firm with a broken or deficient quality system.
Once comments of the team members and upper management have been
incorporated, the overall scope and purpose of the quality plan should be
finalized. Regulations are perhaps the most important consideration, since a
quality system exists, in part, to maintain compliance with the regulations.
In Chapter 4, the elements of cGMP, cGLP, and cGCP are outlined and their
practical applications are discussed in Chapters 6, 8, and 9, respectively.
Next, the quality sections, each focused on a hallmark of quality (Box 5.1)
are written. Not each hallmark applies to every product or quality system,
so certain hallmarks may be omitted from any quality plan. But omission
of a hallmark must be a conscious decision and based upon the fact it is not
applicable in a given case. Again consider that each quality system is unique.
Another important element of a quality plan is the quality organization the
biotechnology firm wishes to use in daily quality operations. Guidelines for
building a quality assurance unit (QAU) (referred to by the U.S. FDA as the
quality control unit) or department within a biotechnology firm will be dis-
cussed in a later section of this chapter but a few points are relevant to quality
planning. The QAU must reflect the size and complexity of the overall quality
plan. Minimal considerations are: quality management, documentation, train-
ing, and auditing. In a virtual biotechnology firm, these tasks may be completed
by a single individual and that person might even be retained as a consultant or
contractor, not a full time employee. However, use of consultants is not always
possible once a functioning quality system is in place and operational. In an
established biotechnology company, a general rule of thumb is the need for one
dedicated quality assurance individual for each 25 employees. Of course this
ratio, 1:25 may change greatly, for example, if the majority of technical employ-
ees are working in development and not basic research. Once again, it is very
important to tailor the quality plan and project quality resource needs to busi-
ness and development plans. A final note is to consider the quality manual as
a living document, one that can be changed if it is not working effectively to
support corporate, quality, regulatory, or technical objectives and functions.
Hallmarks of Quality: Fundamental Criteria
for Building Effective Quality Systems
The term hallmark is derived from the ancient practice of marking precious
metals with a stamp that identified the source and quality of these sought-
after materials. Over time, hallmark has come to mean a distinctive feature
of an item, especially a feature that makes the item stand out as excellent.

160 Biotechnology Operations
In the discussion on quality planning and in Box 5.1 are noted features of a
quality system that make a quality operation stand out: the hallmarks of a
quality system. Apparently, the term quality hallmarks was first introduced
to describe features of the quality system ISO 9001 but today hallmarks are
characteristic of any quality system. Indeed, a novel quality system is easily
built around the most common hallmarks to be described in this section.
Management Responsibility
A quality system cannot exist without the involvement of management,
including executives at the highest levels. Quality principles (Figure 5.2),
promulgated in ISO 9001, provide eight quality management principles:
leadership, involvement of people, process approach, systems approach to
management, customer focus, continual improvement, factual approach to
decision making, and beneficial supplier relationships. These principles, at
the heart of any quality system, are woven throughout an effective biophar-
maceutical development program and each will appear again in later discus-
sions. It is from these principles that a quality system owes its existence.
Quality
management
principles
Leadership
Process
approach
Factual
approach to
decision
making
Mutually
beneficial
supplier
relationships
Continued
improvement
Consumer
focus
Involvement of
people
System
approach to
management
FIGURE 5.2
Eight principles of quality management.

161Quality Systems
In a practical sense, how then is management involved in quality?
Figure  5.3 identifies the primary responsibilities. Leadership is critical.
Indeed, management, both executive management and functional depart-
ment managers, must support quality efforts of their biotechnology opera-
tions. This requirement is so basic to each quality system that regulatory
agencies insist management support be clearly identified, and in writing. We
noted earlier that management involvement in quality must be specified in
the quality plan and other quality documents. The head of the QAU reports
to a high-level executive and this reporting structure is reflected in corpo-
rate organization charts. Operational documents also reflect that the qual-
ity management-to-executive management relationship exists on a daily or
certainly a weekly basis and is functional or even dynamic. This organiza-
tion of quality resources is driven by regulatory compliance and good quality
management practices. Today, inspectors from regulatory agencies such as
U.S. FDA typically inspect, for quality compliance, biotechnology firms in
a top-down manner, reviewing operational management records in the first
hours of an inspection in an effort to learn if management is actually involved
in and committed to quality efforts. If this is not the case, then the firm may
fail the inspection right from the outset. Indeed, when a firm fails an inspec-
tion of a quality system and receives a warning letter, this unwelcome corre-
spondence from the FDA is inevitably addressed directly to the president or
chief executive officer of that firm since this is the person with direct respon-
sibility for all aspects of operations and quality activities (Chapter 4).
Upper management
commitment
Involvement and leadership
Customer
Management
reviews
Quality policy
Quality plan
Delegate
responsibility
and authority
Effective
quality
assurance unit
FIGURE 5.3
Upper management responsibilities in a quality system.

162 Biotechnology Operations
Management responsibility continues largely through management
reviews, a process in which designated upper management considers all
aspects of their quality system to ensure it meets stated objectives and
remains suitable and adequate to meet current operations and future plans.
Management review is especially important for an organization that is
evolving, changing, or growing and whenever new regulatory guidelines,
new products or services, or new markets arise. Thus, management review
identifies opportunities for changes to the quality system.
Guidelines for management involvement are established in writing and the
results are reflected in corporate documents. Executive management under-
stands the biotechnology, regulatory and quality processes to the extent that
they can make intelligent, high-level decisions concerning the overall qual-
ity, safety, and efficacy of their products. Management has set appropriate
goals and objectives, has established standards, and is clearly receiving feed-
backs from their functional area managers. And they are aware of signifi-
cant changes in all areas of operations and how these changes are executed.
Upper management not only empowers quality leaders but also maintains
the authority to make changes when quality issues are brought to their atten-
tion and the record should show this to be the case at their firm. An absentee
or otherwise disengaged executive is not appreciated in the biotechnology
industry, especially by regulatory agencies, since executives often do not
have his or her finger on the pulse of their firm.
Defined Quality System
This hallmark simply means that one or more quality systems have been
established at the firm; this was mentioned earlier in this chapter as an early
step in quality planning. Further, each hallmark identified as part of that
chosen system must be important to operations and to the completion of
the product development life cycle at that firm and for that product. From
a standpoint of compliance, regulators expect each quality system, such as
cGMP, cGLP, cGCP, or ISO 9001, to be identified in the quality plan, any
quality manual and in other quality documents. Also, the chosen quality
system must fit the product accordingly. For example, cGLP regulations
are, by the U.S. FDA definition, a quality system used for nonclinical safety
studies. Yet, cGLP is sometimes applied to systems far removed from this
scope. This leads to organizations touting their compliance with cGLP
when, in fact, they do not even meet the U.S. FDA’s intended scope of the
regulation. This inevitably leads to inappropriate use of the system and con-
flicts between the firm and regulatory agencies or customers. Actual quality
practices must meet procedural definitions as well. Simply stated, if you tell
customers or regulatory authorities your operation is compliant with a par-
ticular quality system then it must indeed meet both the scope and practices
for this system.

163Quality Systems
QbD and Design Control
QbD has, in just a few years, become a key component of biotechnology
operations and most quality systems. Regulatory agencies now recommend
QbD for most medical products just as they demand management involve-
ment in the quality system. It is no coincidence that design control has come
to represent the heart of QbD, an important aspect of development for any
biotechnology product and a requirement for biopharmaceuticals.
Quality by Design
QbD applies quality concepts to the design and development of biopharma-
ceutical products of all types and for every indication. It is based upon cus-
tomer needs for quality products, excellent science, design control, and risk
analysis and management. In the first step and as described in Chapter 1, QbD
identifies product requirements to meet the needs of the user, a patient, and
the health care provider. The next step, designing a product such as a molecule
or a cell, seems counterintuitive. Isn’t biotechnology itself based upon discov-
ery, the concept of developing a product from a new finding? In the real world,
a scientist makes a discovery in the laboratory and then finds investors or
another sponsor to take this new molecule or cell forward to the marketplace.
But QbD does not fly in the face of reality. Instead, it suggests taking this
discovery, new molecule or cell, whatever, and matching it with a require-
ment or a need and a patient population. Sometimes, however, the match
is not good and the molecule must be rediscovered or at least modified to
meet the exact need. For example, a new monoclonal antibody may be dis-
covered using an animal model system such as a mouse. The intended use is
to develop this monoclonal antibody as a therapeutic for human malaria in
man. In mice, the antibody works as a therapeutic, clearing an infection with
mouse malaria parasites. The discoverer wishes to take this finding forward
but, in planning the project, discovers that the mouse monoclonal will not
work against human malaria parasites and that the mouse antibody might
elicit undesirable reactions if it were given to humans. There is no market for
a therapeutic for mouse malaria. This is where QbD might come into play.
In this example, the scientific concept is solid but the initial discovery is not
useful to the intended user. By redesigning the molecule, and this can actu-
ally be done both by generating new monoclonal antibodies or by genetically
engineering the mouse antibody to be a humanized molecule, it is possible
to rediscover the concept and to design a new molecule that will meet require-
ments of the human user.
Indeed, QbD is not the traditional or twentieth century concept of drug dis-
covery, in which thousands of molecules were first generated by an organic
chemist, then screened for any attribute in laboratory and animal studies.
This older concept of pharmaceutical discovery has been compared to a fun-
nel with a fine screen and very narrow opening that often led to nowhere for

164 Biotechnology Operations
most molecules. Very few molecules made it into the development pipeline,
yet many of these were a bad fit for their intended purpose and were consid-
ered great molecules, but needing a useful and marketable indication.
Therefore, QbD was introduced and since 2004 the U.S. FDA has strongly
recommended it as an operational method for modernizing both drug dis-
covery and drug development. It has quickly spread to the biotechnology
industry in part because it works quite well and also because the twentieth
century drug development process, the funnel concept of screening many
compounds to find just one candidate that suited an indication, simply does
not suit discovery and development of large molecule and cellular products.
The funnel concept just did not work for biopharmaceuticals and they often
had to be redesigned to suit a specific need. Another element of QbD is a
need to consider the TPP, discussed in Chapter 1. QbD goes even further than
early stages of discovery and development and continues throughout the
life cycle of the product to ensure continuous improvement and innovation
as critical quality attributes are applied to product development. And QbD
is used with functional areas of development, notably manufacture, qual-
ity control, nonclinical studies, and clinical trials. An example is given in
Chapter 6, where QbD is applied to the design of a manufacturing system for
a biopharmaceutical product.
The concept of QbD was really an idea whose time had come in the early
twenty-first century and both industry and the International Conference on
Harmonization (ICH) deserve credit for encouraging its use. ICH describes,
through their quality or Q series of documents (e.g., Q8, Q9, Q10, and Q12),
QbD concepts and practices for biopharmaceutical products. These arguably
are the leading reference documents on QbD today and should be consid-
ered by any biopharmaceutical firm entering development and establishing
a quality system.
Design Control
QbD applies a formal means and format of designing a biopharmaceutical
product. Design control, at the heart of design, is best designated as the gen-
eral arrangement or layout of a product. It demands both an active process
and results from that process, notably written design documents. Historically,
the process of design control evolved from engineering projects, notably in
the medical device industry, where physical design of a product was seen
as good business practice and, more recently, as a regulatory requirement.
For example, a new heart pacemaker would only function correctly if it was
designed to meet certain specifications. To be effective and safe for the user,
and thus marketable, an implantable pacemaker is a maximum size, very reli-
able, for example, in the accuracy of beats-per-minute, and useable, for exam-
ple, have a battery life that supports many years of use by a patient. If the
management asked for a new pacemaker and did not insist on a user-friendly
certain design, they could find themselves with an implantable device that

165Quality Systems
was 10  cm in diameter, produced 30  ±  20  beats/min, and had a battery life
of 30 min, hardly desirable to the customer or a regulatory agency and quite
difficult to market. Design control takes user needs into consideration before
the product is produced. It is a good business practice.
Today, design control is applied to biopharmaceutical development with
the designer following certain steps, these having been adopted by indus-
try and regulatory authorities. Elements of design control are shown in
Figure 5.4 and further defined in Box 5.3.
Target product
profile (TPP)
Design input
A plan of product
design
Review
Output: A
product design
Review
Product output
and verification
Review
Design
validation
Final
development
and production
Changes to design
and specifications
(Figure 5.5)
Management
guidance
Design
specifications
Engineers
Scientists
experimentation
Design team
Development
team
FIGURE 5.4
Elements of design control.

CR11

166 Biotechnology Operations
• Design control is product specific. The product and indication for the
biopharmaceutical are specified and each design control process
is for one product with a unique indication and no other. Further,
design is repeated each time a change is proposed to that prod-
uct or its indication. As an example, consider again the monoclo-
nal antibody against a malaria parasite. Having a firm grasp that
the concept is feasible from studies in mice, it is now designed as
a humanized monoclonal antibody to bind and kill the human
malaria parasite. The concept of QbD and specifically the process of
design control now impacts future development of this monoclonal
antibody. Further, quality criteria now take on much greater quality
implications, for planning is the first step of development.
• A product design plan is drafted. Design planning is a process much like
product development planning but is much more specific in scope,
focusing on how the design process itself will take place. The design
plan identifies the product and indication and then it outlines vari-
ous elements of design, notably input, output, review, and decision,
serving as an agenda for product design. A product design plan might
have an outline as shown in Box 5.3. But, in addition, it would describe
who is responsible for each step of design, when it would occur, how it
would be completed, and what the expectations might be.
• Design documents and records reflect each step of the design control
process. Requirements for written records are established. These
BOX 5.3 ELEMENTS OR STEPS IN DESIGN CONTROL
• Product is specified: The product and indication are clearly
described (e.g., using TPP).
• Product design plan is drafted: This plan then drives the process.
• Design process is fully documented: Detailed records are kept
throughout the process.
• The process involves the full product development team.
• The design control process begins and these steps may be repeated
until the team is satisfied with the final design.
Input
Review
Output
Verification
Validation
Change

167Quality Systems
include process documents such as agenda and minutes to meetings
and product documents, for example, the draft meeting minutes
and the product’s written draft labeling, user’s instructions, techni-
cal descriptions, specifications, and drawings that have been gen-
erated during the process. Plans are made to keep product- specific
historical files and this is accomplished by the following quality
procedures.
• Design control involves professionals, technical and management,
serving on a design team with responsibility for the product’s
development. Research, manufacturing, sales, marketing, qual-
ity assurance, quality control, senior executives, regulatory, project
management, finance, and personnel must somehow be involved in
the processes referred to as design input, output, review, and change.
If this design review group seems similar to a project team described
in Chapter 2, then you are correct. Very often the members of a proj-
ect design team for a biotechnology company will be assigned to the
development team as well.
• Design input is the next step. The purpose is to enlist opinions of
various individuals regarding how the product should be designed
and this, of course, takes into consideration the operational plans
we have discussed throughout this book. For QbD to be effective,
the quality attributes of the product must be a prime consideration
in design input. Quality professionals should be actively involved
early in the process. Leadership is key to success of design input
and here an effective project manager may lead the team effort and
ensure constructive communication. In a practical sense, design
input involves sitting around a table as a team, speaking, listening,
and learning from each team member. Everyone will, at first, be sur-
prised at differences in individual perceptions for a single product
and indication. Returning to the example of the humanized mono-
clonal antibody to treat malaria, marketing may suggest that it be
manufactured in final form for $50  per dose and be used to treat
three species of human malaria parasites. Manufacturing personnel
may disagree, suggesting that it could only be produced and for-
mulated for over $200  per dose. And clinical affairs might suggest
that initially it can be marketed to treat only one species of malaria
parasite, as initial studies to treat three parasite species would be
prohibitively expensive and time consuming. Consensus must be
achieved in these meetings, otherwise everyone is advocating a
unique design and efforts will be disjointed, at best. In effect, design
input involves soliciting everyone’s opinion and clearly describing a
single route forward, that is, reaching a firm decision.
• This all leads to design output, which, as the name suggests, is
nothing more than producing, in a written report, consensus of

168 Biotechnology Operations
opinions, a feasible product development objective, a consensus on
the design, and an idea of how all these efforts might be applied to
achieve a common objective. Design output provides important key
product specifications, agreed to by the team members and by upper
management. For example, the team might agree the monoclonal
antibody against human malaria infection must target one malaria
parasite species, have a therapeutic effect in 80% of patients, give an
excellent safety profile in infants and children, and be manufactured
for $100  per dose. Of course, the actual output document is much
more detailed but nonetheless the most important attribute is a clear
design for the intended product.
• Design reviews are performed throughout the design and opera-
tional phases of biopharmaceutical development. Once an output
document is established, it should be critically reviewed by a much
larger audience. Using partners, contractors, and consultants is
recommended as is review by the upper management and techni-
cal staff. Reviews often lead to new ideas and improvements and
these then become additional input, thus starting the design pro-
cess over again or leading to changes in design. This might precipi-
tate another round of design review and revised outputs. Hence,
QbD never involves a single meeting, in which everyone agrees
and from which design output is completed days later. Instead, it
requires many meetings, interspersed by additional laboratory and
marketing research, and further input from management. During
this period, the evolving design is reviewed by the team. Once a
design has been agreed, design change is inevitable. Problems are
encountered in development and they must be reviewed at frequent
intervals by the team. Quality issues continually arise and must be
addressed in a revised design.
• Design verification follows. Economics suggests that sooner or later the
design cycle must end and the product must be produced, at least
on a limited scale, and then tested. In biopharmaceutical develop-
ment, this involves manufacture, quality control and studies, both
nonclinical and clinical. The testing aspects are considered design
verification. The product, and hence the design, is tested in labo-
ratory animals and man. It determines where and how the design,
which is really a model on paper, will be developed and produced.
For the monoclonal antibody against human malaria, this might
mean that extensive laboratory testing reveals, for example, the mol-
ecule is stable at a particular temperature, that it can be produced in
a 10 L bioreactor and is not toxic to a small animal. Design verifica-
tion, analogous to Phase I and early Phase II development, involves
laboratory and early clinical experimentation and testing. In the end,
design verification leads to confirmation or rejection of individual

169Quality Systems
components in the design and, if things are not perfect, it can trig-
ger another round of design input, output, review, and verification.
It is at this stage in development that the design team begins to also
function as a product development team.
• Once a product has withstood initial testing and is verified, design
validation may be used to further substantiate the adequacy of the
product’s design. Validation typically has a more stringent defini-
tion than verification. It requires additional testing and might even
involve changes to variables that had not been previously tested. In
biopharmaceutical development we often consider design validation
to be middle or late stage (Phase II or Phase III) development encom-
passing advanced nonclinical and clinical trials.
Design Change
Change is expected during a product’s life cycle. Biotechnology products do
not speed through the full course of development activities without several
changes in TPP, PDP, or product design. While change is expected and even
good in many cases, change must not be a random or uncontrolled event.
Changes in product development must be controlled throughout the design
control process and the development life cycle.
In summary, a hallmark of quality is the concept of QbD incorporating the
process of design control. We think of this in much the same way as plan-
ning development but it has much broader and deeper implications, taking
into account the ideas of design and great technical depth and detail.
Contractor, Vendor, and Consultant Control
Every biotechnology development program depends upon acquisition of
goods and services. A quality system takes great care to source only the
best raw materials, advice, and contract support. It does so by incorporating
corporate policies and procedures for obtaining, by purchase, collaboration
or contract, materials or services. This, along with good business practice
and common sense prevent the purchase of materials or services that might
foul part of the product or development scheme. Unlike large pharmaceuti-
cal firms with in-house resources and procedures available to closely man-
age and control vendors, small- to medium-sized biotechnology firms often
do not have this expertise and capacity. In fact, a virtual biopharmaceutical
firm may rely upon very few employees to manage a significant amount of
the operational effort that is actually performed or supplied by contractors
and vendors. Also, consultants are often retained by biotechnology firms to
provide critical advice or prepare important documents, such as regulatory
submissions or quality plans. The quality of these materials and services is
important to the success of any product development effort but the buyers

170 Biotechnology Operations
must themselves ensure compliance with established quality criteria. How
can this be accomplished in a fast paced biotechnology environment? First,
a buyer establishes specifications for every material or service considered
for purchase or hire. Second, they consider more than one offeror, when-
ever possible, and carefully evaluate each one by reviewing the corporate
history, experience, and references. Vendors or providers passing these ini-
tial screens might then be evaluated in greater depth through audit of their
facilities.
Further to the example of a monoclonal antibody to treat malaria infec-
tions, the manufacturing and quality group considers the need to purchase
saline for formulation of the product. Specifications, such as USP grade nor-
mal saline for injection in 1-L sealed glass bottles, are established. A request
for proposal is sought from three or more contractors. Once the vendor’s pro-
posals have been reviewed, further information, such as copies of Certificates
of Analysis, for the last three lots of saline, might be requested. Additionally,
staff might determine if a supplier complies with a quality system such as
cGMP or ISO or they may request inspectional results from previous ISO
9001 or FDA inspections. Given that the quality of this saline is important
to the overall quality of the monoclonal antibody product, it would be pru-
dent to schedule an audit of the saline manufacturing facility once a likely
supplier has been identified from the list of three candidates. Audit proce-
dures are explained later in this chapter. Once the saline arrives, samples
from each lot might be retested for critical parameters (e.g., sterility, pH, or
concentration of sodium chloride) by the firm’s quality control laboratory
to determine if it does indeed meet the specifications cited in the vendor’s
certificate of analysis. Quality control testing of raw materials is described in
Chapter 7. Upon receipt, saline is kept at the recommended conditions and it
may be tested for stability prior to reaching the expiration date.
Ensuring the quality of consultants or advisors and service providers is
also important to a biotechnology firm. Technical requirements and the
intended scope of work are clearly stated, to include the amount of control,
review and supervision that will be provided by the firm to the consultant.
Resumes of candidates are reviewed and references are checked to ensure
that each consultant applicant is qualified by experience and training.
Service providers, such as contract research organizations (CROs) per-
forming manufacturing, quality control, clinical and nonclinical studies, are
thoroughly examined and references reviewed before contracts and agree-
ments are signed. These are high-cost and high-profile contracts involving
months and years of effort. Potential failure or delay by a CRO has great
negative impact upon a biotechnology development program. The steps
involved in selecting a CRO are no different from those used to select a ven-
dor for raw materials. The purchaser establishes exact specifications and fully
describes the intended scope of work. At small biotechnology firms, it may
be necessary to hire an experienced consultant to write this critical document
and then participate in the selection process. The scope includes a significant

171Quality Systems
amount of detail on technical and quality aspects and a request for proposals
is advertised. Proposals include elements of technical performance, cost, and
quality. Once proposals are received, the candidate list is narrowed to only
qualified proposals. Again, it may be necessary for the small biotechnology
firm to retain consultants to assist in review and to use the expertise of the
project team, to include those with finance and contract responsibilities, dur-
ing the review and selection process. Audits and a check of references is an
absolute must when considering CROs for a major contract. Once a vendor
is selected, they sign both technical and quality agreements. The firm may
assign one or more employees on staff to oversee and manage a major devel-
opment contract.
Quality agreements are based upon the quality expectations and specifi-
cations for the product or service. They define the quality system and pro-
cesses to be used by the CRO or vendor to manage quality aspects of the
material or service contract. The scope of the quality agreement covers the
full scope of quality efforts. The nature of each quality system that applies to
the service or material is clearly stated. The vendor-client relationship, with
responsibilities, procedures for changes to the deliverables and procedures
to resolve disputes, is described in some detail. A responsibility matrix may
be quite helpful. Since ongoing monitoring and auditing are likely to be part
of the contract, the exact nature of these activities is stated in this agreement.
And terms are clearly defined in an effort to prevent misunderstandings. For
example, quality terms that may be confused are substantial deviation or minor
error and these are either not used or they are clearly defined under the scope
of the quality agreement.
Biotechnology firms rely heavily on outsourcing and success or failure of a
service or material provided by a vendor can have a huge impact on an oper-
ation. It is critical to ensure that quality criteria and functions are considered
in all contract, consultant, and vendor agreements and activities.
Product Identification and Traceability
The sponsor must have in place a system to identify all materials and prod-
uct as it moves through the development life cycle. This control applies
in-house and to deliverables from services, such as quality control test-
ing and clinical or nonclinical studies. Product identification and trace-
ability are considered quality responsibilities at a biotechnology firm. For
manufacture of a product, this may be the application of a unique num-
bering system to identify lots of manufactured product and a method to
trace numbered lots through the distribution system. With such systems, a
manufacturer may trace forward to learn where and how the product was
manufactured or to trace backward to identify the origin and handling or
possession of each raw material, facility, and piece of equipment used in its
manufacture. Clearly, product identification and traceability necessitate a
mature and infallible documentation system, a quality function that will

172 Biotechnology Operations
be discussed later. For a service provider, the identification procedures may
be more involved, requiring multiple levels of identification. For example,
a contractor’s quality control testing laboratory has numbering or labeling
systems to track each sample and results as they pass through the test-
ing and reporting process. A clinical or nonclinical study is based upon a
specified and numbered protocol and each segment of that study—animal
or human subject, test product, treatment or procedure—is uniquely iden-
tified to ensure the protocol was executed flawlessly and that each compo-
nent was performed completely and correctly.
Today, the quality function of ensuring identification and traceability
increasingly relies upon barcoding and microprocessor-based systems.
Quality electronic records and signatures are critical to performance require-
ments. Hence, the software and microprocessors themselves must be of the
highest quality and suited to the tasks of identification and traceability. There
are regulatory requirements for microprocessor-based systems (Chapter 3).
Process Control
A phrase common to the pharmaceutical industry is you cannot test quality
into a product. This means quality must be built into the product at every
phase of development and throughout the process, that is at each step in the
production process. Biotechnology products are no exception to this rule and
therefore process controls are used to ensure quality throughout the pro-
cess. Process control relies on clear and concise written documents to guide
operators at each step of biopharmaceutical production. These are standard
operating procedures (SOPs) and batch production records (BPRs) or work
instructions. For nonclinical studies or clinical trials, protocols and SOPs are
used to guide processes.
Protocols, defined by the Oxford English Dictionary as “official formality and
etiquette” (Oxford English Dictionary, 1997), are formal written and approved
instructive documents that describe, step-by-step, how studies and trials are
performed. The BPR, a prospectively generated, formal written and approved
document, guides manufacturing processes. BPRs are also used to collect
information or data as it is generated. Typically, a protocol or BPR gives the
reader broad step-wise guidance and references to SOPs, documents provid-
ing more exact technical instructions and describing exactly how the process
is performed. In a quality environment, such as those mandated by cGMP,
cGLP or cGCP, SOPs are also used to provide a controlled work environment,
thus ensuring products are made in the appropriate atmosphere, to instruct
the use of equipment and utilities, and to mandate exactly how other activi-
ties are undertaken in the manufacturing environment.
The technical staff of a biotechnology firm prepares BPRs, SOPs, Protocols,
and related documents but each one must be reviewed and approved by both
supervisors and quality assurance staff. As approved documents, BPRs, SOPs,
and protocols, are highly controlled and may be changed only using formal

173Quality Systems
processes and with approval of all responsible individuals. Documentation
control and change control responsibilities, a quality assurance function, are
described later in this chapter.
Environmental Controls
A product is only as good as the environment in which it is produced and
biotechnology products are manufactured or tested in a wide range of
environments. Genetically, engineered plants are grown in green houses
or open fields under controlled conditions. Sterile, recombinant proteins
are manufactured and then formulated in highly controlled, indoor, envi-
ronments (Chapter 6). Clearly there is a significant difference between the
controlled environment in a corn or tobacco field and the environment
within a biopharmaceutical manufacturing facility; yet each environment
meets specifications suited for their product’s specifications and intended
use. We think of biomanufacturing endeavors as happening in clean rooms
and in fact this is by far the most common practice. Environmental controls
bring into play the issue of the quality of the facility, the air, the water,
and the personnel. For genetically engineered plants grown outdoors, the
quality of the soil may also be considered. What goes on in the facility is
also important. The flow of raw materials, product and waste, and person-
nel in a biopharmaceutical manufacturing plant can have a major impact
on the quality of the product. The quality of the people is no exception
since their performance is part of the overall environment in a biotechnol-
ogy operation. We discuss in Chapter 7 the laboratory testing efforts that
go into ensuring the quality of all aspects of biotechnology operational
environments. Quality is intricately involved in these efforts and ensures,
through inspection, audit, validation, review, approval, and documenta-
tion, that the environment meets preset specifications and is suitable for
the intended operation.
Inspection or Testing (Quality Control)
Inspection and testing are integral to manufacture of biopharmaceutical
products. As described in Chapter 7, quality control efforts are technical,
performed primarily in laboratories. Today, quality control testing is often
administratively separate from the QAU. Nonetheless, quality assurance
has significant responsibilities for ensuring that testing is fully qualified
or validated for the intended purpose, that testing was performed accord-
ing to procedures and that test results conform to specifications. Unlike
discovery research, quality control testing is a formal process, performed
under SOPs and using highly developed qualified or validated proce-
dures. A distinguishing feature of quality control is the use of specifica-
tions, normally a range of acceptable test values, against which sample
test results are compared (Chapter 7). Quality control testing is performed

174 Biotechnology Operations
on raw materials upon arrival, to ensure that they are, in fact, identical
to claims made on the label or certificate of analysis. In-process testing
is performed on samples taken during the manufacture of biotechnology
products. While quality cannot be tested into a product, in-process testing
in part ensures the quality of products along the production process. Drug
substance and drug product, defined further in Chapter 6, are tested in
the quality control laboratory. Then, once finished and filled into a con-
tainer, final biopharmaceutical product is further inspected and tested.
Stability testing demonstrates that a product remains pure and potent
once it has been stored for a designated time, hence the need to pro-
vide a stated shelf life for each biopharmaceutical. Quality control also
involves the calibration or certification of test and measuring equipment
to ensure that it meets standards or specifications. Just as manufacturing
processes must be validated, so too must analytical tests and measuring
devices (Chapter  7). Quality assurance professionals work closely with
quality control to ensure that adequate test methods are established, that
test results are compared to specifications, and that all testing is fully and
precisely documented.
Release of Material, Service, or Product
Before it is released for use, a manufactured product must conform to all
specifications. This means that raw materials, processes, environments,
identification labels, and results of inspections and testing must meet prede-
termined specifications. Variance from any one specification can, in theory,
lead to the rejection of that product so that it cannot be released for public
use. The jargon used in the biotechnology industry to describe an accept-
able product is conform or pass while an unacceptable product, one that does
not meet specifications, is nonconform or fail. Even though it is the other
operational groups— manufacturing and quality control—that produce and
test product, it is the quality assurance function that reviews the data and
decides whether a product conforms or does not conform to specifications,
that is, passes or fails. Product that does not conform is usually placed into
quarantine while the data is reviewed and the situation investigated. But if
failure is the product’s eventual fate, the product is destroyed or completely
reprocessed if appropriate. Quality assurance has the task of reviewing all
records and, ultimately, deciding whether or not a service or product is
released to the market.
Release applies to services as well as product. For example, written reports
of clinical and nonclinical studies are released to the client only after they
are reviewed, approved, and released by quality assurance. Not only is the
report reviewed but also the performance and compliance of the complete
study, from protocol through data collection, are closely scrutinized to
ensure integrity and correct translation of results to the report.

175Quality Systems
Change Control and Corrective or Preventive Actions
As one might expect, not everything goes perfectly or as planned in a
biopharmaceutical operation. Despite the best intentions or planning, some-
times stuff just happens. If a lot or batch of product or a report does not pass
scrutiny by quality professionals, it is unacceptable for release. Unacceptable
products or reports are considered nonconforming since they do not meet
specifications or were not made according to written procedures; it does not
pass. And quality audits may uncover hidden defects in a process, raw mate-
rial or test program. Nonconforming product may also be identified by cus-
tomers who complain about a product even after it passes and reaches the
market. In cases of nonconformance, corrective and preventive actions must
be taken immediately. These decisions, and the investigations that typically
precede them, are the purview of quality assurance.
A quality system includes procedures to collect and, when appropriate,
review nonconformance issues as they arise. Quality assurance is tasked
with ensuring that timely collection of information, including customer com-
plaints, identifies problems with a product, study, or other service. Quality
professionals then make management aware of the issues and ensure that
appropriate corrective action is taken to resolve the problem and prevent it
from recurring. Corrective plans, instructions for investigating failures or
deviations and initiating corrective action, are written and executed for this
purpose. Investigations are prioritized based upon the risk a problem poses
to the user. A serious problem with a biopharmaceutical, one that might put
patient’s lives or health at risk, is addressed immediately and decisively, per-
haps with a product recall and halt to production. Perceived or unproven
problems are investigated over time as quality assurance staff follow trends
and discuss the situation with professionals such as functional area manag-
ers and consultants or vendors. Corrective actions follow identification of
the root cause and lead to application of controls and additional monitoring
to confirm effective resolution. Quality professionals pass information on to
senior management since, as noted in Chapter 4, executives are ultimately
held responsible for resolving issues. Another result of an investigation and
identification of root cause of a problem is preventive action, a process also
guided by quality assurance. Preventive actions address the root cause of a
problem and are intended to prevent the problem from reoccurring.
Change is normal, often good, for all aspects of a biotechnology operation;
earlier in this chapter, change was discussed in relation to design control.
Corrective and preventive actions may lead to change in a manufacturing
process or clinical or nonclinical study. As manufacturing progresses through
the development life cycle, changes are made to improve product yield and
quality. Clinical or nonclinical protocols require change, mid-study, to correct
omissions or errors that threaten the integrity of the study itself.
Change, a carefully managed process, is referred to as change control
and should always be under the control of employees and change must

176 Biotechnology Operations
be reviewed and approved by quality assurance. A scheme for controlled
change is shown in Figure 5.5. For biopharmaceutical development activi-
ties, the controlled change process is mandated by regulatory agencies.
Change is typically planned and executed by a functional area manager
(e.g., manufacturing or clinical) but the QAU may recommend change and
in any case QAU must approve plans to change and provide oversight and
approvals. Hence, it is always a team effort. Change in a biotechnology
operation, no matter how seemingly insignificant, requires forethought,
extensive discussion, focused decision, and follow through in action and
documentation.
Packaging and Labeling
Each biotechnology product has a container and, adhered to the outside,
a label that exactly identifies its contents. Containers must be appropriate
to hold a given product and to maintain its identity, purity, and potency.
A product label contains very important product information, such as
lot number, exact name, strength or dose, formulation, expiration date,
and warnings or critical instructions to users. Labeling, as described in
Chapter 6, provides additional information on a product as printed matter
that is inserted into the packaging (hence the term package insert). If any
of this information is incorrect, then the product itself is compromised,
misbranded in parlance of the biopharmaceutical industry, because it is
not correctly packaged or labeled. Production and use of packaging and
labeling are highly controlled processes and their quality must be perfect
to prevent omission or error, otherwise the product is considered mis-
branded. Therefore, quality criteria for these processes are every bit as
stringent as they are for making the product itself. Whenever a package or
label is generated, quality assurance approves the printed material before
it is used and they also ensure that the packaging and labeling processes
are performed correctly.
Preservation, Storage, and Handling
A biotechnology product may be perfectly manufactured and labeled but if
it is improperly preserved, stored, or delivered, then it is of no use. In fact,
improper storage renders a product adulterated. Therefore, a quality system
ensures that product is properly handled postmanufacture. For example, if
a protein solution is, by specification, to be kept frozen but inadvertently
warms to room temperature on the loading dock, then it is no longer a qual-
ity product. Biopharmaceutical manufacturers make every effort to ensure
adequate controls are instituted and followed so that only pure and potent
product reaches the customer. Quality procedures and records are used
for all aspects of transportation, storage, and delivery of a biotechnology
product.

177Quality Systems
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178 Biotechnology Operations
Servicing
This aspect of a quality system is seen largely in the medical device indus-
try, where it is essential to provide service, calibration, and technical support
to customers. However, in other areas of biotechnology, such as production
and sales of research reagents, manufacturing equipment or analytical instru-
ments, servicing is also important. While the QAU does not itself provide ser-
vicing to a customer, it ensures that servicing programs are instituted and
they monitor the quality of those efforts.
Customer Concerns and Adverse Event Reports
Every biotechnology firm with an investigational or marketed product
collects and reviews comments from customers and, in the case of medi-
cal products, collects safety data. Trend analysis is often used to prioritize
complaints and identify problem areas. Management and quality assurance
review complaints and establish and maintain programs to address cus-
tomer concerns.
Document Control
Also referred to as record control, documentation is a major element of any
quality system. Keeping quality records is a major endeavor in a biotechnol-
ogy firm and even a small operation generates thousands of critical docu-
ments each month. These records, identified by document type in Figure 5.6,
are used to support regulatory applications, to provide data used in busi-
ness development, to verify compliance with regulations, to demonstrate the
application of appropriate quality systems, to record technical proficiency, to
document and track changes, trends and issues. Records, written, printed,
or electronic, are often legal documents, meaning they can be requested by
a court of law. Each record is reviewed for accuracy and completeness and,
in most cases, signed and dated. Then it is archived, where it is available on
short notice. Document management, as performed by the QAU, is discussed
later in this chapter.
Training
Employees must obtain the appropriate training before they begin work and
that training is kept current during the course of their employment at a bio-
technology firm. Training is directly relevant to an employee’s duties and it
is performed to  assigned procedures. For example, it is acceptable to employ
an individual in a biopharmaceutical manufacturing operation if they
have training and experience in a given skill area but it is not acceptable to
employ a laboratory technician in manufacturing if they do not have train-
ing and experience in that skill area or are not properly supervised during

179Quality Systems
a training period. A quality system ensures that employees are fully trained
and also have on file current job descriptions and documentation of training
and appropriate education before they are qualified to perform a particular
job function.
Auditing
The audit function is key to maintaining a quality system in biotechnology. The
International Standards Organization defines a quality audit as, “A systematic
and independent examination to determine whether quality activities and
related results comply with planned arrangements and whether these arrange-
ments are implemented effectively and are suitable to achieve objectives”
(International Standardization Organization, 1994). There are several impor-
tant phrases included in this definition. First, auditing is systematic because it
is a carefully planned and executed activity. Second, the audit is independent
of the entity being audited to avoid any potential for conflict of interest. Third,
audits focus upon quality aspects and not on highly technical activities. In other
words, an auditor examines whether or not the appropriate quality system is
Policies
or
manuals
Design control
documents
Procedures (SOPs)
Data input and output
(batch records, spreadsheets)
Release, approval, change, and validation documents
Quality record files and archives
FIGURE 5.6
Pyramid of quality documents.

180 Biotechnology Operations
in place to support a particular technology and functional area and has limited
regard for scientific or technological details. Compliance, really the demonstra-
tion thereof (Chapter 4), is of utmost importance in a quality audit. This may
seem counterintuitive for a high-technology industry, but scientists are respon-
sible for technical issues while quality professionals work with technical staff
to ensure the quality aspects of that same operation. Fourth, an audit compares
what was actually performed against planned arrangements. It determines if per-
formance matches instructions. Finally, an audit examines whether or not these
planned arrangements were really appropriate to achieve the stated objective.
For internal audits, a biotechnology firm’s quality assurance auditors
inspect the records of a functional department, such as manufacturing,
within that firm. External audits are performed on contractors, vendors, or
collaborators, operations external to the firm performing the audit. It would
be impossible to audit every function, internal or external, of a biotech-
nology firm or that firm’s contractors and vendors. Restraints of time and
resources allow only for the more critical functions of vendors to be audited
and therefore priorities and audit plans are established. Also, there are many
ways to perform an audit; one method does not fit each entity or situation.
Quality assurance procedures for conducting audits are explained later in
this chapter.
The Quality Assurance Unit
Under the hallmarks of quality section, aforementioned, we discussed the
attributes of a quality system. In most cases, these attributes are managed, at
least to some degree, by a group of professionals within a biotechnology firm
referred to as quality assurance or the QAU or more informally, QA. U.S. FDA
regulations (21 CFR) refer to this entity as the quality control unit but within
the industry quality assurance is the term most widely used. The QAU serves
several important roles at the biotechnology firm. First, it maintains a compli-
ance posture to help the firm meet regulations. This means that QAU works
closely with functional area supervisors and coordinates frequently with
regulatory affairs staff. Second, QAU serves the users of product or clients
of services by ensuring they receive products or services of the very highest
quality. Third, the QAU is the gatekeeper of the quality plan, quality manual
and the various hallmarks of quality for the firm.
By now it has become clear that five aspects of quality operations—
management, documents, training, auditing and change control, and inves-
tigations—occupy much of each quality professional’s time and effort.
These are shown in Figure 5.7. They are also quite important to the success
of a biotechnology firm, especially for those involved in biopharmaceutical
development. These five aspects also garner quite a lot of attention during

181Quality Systems
inspections performed during due diligence or by regulatory agencies. This
section provides more detail on those five important functions, expanding
on descriptions provided in the hallmarks of quality section and describing
how each is handled by a QAU.
Manage the Quality Assurance Function
A QAU is designated under the quality policy and the unit’s responsibilities
and authority are described in the quality plan. A primary function of the
QAU is management of the quality system or systems instituted by the bio-
technology firm under the policy and plan. The QAU is managed by a trained
and experienced quality professional. Indeed, and as noted earlier, this indi-
vidual at a small biotechnology firm may write the Policy and Plan and initi-
ate the QAU. In addition to possessing quality assurance skills, the head of the
QAU understands the technology being developed by the firm and techni-
cal aspects of operational areas falling under the proscribed quality systems.
They also have knowledge of regulatory guidelines, especially as they apply
to the quality systems. In some firms, the quality manger will be responsible
for implementing one or more quality systems. To work effectively with the
product development team and interact with upper management, the head of
the QAU possesses leadership and negotiating skills, as well.
As with other operational units in the biotechnology firm, the QAU is
managed in all respects. Preparing budgets, managing personnel, commu-
nicating, serving on teams, and establishing priorities are but a few of the
routine management tasks. Two quality requirements, communication and
coordination, stand out and, if they are performed effectively, distinguish
the excellent QAU from the mediocre department. These requirements are
Quality
assurance
unit
Document
Train
Change
investManage
Audit
FIGURE 5.7
Responsibilities of the quality assurance unit.

182 Biotechnology Operations
important because quality functions, and therefore quality professionals, are
highly integrated into the daily operations of a biotechnology firm. Quality
professionals provide information, notably advice, to many individuals and
they coordinate their activities with many others at the biotechnology firm.
Functional area managers depend upon communication and coordination
with quality assurance professionals if they are to achieve their objectives.
This means that quality assurance leaders must themselves understand com-
plex technological and regulatory systems and then integrate their quality
processes in a timely and effective manner to meet the objectives of the team.
This makes quality assurance management a unique endeavor and provides
many internal and external challenges to biotechnology development teams.
Control Documents and Manage the Documentation System
Earlier in this chapter, we briefly mentioned documentation as a hallmark of
quality and stated that everything, no matter how seemingly insignificant,
that happens in a biopharmaceutical operation is recorded. Tiers of docu-
ment types were identified in Figure 5.6. The QAU manages and controls
each of these entries as an official record. In this section, we summarize doc-
uments and review quality assurance responsibilities and procedures used
to ensure an effective and compliant documentation system.
Documentation, a process, provides a means to generate, review, use, and
store documents. There is a saying in the pharmaceutical industry, if it was
not written down, then it was not done. Every aspect of biotechnology opera-
tions, including plans and processes generate piles of records, a variety of
documents. While written records are the norm in many firms, the trend
is to move to electronic records and signatures. Most small biotechnology
operations begin by using paper document systems but, with growth of a
firm, electronic systems are adapted and they do provide certain advantages.
Yes, transitioning from paper to electronic records does not in itself simplify
the documentation challenges. Capture, review, audit, and storage of docu-
ments must also be done exactly and these tasks fall to the QAU. Regulatory
agencies expect nothing less. Further challenging documentation efforts for
growing biotechnology firms is operational growth beyond the capacity of
the existing documentation system. Finally, senior management never sees
all these documents and therefore seldom appreciates their volume or the
complexity of properly reviewing, approving then organizing and maintain-
ing all these files.
Who then is responsible for writing, reviewing, and approving each of
these documents? Plans and strategies are written by high-level manage-
ment with the assistance of technical and administrative staff. Protocols are
prepared by investigators or study directors, individuals responsible for
designing and completing a nonclinical or clinical investigation or valida-
tion. Procedures, manufacturing records, and work instructions are written
by technical staff, those who know exactly how a technical procedure must

183Quality Systems
be performed. Finally, each document has considerable detail. Many instruct
what is to be done, by whom, and when. Other documents, so-called forms,
prompt for data entry and thus record what was actually done, who did it,
and when it was performed. Corrections, reviews, and approvals are exactly
recorded on each.
Documents are prepared by operational staff and reviewed by their super-
visors and managers and by quality assurance specialists. Approval, that is
formal signing of each document, is the responsibility of three or more indi-
viduals: the writer, the reviewer, and a representative of quality assurance.
Therefore, in a biotechnology firm, QAU has the major task of reviewing and
approving each document generated by the development team and, in many
cases, by each contractor, consultant, and service provider. This is further
complicated by the fact that documents are often amended, and each change
or amendment must be reviewed and approved by QAU.
To guide documentation activities, the QAU has, you guessed it, their own
SOPs to guide quality activities. Notable are procedures for writing, review-
ing, approving, and changing each type of document. QAU establishes
and maintains an archive in which to place, in an organized manner, those
important documents that customers, regulatory agencies, and investors
might wish to review at a later date. Regulations clearly spell out the length
of time a firm must keep operational documents.
Research activities are generally not under the prevue of a QAU. However,
QAU may be asked to review and maintain laboratory notebooks prepared
in research laboratories, despite the fact they do not come from development
operations. Well kept, accurate and detailed research notebooks are impor-
tant to any biotechnology firm since they are used for intellectual property
applications and because they are valuable to a product development team
as the basis for planning early development activities. For example, and as
noted in Chapter 7, many quality control assays used to support product
manufacture begin as research tools in the laboratory and these records are
helpful if not essential to establishing early specifications. And the exact his-
tory of genetic constructs, as produced in a firm’s research laboratory, are
important documents to support safety claims of products derived from that
research. A prudent biotechnology firm gives research documents the same
care provided to documents from development.
Plans, protocols, procedures, and records instructing biotechnology oper-
ations, defined in an earlier section, are used in each functional area, ensure
consistency of operations, and are required by regulatory agencies. Each
of these documents must be reviewed, approved, and distributed to users.
Most are revised periodically, meaning the process is repeated at least annu-
ally. Data captured on forms or in BPRs are also reviewed, approved, and
archived. Even a small operation generates a large amount of data, further
complicating the documentation task.
The list of operational documents does not end here. Many other docu-
ments are identified in chapters throughout this book. But there is some relief

184 Biotechnology Operations
for the QAU as they do not manage every record generated by the biotech-
nology firm. Notably absent from their responsibilities are financial, human
resources (excluding training), business and marketing records.
Investigate Situations: Manage and Control Change
Change is normal in biotechnology; changes are made to plans, proto-
cols, and procedures. Any document or process or study can be changed
but change is in a controlled, proscribed manner; hence the term change
control. Earlier in this chapter, changes to product designs were discussed.
However, downstream from the design phase, and in all functional areas,
change also occurs and indeed is to be expected. Hence, all change pro-
cesses within a biotechnology operation are managed and ultimately
approved by QAU to ensure the integrity of each study, process or pro-
cedure and to communicate a proposed or completed change to all par-
ties involved. The QAU maintains procedures for change. If a document,
study, or process is to be changed, then the individual with responsibility
for that functional area recommends the exact proposed change to the
product development team. The various departments involved review
the proposal and they then discuss the risks and benefits as well as the
technical issues or challenges associated with the proposed change.
Discussion and agreement to an intended change improves the likeli-
hood of making the correct decisions and thus preventing subsequent
problems with a product or study, as might be the case if change was
made without proper consideration of all factors. A proposal for change
may go through several iterations and extensive discussion before it is
finally approved by each member of the team and by QAU. The change
control process is complex, often lengthy and thus involves careful docu-
mentation and, finally, modification of a document, such as a protocol or
SOP. As implied, a documentation system incorporates levels of interde-
pendencies between documents. Therefore, in many cases a change to
one document may warrant changes to another associated document that
may be affected by this change.
The QAU also manages documents and approves activities such as devia-
tions, the retrospective discovery that a procedure had not been correctly
performed. Variance, change that must be made without proper discussion,
review or approval, is another type of change that is handled by QAU.
Ensure Qualified and Trained Staff
Individuals working in biotechnology must have the training, education,
and experience commensurate with their assigned duties. Operational area
supervisors are responsible for ensuring this is the case for their employ-
ees. However, because adequate training of personnel is critical to the safety
of employees and the quality of biotechnology products, certain training

185Quality Systems
responsibilities are given to QAU. In addition to maintaining training records
for each employee, the QAU provides training on subjects related to quality,
compliance and documentation, and record-keeping. The QAU also ensures
that supervisors are qualified to provide technical training and confirms that
each trainer maintains and follows a training plan and schedule. QAU staff
members also coordinate training activities with senior management and
evaluate training programs to ensure they are effective and compliant.
Perform Audits
Earlier, we noted auditing as a hallmark of quality and its importance to a
quality system. Here, we discuss the performance of quality audits, internal
and external, as managed and conducted by the QAU.
Quality audits support quality operations by applying two types of audits
to compare actual performance and conditions to stated requirements (e.g.,
in SOPs). The external audit is conducted by a company engaged in an agree-
ment or wishing to do business with another company. The vendor audit
is an example. The internal audit is a firm’s own audit of its internal opera-
tions. Virtually everything in the operational arena of a biotechnology firm
is audited internally on a periodic basis or when problems are identified in a
particular area. An example of an internal audit includes the review of study
protocols, study records, and study reports at a nonclinical or clinical study
site to ensure compliance with cGMP or cGCP, respectively, with the firm’s
internal operation. In a manufacturing plant, an internal audit reviews SOPs,
BPs, equipment and validation records, quality control testing, and records
related to raw materials.
External audits may, for a reputable vendor, be performed by checking
credentials, reviewing certificates provided by the vendor or performing
telephone interviews and reference checks. However, for key materials and
services, a quality auditor visits a vendor’s manufacturing facility, nonclini-
cal laboratory or clinical site and carefully inspects to ensure compliance
with expected quality criteria. Reputable product and service providers to
the biotechnology industry are frequently audited by their clients. Therefore,
a vendor or contractor who denies or evades a quality audit without good
reason is suspect.
Several rules apply to the auditing process. The auditor must be independent
of the entity being audited. For internal audits, the auditor and the audited
department typically have parallel but independent reporting schemes in
the corporate structure. In a practical sense, this means the QAU performs
the audit and reports directly to a senior executive in the biotechnology firm.
Even small firms may have a person trained to perform audits. Hence, audits
and audit reports have become a major means of ensuring quality of a prod-
uct or service. Careful preparation is important for success of any audit and
usually outlined in a prospectively developed audit plan to include the pur-
pose and scope of the audit. The format, length, and organization of the audit

186 Biotechnology Operations
report should also be considered before the audit is initiated. The auditing
firm must address several questions in the audit plan. For example, what
is the purpose of the audit? Are there specific issues or problems with the
system being audited? What is being audited and who are the individuals
involved? The auditor should be carefully selected by QAU. It would not be
correct to send a very thorough and detail-oriented individual to perform
an audit that was intended as a superficial overview of a vendor’s quality
system. Alternatively, if there was a need to examine in detail any aspect of
that quality system, for example, a biopharmaceutical aseptic fill operation,
then an expert in this area should perform the audit.
Audits performed by QAU typically focus upon whether procedures exist
and if they do, whether or not a technical operation is following that pro-
cedure. However, during the audit it is not uncommon for the auditor to
observe a technical aspect of the actual operation that the auditor, in their
opinion, believes to be erroneous. For example, an auditor observes that a
manufacturing procedure is being performed according to SOP, that the
operator is properly trained and keeps accurate and proper documentation.
Yet, the auditor disagrees that this procedure, as written and performed, is
not technically incorrect or unclear. Such issues arise in many audits and
they beg the question of whether or not the auditor is justified or even quali-
fied to critique the technical approach being used. If the auditor was from
the U.S. FDA, we might say yes, the auditor may critique technical aspects,
since U.S. FDA inspectors evaluate both technical and quality aspects of an
operation. But what is the answer if the audit is internal or if the audit is per-
formed by a biopharmaceutical firm on their contractor? One key to avoid-
ing such issues is for QAU to be very specific about the scope and purposes
of each audit, internal or external. Nonetheless, such issues frequently arise
and they often lead to conflicts, which can be troubling, time consuming,
and often difficult to fully resolve.
Performance of the audit is important to maintaining validity of the
outcome. QAU is responsible for ensuring audits are planned, performed
to standards and procedures and fully reported with proper action taken
to resolve findings. The audit process is outlined in Figure 5.8. An audit
begins with extensive planning. For an external audit, QAU seeks input
from management and functional area supervisors. An audit plan identi-
fies the purpose and scope, standards and procedures and, in the case of
external audit, the plan is typically reviewed with internal managers before
the audit is conducted. Most audits begin with an introductory meeting at
which agenda, participants, purpose, and scope are confirmed. It is com-
mon for such audits to adopt a systems approach to obtaining information in
an audit. Most audits focus upon documents, both prospective and instruc-
tional documents such as SOPs and performance or data records. The facil-
ity, such as laboratories or a manufacturing area, may then be inspected by
the auditor. This is critical because a quality audit is performed largely to
demonstrate that procedures were in fact performed according to written

187Quality Systems
procedures or requirements. Notes are taken by the auditor. At the conclu-
sion of the audit, a closing meeting is usually held so both parties have an
opportunity to discuss the findings of the audit and perhaps resolve any
apparent discrepancies or misunderstandings.
Every audit results in a written report, prepared by the QAU’s auditor, to
relate important and relevant findings or discoveries. The report states the
facts and cites regulations or guidelines, but without being judgmental or
finely interpreting regulations. The audit report may also make recommen-
dations when deficiencies are found, but it should not mandate exact pro-
cedures to resolve these issues. Functional area supervisors at the audited
Plan
Identify scope, standards,
and procedures
Conduct
Meet
Ask questions
Inspect
Review
documents
Examine
situation
and
facts
Prepare
listing of
findings
Report
Findings
Communicate
Write
Meet
Concur
Complete report
Actions
Input
QAU procedures and
management
recommendations
FIGURE 5.8
The quality audit processes.

188 Biotechnology Operations
entity are left to take corrective action but the auditor follows up to ensure
that corrective action has adequately addressed the original issues.
There is another side to viewing audits, since every biotechnology firm
will, at some time, be audited by an outside party. This may happen as
due diligence, the result of pending business arrangements, as an inspec-
tion by an interested party, such as a regulatory agency, a client or a col-
laborator, or it may be a potential or active customer, someone wishing
to purchase materials or services. When audited, employees should make
full disclosure of any records requested by the auditor but they are not
responsible for volunteering additional materials or information that is
not specifically requested. It is in everyone’s interest to be ready with
complete, unambiguous, and well-organized materials for the auditor to
examine. The entity being audited typically sets the tone for the audit and
a positive, cooperative tone is especially important since, like a dental
appointment, getting it completed quickly and painlessly is in the best
interest of all parties.
Initiate a Quality System for a Biotechnology Operation
Once a biotechnology firm decides to develop a product and enters the regu-
lated or quality compliance arena, it finds an effective means to build qual-
ity systems into the proposed development operation. As described earlier,
quality planning, specifically the quality plan and quality manual are key
to initiating an appropriate and effective quality system, one that will grow
with the operation. Quality manuals and quality plans are living documents
and may be changed as operations increase in development phase or scope.
Thus, while quality assurance is seldom the first operational element at any
biotechnology firm, it must be adopted early and then it soon becomes an
integral part of the development effort. The excitement and expense of enter-
ing into product development sometimes obscures this immediate need for
quality function; if this happens, quality efforts lag and adequate systems
will likely suffer as a result. Nonetheless, senior management support of the
QAU enables the establishment of an effective quality system, which sup-
ports the other operational areas.
Therefore, where does one begin in this quality assurance process? The
need for a quality assurance role at the biotechnology firm is often spelled
out by a consultant or by a new employee who has worked in a regulated
biotechnology environment and is familiar with quality and compliance.
Or, it may be stimulated by recurring problems in operations. However, it
begins, the growing biotechnology firm may retain an experienced quality
consultant, someone who has built a quality department within a growing

189Quality Systems
operation, or they may elect to hire a quality professional with the same
experience. Few succeed in establishing an effective quality assurance func-
tion by relying solely on tasking an inexperienced or untrained staff member
with such responsibilities.
The next step is to generate management support and then quality poli-
cies and plans that focus on the mission and operations of the organization.
Budget must be considered, of course. The quality function is tailored to
fit the PDP and it is written in parallel to, or as part of, that overall plan.
Establishing a QAU is especially challenging for the virtual biotechnology
company, the firm with less than 10  full-time employees managing a full
product development program. Here, it may be necessary to delegate, over
a long period, quality functions or oversight to a contractor or consultant.
Many quality issues face the maturing or fully mature biotechnology
firm, defined here as 100 or more employees working in a fully opera-
tional environment. First is the inability to sustain growth of the qual-
ity function. Unlike the virtual or start-up operation, the maturing firm
already has a QAU but it may be woefully understaffed, with manage-
ment emphasizing rapid product development over quality programs.
Indeed, mid-sized firms can, for various reasons, have less concern for
quality functions than do smaller, younger companies. Functional man-
agers may ignore quality; or quality staff may experience burn-out and
lose interest. A weak QAU is easily detected during business due dili-
gence or inspections by regulatory authorities and this lowers the value
of a biotechnology firm in the eyes of potential business investment, part-
nership, or purchase.
Operational growth requires additional resources for quality efforts and
so growth in quality requirements are planned and budgeted. Growth
requires addition of specific quality skill sets. A documentation specialist
may be needed or an experienced individual is required to perform audits.
Mundane issues, such as space to work and secure files in which to archive
all those documents, face the growing quality operation. The key to success-
ful growth of the QAU in a mid-sized biotechnology firm is effective and
timely quality planning through revision of the quality plan.
Unfortunately, some biotechnology firms have, for whatever reason, a
largely dysfunctional QAU. This situation often results from inadequate
planning, poor management support and, unfortunately, ineffective lead-
ership. Such QAUs first require immediate management involvement and
support. This does not mean simply throwing money at the problem but
more often requires investigation followed by organizational change or res-
olution of issues, for example, resolution of interdepartmental squabbles, by
upper management. Inability for any one department to operate effectively
is often a reflection of an ineffective project team. Once the root cause has
been identified, then senior management begins to repair the quality sys-
tem and QAU.

190 Biotechnology Operations
Unique and Effective Approaches to Quality Management
Risk-Based Approaches to Quality Systems
Risk-based approaches are a popular and effective means of ensuring
quality in development. The U.S. FDA recommends risk assessment and
management as a means to enhance and modernize pharmaceutical and
biotechnology manufacturing and product quality. This initiative uses a
scientific framework to find ways to mitigate risk posed by medical devices,
drugs and biotechnology products.
Risk-based approaches in quality assurance examine a biopharmaceutical
operation as a process and then identify those areas within that process that
pose the greatest risk to the product. They also examine issues or problems
associated with a product or particular type of study, such as number of fail-
ures. It focuses efforts early in the life cycle of product development. This
naturally fits with quality development efforts for any product. Risk man-
agement then uses the scientific method to examine the risks and to address
and lessen those risks using appropriate quality systems. Continuous, real
time quality is a hallmark of this approach.
Risk analysis and management procedures are described further in
Chapter 2, since they typically involve multiple operational areas and
often fall largely within the purview of project managers. Nonetheless,
the QAU has critical functions and plays an important in this area, often
identifying risks and recommending that risk approaches be initiated or
completed.
Total Quality Management
TQM aims at customer satisfaction. It has been adopted by many firms,
including large biotechnology companies, and is especially popular with
sales and marketing groups. It is a structured system for satisfying internal
and external customers and suppliers by integrating the business environ-
ment, stressing continuous improvement, refining development processes,
encouraging maintenance cycles, and changing, for the better, organizational
culture. The term structured system relates to the fact that TQM relies upon
principles of quality systems and an environment that fosters such systems.
TQM has three cornerstones:
• Everyone and Everything: total quality involves every individual and
all activities in the company.
• Quality: conformance to Requirements (meeting Customer
Require ments).
• Management: quality can and must be managed. As one might imag-
ine, TQM must be driven from the top.

191Quality Systems
Six Sigma
This program has been adopted by many firms worldwide as an avenue to
produce quality products and reduce customer complaints. A major objec-
tive is to incorporate a quality system that is so effective that less than 5% of
a firm’s revenues are used to address and repair quality issues. Specifically, it
aims to reduce product or service failure rates. The six sigma process encom-
passes all aspects of a business, including management, service delivery,
design, production, and customer satisfaction. As compared to an operational
quality system such as cGMP, in which only the manufacturing and control
departments are directly affected, six sigma involves every aspect of a firm.
Statistics in Quality Assurance
Quality Assurance collects a significant amount of data and uses this data
to map trends and plan future endeavors or prevent recurrent problems.
Statistical analysis of that data is critical to making decisions and taking proper
actions. One outcome of statistical analysis is a control chart, which graphi-
cally represents one or more aspect of the quality process and identifies varia-
tions beyond limits. As such, statistics is a tool that allows the QAU to monitor
processes and provide meaningful information to functional area managers
and upper management. The quality programs described earlier, such as six
sigma or TQM are dependent upon statistical analysis of quality data.
Quality Systems for Research
What quality considerations should be given to (or imposed upon) biotech-
nology research laboratories? Is there a compelling business reason to estab-
lish a quality system for research efforts in any context? Application of a
full quality system may be helpful for research quality, but in many cases it
may actually hinder research endeavors. Research laboratories are for dis-
covery and not for structured development. Research results must be of high
quality but discovery research does not directly lead to products or to users.
Particularly frustrating are attempts to impose on a discovery research labo-
ratory a formal quality system, such as cGLP, when it is neither needed, by
regulation, nor helpful to achieving objectives. The quality standards applied
to research and development or commercial applications are different.
However, some hallmarks of quality, mentioned earlier in this chapter,
can be very effective at improving productivity and reproducibility in the
research environment, at least if they are applied correctly. For example,
vendor control is an excellent way to save time and money. An effective
documentation system can be very supportive of patent applications and
improve records upon which future product development efforts depend.
Hence, the small firm, engaged exclusively in research, is encouraged to
institute quality hallmarks that help their laboratories achieve success

192 Biotechnology Operations
without burdening research efforts through establishment of a fully com-
pliant quality system. Other hallmarks of quality, notably training, man-
agement responsibility, design of experiments, or vendor control to name a
few, can greatly improve the productivity of a research laboratory.
Resolving Quality Issues or Problems
The quality assurance professional takes on huge responsibilities and has
great authority within a firm. He or she reports to upper management and
may be more influential than many other functional area managers. One
example is in a growing biotechnology firm, where the QAU might ques-
tion whether a clinical study site is fully qualified, under cGCP, while the
clinical manager strives to meet an already challenging schedule to begin
a study. Another example, this in manufacture and control, is whether or
not to release product for a clinical study because a specification was not
fully met by analytical results. Specifications for product in early develop-
ment can be ambiguous in certain respects and individuals on the product
development team may disagree on whether it passes or fails. In another
example, the QAU may question the validity of an important aspect of an
expensive nonclinical study and suggest that portions be repeated. Note
that repeat testing has a set of regulatory guidelines around the nature and
frequencies of appropriate retests to be conducted. These quality opinions
have a tremendous effect on day-to-day operations, expenditures of time
and money and, in the end, the success or failure of a biotechnology firm.
Such situations often leave the quality assurance professional in the hot seat.
Since disagreements are not infrequent and because operational depart-
ment directors disagree on important matters, tempers may flare or dis-
agreements linger and fester.
Well-led project teams are perhaps the best means of resolving differences
while ensuring correct decisions. Several guidelines must be considered. In
the biopharmaceutical industry, FDA regulations make it clear the QAU has,
under cGMP, cGLP, and cGCP, the final word on matters relating to quality,
even though a high-level manager can and has been known to reverse QAU
decisions. Second, individuals with different backgrounds often perceive the
same situation or interpret the same data quite differently. We see this poli-
tics, in the mass media and at scientific meetings; science is not immune to
disagreements. These differences can lead to animosity and disregard for
the other’s opinion. Great quality professionals are therefore good at nego-
tiation, which is based upon understanding the other person’s point of view
and then trying to work within that opinion to reach a solution or common
understanding. This, in turn requires them to listen carefully and to be
patient. They also must clearly explain the reason for a judgment and they
are well advised to seek the opinion of regulatory professionals. Again, a
good project management team with a strong project manager is wonderful
at facilitating negotiations, if only by setting a positive environment.

193Quality Systems
Upper management plays a major role in preventing hostilities and
resolving disagreements between the QAU and other operational areas.
Misunderstandings in biotechnology operations are often the result of
poorly-established or undercommunicated corporate and product devel-
opment objectives. In these all-too-frequent instances, upper management
bears responsibility. Management is responsible for defining quality policy
and responsibilities and authority in the quality manual. Further, upper man-
agement must recognize when communication has broken down between
quality assurance and another department manager and then make every
effort to resolve the differences and have each faction work together toward
a common objective. To perceive developing issues in a timely manner, upper
management must always be involved and alert. Project managers and team
members ensure that management is engaged in development activities,
including professional roles and disagreements.
Why is it so vitally important to identify quality issues in a biotechnology
firm? First, these small firms are so fragile, very susceptible to failure for a
number of reasons. Second, they have little depth—fiscal resources, product
line, facilities—to rely upon in times of trouble. Third, the team has worked
together for a brief period, as most firms are relatively new and develop-
ment may have just begun. Differences related to quality aspects of the oper-
ation could spell the difference between success and failure, especially in
biopharmaceutical endeavors. Often, quality issues are for a variety of rea-
sons invisible to insiders but most obvious to outsiders, such as consultants
and auditors. Indeed, seasoned professionals in biotechnology have said that
by examining the roles, authorities, and responsibilities of a QAU, one can
quickly surmise a key indicator or success versus failure at a young biotech-
nology firm.
This section on resolving quality issues has provided no magic solutions
for problems one might encounter in a biotechnology operation. It aimed
instead to summarize but a few of the situations one might encounter in
the operational environment of a biotechnology firm. By understanding
quality systems and through careful planning and effective management,
quality functions and the quality professional who manage them, the qual-
ity endeavors can be a valuable asset to any biotechnology development
team and the firm they represent.
Summary of Quality Systems
Quality assurance is a planned and structured function designed to ensure
each product or service provided by a biopharmaceutical firm will meet
established requirements and user expectations. Quality planning and insti-
tution of a quality system early in biopharmaceutical product development

194 Biotechnology Operations
is critical to the success of any biotechnology operation. Hallmarks of quality
are distinctive features of excellence comprising any quality system and
include features such as management responsibility, definition of the qual-
ity system, design and design control, contractor control, product identifica-
tion and traceability, process control, environmental control, quality control
or testing and release, change control and corrective or preventive actions,
packaging and labeling, preservation, storage and handling, servicing, and
customer concerns. The quality assurance function focuses on the quality
attributes of quality management, documentation, investigation and change
management, training, and auditing. Effective quality systems are devel-
oped specifically for each process and product or service and established
quality systems, such as cGCP, cGMP, ICH Q-10, cGMP, or ISO 9001, are often
required of biotechnology firms, especially for biopharmaceutical develop-
ment. Today, several unique and effective approaches can be adopted to bet-
ter manage a quality system but their benefit must at least equal their risks.
The QAU is the functional area that manages a quality system within the
overall biotechnology operation. The QAU controls documents and manages
the documentation system, investigates situations, manages and controls
change, ensures all staff are qualified and trained to perform their duties,
and performs audits, internal and external. Initiating or maintaining an
effective and respected quality system in a biopharmaceutical operation is
a challenging task and relies in part upon both technical and social skills
of quality professionals. Further, the quality system must be matched to
requirements and be always balanced with the operation, solving and not
creating problems or issues.
References
British Standards Institute. 1991. Standards of BSI, BSI, Herndon, VA.
International Standardization Organization. 1994. ISO 9001, Geneva, Switzerland.
Oxford English Dictionary. Oxford University Press. 1997. University of Oxford,
Oxford, UK.

195
6
Biomanufacture
Overview of Biomanufacturing Requirements
The biotechnology operation focuses on the development of a specific prod-
uct. This concept carries with it the need to plan and then develop a bioman-
ufacturing process to produce a biological substance of high quality and in
amounts required for testing and marketing. Further, the biomanufacturing
materials, processes, and the resulting product must be compliant, that is,
they must satisfy regulatory agencies through application of good science
and a quality system: current Good Manufacturing Practices (cGMP). To
achieve these objectives, the biotechnology operation must develop a bio-
manufacturing plan.
Even for the simplest product in the hands of experienced bioprocess engi-
neers, biomanufacturing is a demanding endeavor and requires consider-
able planning, time, and financial and human resources. False starts in the
biomanufacturing pathway, which is usually the result of inadequate plan-
ning, often lead to project failure and termination.
Hence, biomanufacturing planning begins early in the product develop-
ment cycle and is based on the exact understanding of the product’s nature
and intended use or indication. The overall product development plan (PDP)
(Chapter 1) coordinates the manufacturing plan with plans for the quality
assurance, quality control, and regulatory, clinical, and nonclinical aspects
of product development. To ensure this integration, the biomanufacturing
planning process requires leadership from biomanufacturing experts, con-
siderable time and effort, and frequent interactions between individuals
from various departments.
This chapter on biomanufacturing considers design and planning, pro-
duction technologies, compliance and quality, major stages and steps of
manufacturing for various types of biotechnology products, and the manu-
facturing facility.

196 Biotechnology Operations
Design in Biomanufacture
At the heart of a biomanufacturing plan is the manufacturing design or
scheme—pictured from beginning to end—with the various control testing,
quality, and regulatory elements that impact product production. The objective
of biomanufacture is to produce a product that has the attributes, for example,
strength, identity, purity, potency, and safety, commensurate with the intended
use. Product attributes are further defined in Chapter  7. Each biotechnology
product is unique and is, or will be, produced using both well-characterized
and well-known commercial processes and special methods developed for that
particular product or class of products. The flow diagram shown in Figure 6.1
is a general format or template used to design a product-specific biomanufac-
turing scheme. Three stages of biomanufacture—(1) upstream processing;
(2) downstream processing; and (3) formulation, fill, and finish—are the back-
bone of a biomanufacturing design. In the first stage, upstream processing, the
product is produced from raw materials by using process technologies such
as cell culture, fermentation, and synthesis. The second stage, downstream
processing, involves purification of the desired product by its separation from
impurities and contaminants. In biopharmaceutical processing, the output is
referred to as the bulk (drug) substance (BS). For a biopharmaceutical, the BS
is also the active pharmaceutical ingredient, which means that it has the ther-
apeutic activity. Stage 3 processing ensures that the product is fit for use, by
applying the processes of formulation, filling into a container, packaging, and
labeling. The result is a final product (FP), which is ready for use.
Quality by design (QbD) is a concept applied to all product development
endeavors (Chapter 5) in biotechnology and is a critical and early aspect of
biomanufacture. It evolved in part from regulatory and quality initiatives
in the medical device industry late in the twentieth century. More recently,
the principles and practices of QbD have been adapted to biopharmaceutical
development. Product development or manufacturing QbD is driven in part
because regulatory agencies have provided evidence that, when followed,
QbD consistently leads to high-quality products. This represents a para-
digm shift from the previous practice of simply testing samples of the FP for
quality in the hope of demonstrating quality. Indeed, this past practice was
referred to as testing quality into a product. This practice has now changed, and
although quality control testing (Chapter 7) remains an important aspect of
product quality, it is now recognized that production design and process
practices play equally important roles in ensuring a pure, potent, consistent,
and predictable quality product every time. In other words, quality should
be built into the product by design rather than by solely relying on the FP
testing. The biotechnology industry also recognizes that QbD makes good
business sense. Janet Woodcock of the FDA defined QbD in 2005 as “a maxi-
mally efficient, agile, flexible pharmaceutical manufacturing sector that
reliably produces high quality drug products without extensive regulatory

197
B
iom
an
ufacture
Stage 1
Upstream
processing
Raw
materials
Target
product
profile
Stage 2
Downstream
processing
Stage 3
Formulation, fill,
finish, and labeling
Genetic construct,
expression vector
Package
label
Aseptic fill
Formulation
Design of manufacture
in a product
development strategy
Initial product
capture
Purification 1
Purification 3
Testing
Staff, quality
assurance, and
quality control
Purification 2
and andandand
Research seed
Cell banks
Cell culture
fermentation
Initial recovery
Testing
Testing
Testing
Cell paste
Stages:
Processes:
Outputs:
Bulk
substance
(BS)
Final
product
(FP)
Testing
Testing
Testing
Testing
Testing
Testing
Inputs:
Facility,
utilities, and
equipment
FIGURE 6.1
General outline of biomanufacturing activities by stages or steps of biomanufacture. This flowchart traces the biomanufacturing scheme applied to
many biotechnology products. Boxes in the upper row define inputs, that is, the resources required to begin biomanufacture of a product. The flow-
chart below is divided into three stages typical of a complete manufacturing process and describes outputs (or results) from each of the three stages.
Process elements, shown in the shaded flags, are typical for production of a recombinant protein product.

198 Biotechnology Operations
oversight.” Yet again in 2014, elements of QbD were highlighted by Margaret
A. Hamburg, Commissioner of Food and Drugs, in her outline of FDA stra-
tegic priorities for the next 4 years. She stated that the continued use of risk-
based approaches would help to ensure product quality in pharmaceutical
development. (Hamburg 2015)
In its simplest form and as provided in the International Council for
Harmo nization of Technical Requirements for Pharmaceuticals for Human
Use quality guidelines (Chapter 4), the concept of QbD instructs the devel-
oper to design a product, so it consistently meets the desired performance
criteria and always meets expected quality attributes. This definition
demands much of a product’s sponsor. First, it identifies the need to inte-
grate a manufacturing plan into the overall PDP, described in Chapter 1. It
also directs the use of formal manufacturing design process, in which the
product designer considers and documents both the expected performance
and quality attributes of the product.
QbD, as it applies to biomanufacturing and the biomanufacturing porting
of the PDP, is shown in Figure 6.2. The nature of a biotechnology product,
Processes
Design space
QC plan:
TPP
User needs Regulatory requirements
Available manufacturing:
–Raw materialsAvailable tests:–Assays
Input
Specifications
Limits
Specifications
–Processes
–Facility and equipment–Laboratories
Quality by design
Input risk analysis
Review output
Document
Manufacturing plan
Quality
requirements
Management
Financial
Personnel
LimitsLimits
Specifications
–Tests
–Specifications
–Release
–Stability
–Validation
Manufacturing process plan:
–Stages
–Steps
Facilities, equipment, and
validation
Output and PDS
FIGURE 6.2
Quality by design in biomanufacturing. Manufacturing design begins with inputs, notably
user needs and regulatory requirements, that are synthesized into a targeted product profile
(TPP). A design space has specifications and limits as boundaries and quality requirements,
available manufacturing resources, tests and management, and resources as inputs. This
allows the manufacturing process to be designed within a design space, represented in the
center. Outputs of the design process include a quality control plan; the manufacturing process
plan; and a strategy for facilities, validation, and other requirements.

199Biomanufacture
as provided in a targeted product profile, must be carefully considered in
the manufacturing design. QbD requires that a manufacturing process be
designed using scientific approaches, quality criteria risk management,
and design space. QbD prompts the need for application of design con-
cepts: input, output, reviews, design space and specifications, and ranges of
acceptable values or limits. These are discussed in Chapter 1. Design space,
as it refers to biomanufacturing and control activities, is the requirement to
design within limits or boundaries. For planning biomanufacture, the limits
are imposed by constraints of manufacturing technology and by product
specifications. Specifications, further defined in Chapter 7, are measurable
quality criteria for a product. Although these boundaries on design space
restrict initial manufacturing design, they allow later changes in the manu-
facturing processes, with minimal justification, as long as the changes are
implemented within the design space and consider the specifications. This
allowance is based on the premise that changes to a biomanufacturing pro-
cess made within a reasoned design space will most likely not change the
quality of the biopharmaceutical product. Planning under QbD helps to
ensure that a biomanufacturing process will be robust and the product will
predictably be consistently of high quality throughout the life cycle.
As shown in Figure 6.2, QbD also applies the concepts of input and out-
put to biomanufacture. Input, notably user needs, the nature and the profile
of the product, and manufacturing and control technologies are considered
within the confines of design space. Within the design space, active manu-
facturing design and review lead to the output, notably plans for the appro-
priate processes, facility, and quality control tests.
In manufacturing design, the concept of risk analysis, elaborated upon in
Chapter 1, is considered in light of how the manufacturing process and the
design space might impact the user of the biopharmaceutical product. Any
biotechnology product carries some inherent risk to the user or to the public.
Other risks are imparted through the product’s manufacture. Both design
and manufacturing planning activities identify and attempt to mitigate all
risks. QbD considers these risks in a logical manner and demands that the
design should take into account any possible risk.
Biomanufacturing design and planning are greatly impacted by affordable
biomanufacturing technologies available for a given type of product and
process. For a given product, the biomanufacturing planner has a variety
of process methods from which to choose. The process technologies chosen
during design are added to the plan and referred to as input. The results of
these technological applications are known as output, which also become
embodied in the manufacturing plan. Consider, throughout the remaining
chapter, the need to design a biomanufacturing scheme, applying product
limits and specifications, the inputs and outputs of a design, and the need for
understanding process and product risk at every point. These concepts help
us better understand why products are manufactured in a certain manner.
Finally, some biomanufacturing process change is inevitable for even the

200 Biotechnology Operations
best conceived biomanufacturing plan. Consideration is given for making
changes in a manufacturing plan as long as the changes are kept within the
boundaries of design space and the risk is carefully considered.
Technical Considerations for Biomanufacture
Biomanufacturing is a relatively new field, which has expanded, by quan-
titative and qualitative measures, rapidly. Before 1970, the manufacture of
biological products was accomplished largely by purification of biologically
active molecules from various natural sources. For example, albumin was
precipitated from human plasma and then separated from other blood pro-
teins. Some years ago, vaccines were strictly natural products, such as sub-
units of viruses or protein toxins, derived from microbes grown in culture.
Although these methodologies are still considered biological technologies,
the advent of genetic engineering led to the endeavor we now refer to as
biotechnology. Genetic engineering made possible the transfer of genes from
one organism to another, and this science allowed us to genetically modify
bacteria or mammalian cells, which in turn led to biomanufacture, the pro-
duction of small amounts of recombinant proteins or nucleic acids. To make
large amounts of these recombinant products, first for further evaluation
and then for commercial use, biomanufacturing protocols and technologies
or methods were expanded. Biomanufacturers soon discovered that product
quality was important to ensure proper function. If a molecule were of poor
quality, it would not perform as intended, when used in critical test proto-
cols, such as in animal or clinical studies. Notably, when a manufactured bio-
technology product failed to perform consistently, the sponsor was left with
little product value and doubts about the product’s utility and marketability.
It was also recognized that quality control testing of a manufactured biotech-
nology product, alone, did not ensure product quality. Indeed, product qual-
ity reflected both the processes and the technology applied to biomanufacture,
such as facilities, utilities, and equipment used in biomanufacture. Consistency
of manufacture was also critical to achieving the desired attribute; the biophar-
maceutical product had to be the same every time it was manufactured. Hence,
commercial biomanufacture demanded attention to product consistency.
Rapid advances in biotechnology have challenged the young field of bio-
manufacture in other ways. Many organisms capable of expressing recom-
binant proteins expanded greatly in just three decades, and the types and
classes of biotechnology products that must be manufactured by our indus-
try continues to both expand and diversify. More advanced expression
systems that utilize somatic or stem cell engineering are the examples of
rapidly growing scientific methodologies that have brought about the need
to develop and apply new biomanufacturing technologies. Transgenic plants

201Biomanufacture
and animals have become commonplace, and biologically active molecules
are now regularly processed from these sources. Synthesis of biologically
active molecules is a field that continues to expand. Other challenges include
developing novel products, refining old processes to produce currently mar-
keted products in a more economic way, improving the quality or consis-
tency of investigational and marketed products, and engineering production
of generic or follow-on biopharmaceuticals—new products that are safe and
effective, exactly like a predicate product.
To meet these challenges, careful planning and development of new manu-
facturing technologies, analytical tools, and processes continues unabated.
Biomanufacturing scientists continue to invent, apply, adopt or adapt processes,
procedures, and skills to meet this increase in demand. Facilities and equip-
ment have been designed or redesigned, built, validated, and commissioned
to house and support these more sophisticated processes. In summary, there is
much activity in the field of biomanufacture, which is leading to excellent mar-
keted products, and each success is based on proper manufacturing planning
and design and the ingenious application of existing and novel technologies.
For the remaining chapter, we present an overview of the stages and
steps used in biomanufacture, identify technical considerations for vari-
ous processes, and integrate quality and compliance into biomanufacturing
schemes. We then apply biomanufacturing criteria and technologies to sev-
eral classes of biotechnology products, highlighting differences and similar-
ities of various products. At the end of this chapter, we describe the design,
use and validation of biomanufacturing facilities, utilities, and equipment.
Quality control and quality assurance activities are closely associated with
biomanufacture, and these are discussed in Chapters 7 and 5, respectively.
Phases and Scale-up: The Biomanufacturing Life Cycle
Biomanufacturing is performed throughout the life cycle of a product. We iden-
tify phases in the life cycle and further ask the biomanufacturer to ensure that a
product possesses particular qualitative and quantitative attributes or traits in
each phase of development, with process and product specifications becoming
increasingly more stringent as the cycle progresses. For biopharmaceuticals,
manufacturing phases of development follow those applied to clinical studies
(Chapter 9): Phase 1 (early phase), Phase 2 (mid phase), Phase 3 (late phase), and
Phase 4. This approach makes sense because we use product in greater amounts
as the number of human subjects increases at each clinical phase. At Phase 1,
requirements are in the hundred of doses, but as product approaches the mar-
ketplace, product might be needed for millions of users. Compliance issues,
specifically adherence to cGMP, also increase in intensity and importance,
as biomanufacturing development increases through the phases. Both total

202 Biotechnology Operations
amount of product and quality criteria undergo change, as the product is used
in a greater number of individuals. These relationships between clinical phase,
biomanufacturing phase, product quality, and product quantity are shown in
Box 6.1, and these relationships must each be considered in a manufacturing
plan. A greater amount of product is produced in each subsequent phase, and
along with this comes the need to better characterize the product and to meet
ever greater compliance standards through improved quality and production
systems. Phased product biomanufacturing development is a dynamic process,
and change is desirable and normal. How this change is anticipated, planned,
controlled, and executed in the manufacturing plan is critically important to
the overall product success.
The quality of product required at Phase 1 clinical studies is, to some
extent, mandated by cGMP, but it is also a function of the indication,
intended use, and proposed manufacturing process. In planning and devel-
oping the process, it is important to consider that greater amounts of prod-
uct will be required later in the development; therefore, a biomanufacturing
process must be amenable to change to accommodate scale-up and to meet
more stringent quality and compliance criteria. Process control, quality con-
trol testing, and consistency of manufacture are important measurements
that can demonstrate product quality at Phase 1. Hence, the process is well-
defined and some quality control assays are established before initiation of
biomanufacturing. In early phase development, it is best to produce multiple
batches of BS and multiple lots of FP to understand and control variation.
This information can be used to ensure consistency of manufacture—
amount and quality—from batch to batch and from lot to lot.
At mid-phase development, the quality objectives are to confirm and extend
the findings of early phase biomanufacture. Biomanufacturing scale-up at
mid-stage further tests the application of quality criteria to the process and
to the end product. Product manufactured at mid stage is used in those clini-
cal studies, and it is also applied to refinement or qualification of analytical
tests. Process improvements are often implemented and tested to ensure that
changes yield a product with the same or better quality attributes than those
seen at early-stage development. The ranges of acceptable values for product
specifications, both process and quality control, are often narrowed at Phase
2. Mid-phase biomanufacture provides a product that is used for the qualifica-
tion or validation of analytical tests and also for additional stability studies.
It is not uncommon for a biomanufacturer to miss critical mid-phase manu-
facturing objectives. Failure in achieving consistent manufacture may result
in the need to abort the ongoing processes. In such situations, it is important
to review the manufacturing plan, make changes, and then repeat mid-phase
biomanufacturing before progressing to scale-up or late-stage manufacture.
Unfortunately, such advice is too often ignored, leading to biomanufacturing
failures at Phase 3, which result in the need to repeat manufacturing devel-
opment, often including Phase 2 and Phase 3 biomanufacturing and clinical
studies, both very expensive propositions.

203Biomanufacture
BOX 6.1 BIOMANUFACTURING ACTIVITIES BY
PHASES OF BIOPHARMACEUTICAL DEVELOPMENT
Phase
Design and
Plan
Manufacturing
Processes
Quality
Control
Laboratory
Quality and
Compliance
Planning (0) Targeted
product profile
Product
development
strategy
Technology
Documentation
system
Develop constructs
Technology
transfer from
R&D laboratory
Cell bank
development
Research
laboratory
development
of critical
analytical tools
QC constructs
and cell banks
Identify
regulatory
guidance
Establish quality
plan and basis
for quality
system
Ensure quality
assurance
activities
Early phase (1) Implement
design and
process
development
schedule
Accept constraints
Produce clones
and cell banks
Perform the
process two times
or more
Produce product
for nonclinical
and Phase 1
studies
Establish
certificate of
analysis with
product
attributes,
tests, and
specifications
Test Phase 1
products
Evaluate final
product
stability
Institute Phase 1
cGMP
compliance
Formalize
training system
Manage
documentation
system by using
version-
controlled
documents
Mid-phase (2) Refine plan
based on
findings
Scale-up for
multiple batches
and lots
Phase 3
requirements
Adjust process and
refine steps
Further develop
assays, qualify
critical tests,
refine
specifications
and add new
tests
Test Phase 2
products
Expand viability
testing
Increase scope
and depth of
cGMP
compliance
Implement
more
qualification
activities
Perform internal
and external
audits
Late phase (3) Plan commercial
process and
validation
activities
Execute multiple
lots at or near
commercial scale
Validate process
and facility
Validate or
verify each
assay
Test product at
scale-up and
for Phase 3
Expand viability
testing
Come to full
cGMP
compliance, as
applied to
commercial
production
Approve
validation
Postlicense/
commercial
(4)
Plan and
document all
change
Manufacture for
commercial
market
QC for
commercial
product
Approve change
Maintain full
cGMP

204 Biotechnology Operations
Mid-phase is the best time to make significant and necessary process
changes. Process changes may result in changes to the purity or potency of
the product, and these may negate the validity of nonclinical and clinical data
generated during Phase 1 and Phase 2 studies. For these reasons, every effort
is made at this time to improve the process without changing the molecular
or cellular nature or the quality profile of the biotechnology product.
Late-phase biomanufacturing development focuses on preparing material
for Phase 3 clinical studies. Production at this stage also ensures a robust
process at greater scale, and the late-phase product is used for further assay
development or validation and stability studies. Biomanufacturing process
validation, described later in this chapter, is another objective of the late-
stage biomanufacturing program. Manufacturing changes, in addition to
scale-up, can be instituted at late stage, but they must be thoroughly ana-
lyzed to ensure that the product remains consistent in quality with the mate-
rial made in the earlier phases and used in clinical and nonclinical studies.
To this point, we have discussed changes to biomanufacturing processes
as a qualitative perspective. We now cover the subject of quantitative manu-
facturing changes or scale-up. Early phases of biomanufacture yield limited
amounts of product, certainly not a sustainable quantities required to address
market demand. Biomanufacturing scale-up, depicted in Figure 6.3, is used to
gradually increase the total amount of the product that is available from each
production run. A production run is a distinct series of processes, and each
run results in one batch of BS or one lot of FP. Scale-up must be considered
within the confines of design space. One should, at the outset, have an idea
of commercial requirements and then base a scale-up plan on these require-
ments. Scale-up may follow several pathways. Scale-up may be achieved by
increasing the yield (1) by increasing the amount of product produced within
Nonclinical
Phase 1
Appl
icabil
ity of
good
man
ufact
uring
prac
tice r
egula
tion
Prod
uct c
harac
teriza
tion
Phase 2
Phase 3
Quality systems
FIGURE 6.3
Manufacturing by phase of development. Simultaneous increases in product quantity, quality,
characterization, and regulatory compliance through phases of biomanufacture. (Courtesy of
Anthony Clemento, 2008.)

205Biomanufacture
a batch or a lot; (2) by upping the scale of manufacture for each batch or lot;
and (3) by increasing the number of manufacturing runs. Often, a scale-up
plan considers two or even all three methods to increase the total amount of
BS and FP. This is particularly important with therapeutic biopharmaceuti-
cals, as small changes in molecular structure may have significant implica-
tions on function, thus translating to safety or efficacy outcomes for patients.
Scale-up is an expensive process, which is not typically initiated until suc-
cess has been demonstrated in clinical or field studies. Scale-up has a greater
impact on the production of BS than on the production of FP. Scale-up of final
drug product manufacture often involves increasing the size of the formula-
tion batch by using larger vessels, by building another production facility, or
by outsourcing to a qualified contract manufacturing organization (CMO)
that has the capacity to perform a greater volume of fill, finish, and labeling.
Of importance, scale-up to obtain greater amounts of BS most often cannot
be economically accomplished by simply multiplying the number of bioreac-
tors or fermenters, or by installing several rows of chromatography columns.
Instead, scale-up typically involves developing new or modifying existing
technologies to produce much greater volumes in a single batch (i.e., a single
large fermentation vessel). Biomanufacturing systems are often finicky when
it comes to scale-up; hence, there is a need for imagination and extensive
experimentation. At large scale, the experimental processes require a sig-
nificant investment in equipment, are costly to perform, and require large
amounts of raw materials. This scaling process forces the operator to devise
BS production systems that appear to be quite different from the smaller sys-
tems used in early phase biomanufacture. Process and laboratory controls
are continually applied during the scale-up process, as changes to yield are
likely to impact product quality. It is an important consideration to limit all
changes to a process within the designated design space.
Raw Material Considerations
Like any other manufacturing endeavor, biomanufacturing requires raw
materials, which are sometimes referred to as components. These provide
the structural building blocks, dynamic metabolic energy source, and bio-
manufacturing environment for every product and process. Raw materials
such as water, gases, salts, and nutrients are critical elements employed at
every phase of the biomanufacturing process. The quality of each raw mate-
rial should remain unchanged throughout the manufacturing cycle, but
amounts increase with scale-up. Requirements and specifications of raw
material are included in a manufacturing plan.
Box 6.2 presents a list, albeit incomplete, of raw materials that may be used in
upstream production, such as fermentation of yeast, to produce a recombinant

206 Biotechnology Operations
protein. Box 6.3 provides a list of raw materials that may be used in downstream
production, that is, in the purification process of that product. Since the quality
product output is, in part, a reflection of the quality of the input raw materi-
als, biomanufacturers, and regulatory authorities take the source and quality
of each raw material very seriously, no matter how insignificant it may appear
to the process or application. Special consideration is given to the raw materi-
als that contact or are incorporated into a FP. Raw material specifications and
acceptance criteria are critical to consistently meeting the standards set forth in
manufacturing plans and procedures. The possibility of raw materials contain-
ing toxins or adventitious agents is especially noteworthy, because these impu-
rities present risks to the user, and because once introduced into the product
stream, these may be difficult to detect and remove.
A raw material for biomanufacturing may be purchased from a ven-
dor or it could be produced in-house, by the product manufacturer. For
example, sodium chloride is typically purchased, whereas highly purified
water, water for injection (WFI), is often produced in the sponsor’s facility.
As you might expect, raw materials are a highly controlled commodity in
the biomanufacturing arena. To prevent misidentification or contamination,
BOX 6.2 EXAMPLE OF A MATERIAL LIST:
UPSTREAM FERMENTATION
Material
Number
Description and
Attribute Source Specification Comment
H2–115 Working cell bank TA
Biotechnical
CoA Manufactured
01/11/2010
145621 Yeast nitrogen base
without amino acids
DB/Fidco CoA No animal
product
RX001 Glycerol Spectarm USP No animal
product
LC1121 8 N Ammonium
hydroxide
TJ Booker USP Not applicable
31772 Glucose/dextrose TJ Booker USP Not applicable
32274 Tryptic soy agar
plates
Remel SOP QC-1181 Passed
32371 Yeast peptone
dextrose plates
Remel SOP QC-1181 Passed
16–2010 Water for injection TA
Biotechnical
SOPs MF-1141
and QC-1832
Passed
4–115 Biotin Spectrum CoA Not applicable
Note: Manufacturer’s material number: CoA, manufacturer-provided certificate of anal-
ysis; SOP, internal testing by QC laboratory standard operating procedure with
specification and passed by QC and QA; USP, U.S. pharmacopeia-grade material;
animal product, manufacturer- provided certificate ensuring that no animal prod-
uct was used in this material.

207Biomanufacture
vendor-supplied raw materials are inspected, clearly labeled, sometimes
retested, and then kept in controlled storage areas of the manufacturing
facility until used in the manufacturing process. Quality of raw materials is
further discussed in Chapters 5 and 7.
Compliance and Quality in Biomanufacture:
Current Good Manufacturing Practices
Quality considerations for biomanufacturing begin with design and plan-
ning and continue throughout the life cycle of a product. In the United States,
biopharmaceutical manufacturing quality is promulgated in a set of regula-
tions known as cGMP. Other countries also have manufacturing guidelines,
BOX 6.3 EXAMPLE OF A MATERIAL LIST:
DOWNSTREAM PURIFICATION
Material
Number
Description and
Attribute Source Specification Comment
1–110 Clarified fermentation
supernatant
TA technology Batch production
record-661–00
Manufactured
01/10/2011
040721 Water for injection TA technology SOPs master
formulation-1141
and QC-1832
Passed
SF-1418 Sodium phosphate
(monohydrate)
TJ Booker USP Not applicable
TM0012 Sodium hydroxide,
pellets (NaOH)
Spectarm USP NF Not applicable
SF-1416 Sodium phosphate
(dibasic)
heptahydrate
TJ Booker USP Not applicable
C3HN5–
9990
Millipak-20 filter units
(0.22 µm)
Milepour CoA Meet
specifications
65 SD105 Superdex
200 chromatography
gel
EG healthcare CoA cGMP grade
30 SO672 Sepharose high
performance
chromatography gel
ED healthcare CoA cGMP grade
Note: Manufacturer’s material number: CoA, manufacturer-provided certificate of anal-
ysis; SOP, internal testing by QC laboratory standard operating procedure with
specification and passed by QC and QA; USP, U.S. pharmacopeia-grade material;
and NF, national formulary.

208 Biotechnology Operations
and for the biotechnology firm intending to export a biopharmaceutical,
attention must be paid to directives from European, Japanese, Canadian,
World Health Organization, and other national and international agencies or
organizations. In addition, the International Conference on Harmonization
of Technical Requirements for Pharmaceuticals for Human Use has guide-
lines on manufacturing quality. These references are further identified and
discussed in Chapter 4.
Biotechnology products and raw materials, those other than biophar-
maceuticals, also have manufacturing and product quality criteria, either
known as an industry standard or established by industry trade organizations,
national or international bodies, and regulatory authorities. For example, the
International Standards Organization guides activities and establishes stan-
dards for biomanufacturing and thousands of other industrial endeavors.
Good manufacturing practice guidelines, no matter what the standard or
guideline source, are followed by biomanufacturers, first because they are
regulatory requirements and second because they are logical for business
development, product marketing, financial stability, and product liability
reasons. Product recalls are expensive for a biotechnology firm, and adverse
events due to production and release of substandard product can devastate
the reputation and lead to financial ruin, even for a large company.
The objective of cGMP is to consistently produce and deliver the highest-
quality product to the user. Today, cGMPs apply beyond production activities
in a biomanufacturing facility. They encompass the concepts of biopharma-
ceutical design, risk analysis, and manufacturing planning, the functions that
begin well before the product even enters the facility and extend to warehous-
ing, an activity found at the far end of the biomanufacturing development
pathway. The full embracement of cGMP, the U.S. FDA regulation, is phased
into the manufacturing plan, as shown in Figure 6.3. Phase 1 manufacturing,
under cGMP, ensures that raw material and process hazards are identified.
Steps are also taken to ensure that these hazards do not endanger human
subjects during any phase of clinical investigation. However, FDA recognizes
that not all aspects of cGMPs apply to a given product, especially in early
development, and FDA offers additional guidance for Phase 1 manufacture
of biopharmaceutical products. Application of cGMP requirements is consid-
ered in a manufacturing plan by focusing on product-specific attributes and
quality issues that might affect biopharmaceutical manufacture for nonclini-
cal and Phase 1 clinical studies. Risk analysis of the manufacturing plan is
one way to identify quality issues, and apply cGMPs to production processes
in a rational manner.
The plan also considers manufacturing compliance as product moves
through subsequent phases of manufacture and greater numbers of subjects
are exposed to a product. Now, cGMP application has broadened and has
become increasingly stringent for each stage of biomanufacture. There is
a heightened level of importance and need for cGMP in certain processes
such as aseptic technique or sterile fill, because these processes are critical to

209Biomanufacture
ensure product safety. With respect to sterility and several other manufac-
turing controls, there is a single interpretation of cGMP and it applies from
Phase 1, or early phase biomanufacture, to commercial manufacture. Yet,
less risky processes have little impact on safety and thus are of lesser con-
cern. Therefore, the concept of cGMP application is considered a gradient,
beginning with cGMP at Phase 1 biomanufacturing and increasing through
commercial production and weighing the risk of a practice or material at
every phase. The cGMP regulations are outlined in Chapter 4. Specific exam-
ples of quality criteria and application of cGMP are provided in subsequent
discussions of biotechnology products and biomanufacturing technologies
in this chapter.
Biomanufacturing Processes for Biotechnology Products
The discussion on biomanufacture now shifts from general subject matter
to focus on various biotechnology products and the technologies used to
manufacture those products. We begin by reviewing standard production
methods used to biomanufacture recombinant proteins in living cell-based
systems and follow with a discussion on the use of transgenic organisms.
The information then moves to the field of stem cell or somatic cell and tis-
sue production, delves into technologies such as the synthesis of biologically
active molecules, and introduces the growing field of combination products,
where biopharmaceuticals are merged with medical devices or pharmaceuti-
cals (drugs). There is a great diversity of biological products, so it is impossi-
ble to mention each one or even to discuss each class. However, the examples
should provide the reader with an idea of what has been achieved and, in a
few instances, what could be done in the future to manufacture biotechnol-
ogy products.
Expression of Recombinant Proteins and Nucleic Acids
Production of Recombinant Molecules from Expression Vectors
Laboratory methods to manipulate living organisms and the biological mol-
ecules they produce are at the heart of biotechnology. Operational endeavors,
including biomanufacturing, flow from discoveries made in basic research
laboratories where tools or methods are devised and first applied in discov-
ery research. Hence, it is no wonder that the first step in biomanufacturing
is the discovery or invention of an organism or molecule that expresses a
desirable trait or otherwise serves a useful function. To date, thousands of
discoveries or inventions have enabled genetic engineering of nucleic acids
and living organisms. Research laboratories have capitalized on a wealth of

210 Biotechnology Operations
information regarding recombinant DNA, cell metabolism, and the basis for
life itself. But there is a caveat in all this. Although it is these scientific find-
ings that constitute the foundations on which we base product development,
it is the biomanufacture, the production of large amounts of high-quality
biotechnology product, that brings the product to the market and the user.
Today, the production of recombinant molecules, notably proteins and
nucleic acids, represents, by volume, the bulk of biomanufacturing capacity.
A variety of active recombinant molecules; proteins such as insulin, human
or bovine growth factor, monoclonal antibodies, and vaccine antigens; and
nucleic acids for genetic therapy and diagnostic purposes have entered the
marketplace. Some are sold in large quantities and represent blockbuster
products in the marketplace. Today, biomanufacture of recombinant proteins
and nucleic acids meets the growing demand and represents an important
economic sector of the biotechnology industry.
Genes, Vectors, and Host Cells
The first stage in biomanufacture of a recombinant product involves three
processes: gene isolation, cloning and development of an expression vec-
tor, and production of cell banks. The process and controls of this first stage
are outlined in Figure 6.4. First, a gene of interest is identified and isolated
through molecular cloning, most often by using polymerase chain reaction
and other technologies. Alternatively, the gene may be selected from an
established library of cloned DNA. The gene is characterized by molecular
weight determination and DNA sequencing. A vector, available from public
or private vector libraries, is selected, based on suitability for biomanufac-
ture of the designated product. The attributes for selection include ability
to adapt and function in a suitable host, replication, promotion of protein
expression, protein chain termination, and absence of undesirable character-
istics, among others. The gene is then inserted into a selected vector by using
methods such as recombinase-based cloning or restriction-ligase cloning.
Next, the vector is transformed into a host cell: bacterial, yeast, insect, or
mammalian cell. The host must also be carefully chosen so as to be compat-
ible with the vector. Each type of cell has particular attributes, and no cell
is universally well suited for expression of every recombinant DNA or pro-
tein molecule. After transformation, the vector must be stable, that is, held
within the host, and be maintained as one or more copies of the vector over
many generations, as the host divides. Methods are applied to increase the
chances of successful transformation, but many attempts may be required
before a stable and fruitful match between the host and the vector is
achieved. Ultimately, transformed hosts are produced and one single isolate
is selected. This selection is done only after performing extensive testing to
ensure that all qualities have been achieved. Once the transformed host is
deemed acceptable, it is cloned by limiting dilution to ensure that all future
transformed cells are derived from a single cell. The progeny of this host

211Biomanufacture
Procedure Information and testing
Step 1. Isolate and characterize gene
Step 2. Clone gene into expression vector
Step 3. Transform host cell with vector
Step 4. Clone host cell
Step 5. Select and expand cell to research seed
Transfer to development
Step 6. Produce master cell bank
Step 7. Produce working cell bank
DNA and gene
Source and derivation
Homology of sequence (genome database)
Molecular weight
Vector
Derivation, history, and map
Sequence
Selectable markers
Signal sequences
Cloning site
Cloned vector
Sequence
Map
Alignment of gene in vector
Host cell
Derivation and history
Purity and morphology
Raw materials (medium,
supplements, etc.)
Transformed host cell
Copy number
Stability
Expression gene
Purity and morphology
Cloned host cell
Purity and morphology
Copy number
Gene expression
Research cell bank
Purity and morphology
Copy number
Gene expression
Viability
Sterility
Master and working cell banks
Identity of host, vector, and gene insert
Viability, sterility, purity, and morphology
Vector copy number
Molecular markers
Annual testing of cell banks
Sterility and copy number
Expression and stability
Vector
Box II
Box I
Box III
Box VI
Box V
Box IV
Box III
Box II
Box I
Box I
FIGURE 6.4
Production and testing of a recombinant molecule in an expression system and production of
cell banks. This flowchart serves as an example of the steps that are taken in the early develop-
ment and biomanufacture of a recombinant molecule in an expression vector. The expression
system is constructed by genetic engineering and then produced as cell banks. Quality testing
is performed throughout the process.

212 Biotechnology Operations
cell are considered a research seed, and this seed is characterized for purity of
cell line, retention of vector, and other traits or attributes (Chapter 7).
Selection of the host cell is worth additional mention. There are many
species to choose from, and within a species, there are several strains, each
of which is well characterized. Bacteria, notably Escherichia coli, and yeasts,
such as Pichia pastoris, are common choices of prokaryotic host cells.
Host cell lines are purchased from a reputable source, such as the American
Type Culture Collection, or a biological supply house. These vendors main-
tain several strains or lines of cells, each with a full genetic history, and the
buyer expects and should receive only top-quality, highly characterized cell
lines. Much like pedigreed horses, cell lines and strains are noted for various
attributes, such as a posttranslational capacity or large yields of recombinant
protein, under specified conditions. In addition, they may also have known
limitations or deficiencies, such as slow growth or stringent nutrient require-
ments. Although several species and strains of host may be able to express
a given recombinant protein, there are caveats; thus, great care is taken in
selection of any expression system.
Bacterial Cell Expression Systems
Bacteria are often chosen as host cells because they express large quantities
of a wide variety of proteins very economically. Escherichia coli has been used
for decades because its genome has been sequenced, its laboratory strains are
plentiful and very safe, and this bacterium is receptive to accepting, hold-
ing, and expressing recombinant genes from vector plasmids. Escherichia coli
is often the first choice when biomanufacturing is considered. Within the
species of E. coli, there are many strains to choose from, and each strain has
particular attributes and advantages as well as disadvantages. For example,
some strains are best suited to secrete the desired recombinant protein into
the culture media during fermentation, which can simplify downstream
processing. However, production by E. coli can also have drawbacks. With
certain proteins, E. coli does not secrete but instead harbor protein internally,
within inclusion bodies. To obtain recombinant product from inclusion bod-
ies, cells must be split open, that is, lysed, which adds extra steps, compli-
cates purification, and possibly adds unwanted impurities to the product
stream. Protein from inclusion bodies may not be properly folded, neces-
sitating refolding steps. However, purification from inclusion bodies may be
easier and more productive than purification from cytoplasm.
Another disadvantage to bacterial expression systems is the inability
to make or correctly complete certain posttranslational modifications to a
recombinant protein. Bacteria do not add carbohydrates to proteins, as do
eukaryotic cells. Hence, if glycosylation is required for bioactivity, a bacte-
rial host cell may not be the best choice. Another issue with bacteria is the
production of undesirable contaminants, which are released into the process
stream. Gram-negative bacteria have certain molecules such as the cell wall

213Biomanufacture
component called endotoxin. If molecules like endotoxin cannot be readily
separated from the desired protein, then such organisms are not good can-
didate hosts. These examples demonstrate the importance of identification
of the proper host before beginning experimentation. Alone, this aspect of
planning may save considerable time and resources.
Yeast Cell Expression Systems
Yeasts are eukaryotic, unicellular organisms that offer both advantages and
disadvantages as host cells. Two species are commonly employed, but other
species are available. Saccharomyces cerevisiae, brewer’s yeast, is well charac-
terized as an expression host, as is P. pastoris, which has the advantage of
secreting recombinant proteins into the culture medium. Yeast cells grow
rapidly and economically in commonly defined medium, even in large ves-
sels up to 10,000 L or more, which can enhance protein expression scale-up.
Yeasts are very efficient in producing some recombinant proteins, and the
fermentation of yeast cells is usually inexpensive. Both yeast and bacteria
can be grown in the same types of fermentation vessel, and the equipment
is standard, reusable, and comparatively inexpensive. Yeast host cells are
available in many strains, allowing selection based on attributes. As with
bacterial host cells, and unlike mammalian cells, yeast growth medium is
very well defined, so there is little concern about the introduction of host cell
adventitious agents, such as human or animal retroviruses, with yeast cells.
In contrast to bacterial cells, yeasts have the capability to correctly add and
process many posttranslational modifications. Yeast strains commonly used
in fermentation are genetically engineered to be inducible. This highly desir-
able trait means that the yeast cells can begin to produce greater amounts of
a recombinant protein when a simple chemical, such as glycerol, is added
to the fermentation chamber or when an exact environmental condition is
established in the chamber. It allows for greater control of fermentation.
Hence, production in a yeast cell system provides an opportunity for pro-
duction of recombinant molecules.
Mammalian or Insect Cell Expression Systems
Mammalian or insect cell expression systems are increasingly selected by
sponsors for biomanufacture, especially for production of high-value human
recombinant proteins, such as monoclonal antibodies. Although transforma-
tion of a mammalian or insect cell line with a genetic construct can prove more
difficult, as compared with bacteria and yeast cells, these cell systems have
the advantages of accepting and expressing a large gene and completing most
posttranslational modifications. Because large proteins with glycosylation
(such as monoclonal antibodies) are common to the world of biopharmaceuti-
cals, mammalian cells are frequently chosen as an expression and production
system. However, as compared with yeast or bacterial cells, mammalian or

214 Biotechnology Operations
insect cells are often less robust and more fragile, grow more slowly and may
be more fastidious, thus requiring more stringent environmental controls.
They may require a continuous flow of complex medium to deliver nutrients
and may demand continuous waste removal. Concerns regarding the pres-
ence of latent virus in mammalian cell lines, specifically cells from a new and
poorly characterized cell clone that might harbor and then shed viral parti-
cles into the product stream, have slowed the introduction of new cell lines.
However, advances in mammalian and insect culture techniques and extraor-
dinary characterization efforts have overcome some of these difficulties, and
today, there are several effective cell bioprocessing systems in the market.
Several factors enter into the choice of a mammalian cell line intended for
protein expression. The ease of transfection with a particular gene or transfec-
tion technology, cell growth and protein secretion profile, and environmental
requirements, all enter into the decision. Hence, the key to selecting the cor-
rect cell line for the expression of a given gene is experimentation with several
highly regarded lines, which allows comparison with a particular construct.
Cells are named by their derivation. Cells of the epithelial origin are the most
used for biomanufacture. The mammalian Chinese hamster ovary (CHO)
cell, a cell line in use for more than 50  years, is high on the list of choices.
This cell line was widely used first in virology and cancer research laborato-
ries and later in biomanufacture. The CHO cells are very well characterized
and certified free of adventitious agents (with the exception of endogenous
retrovirus-like particles). Products derived from CHO cell production have
been used for decades without safety problems. Other cell lines chosen for
biomanufacturing are African green monkey kidney (Vero), Madin–Darby
Canine Kidney (MDCK), human embryonic kidney (HEK-293), baby hamster
kidney (BHK), human retinoblast (Per C6), and murine myeloma (NSO).
The process of establishing an expression vector, referred to as transfec-
tion, is outlined in Figure 6.5. The expression gene is isolated and cloned into
an expression vector by using methods described earlier in this chapter and
technically in the manner described for yeast and bacterial hosts. The method
of delivering that gene to mammalian cells, transfection, differs noticeably
from the methods applied to bacteria or yeast cell. One of the several transfec-
tion methods, most commercially available, may be used to transfect a gene
to a mammalian cell. Notably, the method must deliver the intended gene
directly into the nucleus and integrate it into the chromosome of the target
cell. Most transfection methods rely on chance, that is, the probability that a
gene will enter into the nucleus, integrate into the genome of a mammalian
cell, and result in a stable and productive transformed cell. In practice, this
means treating a large number of cells and using cloning and selection meth-
ods to determine which cells are stably transformed. Additional experimen-
tation is performed to characterize cell lines, and the best cell line is chosen
for expansion to become the transformed cell research seed (Figure 6.5).
Transfected insect cells are also used to produce recombinant proteins,
often times large quantities of proteins that could not be well expressed in

215Biomanufacture
Choose transfection method
Mechanical: Gene gun, electroporation,
and optical laser
Chemical: Calcium phosphate, liposomes,
and cationic polymers
Infection: Adenovirus and lentivirus
Others: Nucleofection and impalefection
Construct expression
gene (selection and
marker)
Transfection
Stable transfection
With matched cell
line+transfection
method+genetic construct
Genetic construct
Select stable
transformed
clones
Expand clonal
cells
Master cell
bank
Working cell
bank
Evaluation
Establish gene
Establish cell line:
Mammalian or
insect
Mammalian or insect
cell line
Expand cells
Established cell
line
Test:
Transient transfection
Evaluate transfection
Efficiency
Test expression level
Test:
Stable
transfection
Test:
Stable cell
line
Test:
Purity and
viability
FIGURE 6.5
Process of gene transfection for mammalian cellular expression. An established cell line is
selected and tested in the left panel, and a gene is engineered in the right panel. By using a
carefully chosen transfection method, the cells are transfected with the gene, and after evalu-
ation, these cells are used to produce the expression product.

216 Biotechnology Operations
other systems. The process of gene transfer to insect cells is quite differ-
ent from that applied to mammalian cells. The gene of interest is inserted
into the genome of baculovirus, a virus that normally infects insects, which
then acts as the delivery vector. The cell lines used as targets are derived
from insects and are thus free of potentially harmful human viruses but
are capable of hosting baculovirus. Insect cells are also desirable because,
like the mammalian cells, insect cells also perform complex yet accurate
posttranslational modifications of the expressed proteins. On infecting an
immortalized insect cell of a well-characterized cell line, genetic information
is transferred from the virus to the cell nucleus; some insect cells are stably
transfected. The transformed cells are then identified, selected, character-
ized, cloned, and expanded to produce a research cell seed. As the trans-
fected insect cell grows and multiplies, it expresses the recombinant gene of
interest and the gene product, a recombinant protein, is produced, which can
then be harvested.
Production of Master Cell Banks and Working Cell Banks
Research seed, described earlier, is transferred from the research laboratory,
where it is produced, to a development laboratory, where it undergoes addi-
tional examination and characterization. The complete history of the con-
struct, to include descriptions and sources of all raw materials and detailed
summaries of the procedures used to derive the seed, is archived as the
research seed history. Once development scientists are satisfied that the seed
is adequate for production of the intended product, master and working (or
production) cell banks (WCB) are manufactured.
Cell banks provide a uniform stable stock of cells with genetic inserts.
These stocks are available to production for future use. Cell banks include
the master cell bank (MCB), which is derived from the research seed, and the
WCB, which is derived from the MCB. To produce an MCB for bacterial or
yeast cells, a single clone of research seed is expanded in the culture and then
transferred for further growth in a shake flask or small fermenter, as outlined
in Figure 6.4. A limiting dilution step may be employed before transfer to
ensure that a single cell is indeed the ancestor of the MCB. The cells are har-
vested and counted, and specified numbers are aliquoted into several hun-
dred vials; these are labeled, cryopreserved, and placed into secure storage,
usually divided between two or more storage sites. The vials of an MCB are
the ultimate source from which the product is derived for decades to come.
Since an MCB is limited in the number of vials produced while demand for
MCB could be great, a WCB is produced from a vial of MCB. Procedures used
to produce and control WCB are very similar to those used to produce and
control MCB. Biomanufacturing uses stock from WCB, until it is exhausted,
and then another WBC is produced from a vial of MCB.
To ensure identity, purity, and safety of MCB and WCB, samples taken
immediately after production from both MCB and WCB are extensively

217Biomanufacture
tested, as outlined in Figure 6.4 and described in Chapter 7. Tests of MCB and
WCB samples are repeated at specified intervals (e.g., annually). This strin-
gent test regimen once again emphasizes the need to develop analytical tools
early in development, even before biopharmaceutical production begins. At
this stage, a significant resources have gone into the development of the con-
structs, MCB and WCB, as such they represent a precious commodity and
should be treated as such. Secure storage of all cell banks is critical. The pro-
cesses of producing research seed, MCB and WCB, are often both rewarding
and instructive to a new biotechnology operation and often represent their
first introduction to biomanufacture under cGMP. There is great satisfaction
in having completed the first stage of biomanufacturing by having produced
the foundation for later production efforts.
Biomanufacture of Recombinant Proteins
Planning Production of a Recombinant Protein
In a product-specific manufacturing plan, most processes have at least three
stages, and each stage is further divided into several technical steps, as out-
lined in Figure 6.1. We previously covered the steps of Stage 1. Now, we
will consider Stage 2, upstream processing, which involves the production
of recombinant protein in the expression system, and Stage 3, downstream
processing, which involves the purification of recombinant protein product
as BS. A good biomanufacturing plan goes beyond the initial process outline
and also considers facility, utilities, equipment, raw materials, quality con-
trol testing, staff requirements and compliance, or cGMP for both upstream
and downstream processing.
The initial attempt at biomanufacturing using a new process or for a new
product is referred to as pilot production. Pilot production involves per-
forming defined, sequential runs in an attempt to develop the process and
to eventually get it right; that is, to make a safe, pure, and potent product.
Indeed, pilot production is much like research experimentation because it
involves trial and error, tweaking various systems, and even making sig-
nificant changes in process protocols and procedures. Pilot production may
precede Phase 1 production, described above, or it may overlap or be syn-
onymous with Phase 1 production. The term run is used in biomanufacturing
to describe the performance of one defined process, such as all steps in the
upstream fermentation stage or a full set of process steps and fermentation
followed by purification, from beginning to end. It also demands repeat-
ability to confirm that the system is performing properly and consistently.
It is not unusual for a biomanufacturing operation to attempt a new pro-
cess in 5 or even 10 runs before it is considered reproducible and robust.
Hence, no matter what the biotechnology product is, pilot production can be
a long, arduous, and expensive endeavor, stretching over several phases of
development.

218 Biotechnology Operations
Upstream Process: Production by Bacterial or Yeast Cell Fermentation
Fermen tation is an ancient process, best exemplified by brewing of beer in the
presence of yeast. It is a skill that has developed over the ages. Substrates for
fermentation of biomolecules remain simple and include well-characterized
materials such as water, salts, and sugars. In some instances of biomanufac-
ture, more complex nutrients, such as soy extracts or vitamins, may be added
to the fermentation vessel. Animal materials such as liver powder or serum
supplement are used in the fermentation of a biopharmaceutical product only
in special circumstances where they are essential to the success of a process.
Such materials can harbor adventitious agents that can contaminate FP, and
thus, their use is discouraged.
Fermentation to produce biotechnology products is performed in a fer-
menter, a closed and sealed glass or stainless steel vessel with a series of por-
tals, stirring devices, and tubes entering the chamber (Figure 6.6). To begin
the fermentation process, one must have raw materials of the highest quality,
including a growth medium, gasses, a seed of recombinant bacterial or fungal
FIGURE 6.6
Equipment for microbial fermentation. This picture shows fermentation equipment in a bioman-
ufacturing suite. The operator in the center is programming the microprocessor controller in the
square unit. On either side of the controller stand two fermentation vessels, a small one in the
background on the bench top and a medium one behind the controller. In the foreground is a
large, cylindrical storage vessel made of stainless steel. (Courtesy of Waisman Biomanufacturing,
University of Wisconsin, Madison, Wisconsin. www.gmpbiomanufacturing.org.)

http://www.gmpbiomanufacturing.org

219Biomanufacture
organisms, and a means to control the process. Seed material, formed of bil-
lions of organisms that are capable of active division, is derived from a vial of
WCB that has been expanded in a flask containing the defined medium. This
is called the inoculum. The environment inside the vessel is controlled by
human intervention or, when on auto-pilot, by a microprocessor. The fermen-
tation process is initiated once all ingredients and the seed have been added
together in the vessel and the fermenter has been closed and sealed. Once
the environment inside the chamber is optimal, the cells replicate and are
active manufacturers of the recombinant protein product. The chamber is
typically stirred or otherwise agitated in an effort to evenly distribute gasses
(particularly oxygen), nutrients, organisms, and, if secreted, the recombinant
protein product. As stirring and movement of gasses may cause foaming,
addition of antifoaming agent, a chemical designed to reduce microbubble
formation, is often helpful. High shear stress and foaming contribute to pro-
tein denaturation and as such is avoided whenever possible. Cell growth and
product production are monitored by taking samples from the chamber, and
critical measurements, such as pH, gas tension, and osmolality are measured
by probes placed directly in the chamber. This in-process testing allows the
operator to follow the progress and correct variables if deviation from the
specified limits is required. For example, if pH drops out of range, sodium
hydroxide may be added to raise the pH, thus getting the fermentation sys-
tem back into an optimal pH range.
Division and growth are separated into several phases, as shown in
Figure 6.7. First is the brief lag phase, during which organisms adjust to the
culture environment. Next is the exponential growth phase, during which
organisms divide rapidly and hopefully produce large amounts of the
intended recombinant protein product. The deceleration phase represents
a slowing in growth, and the fourth phase is stationary, with little growth
or even increasing amounts of death. The final phase, decline, represents
reduction in metabolism and is indicative of large amounts of organism
death. Once measurements determine that the organisms have grown to
the required optical density, or that the death is extensive, or that sufficient
product has been produced, the fermentation is halted by radically chang-
ing the pH, by rapidly cooling the chamber, or by some other interven-
tion that is conducive for being gentle on the recombinant protein product
and facilitating high yields. It is important to identify the optimal growth
and harvest phases of the formation process. Termination of the growth
phase is important in an effort to maximize the production of the recom-
binant protein while reducing the number of unhealthy or dying organ-
isms. Dying organisms release destructive enzymes (e.g., proteases) and
debris or impurities into the medium; continuing a controlled fermentation
beyond that point can interfere with product purity or complicate down-
stream purification. The result of a successful microbial fermentation is a
vessel filled with slurry of organisms, debris, expended medium, and large
amounts of the intended recombinant protein product.

220 Biotechnology Operations
Certain cells, notably yeast used in some fermentation systems, may be
cued or induced to begin the production of recombinant protein. To engineer
an induction system, a gene or genes are inserted into the vector construct
for the purpose of controlling protein production by the expression system.
These inducible genes are active in the presence of certain environmental
cues or products. An example is selective induction of recombinant protein
production by P. pastoris on addition of glycerol to the fermentation vessel.
Upstream Process: Production by Mammalian or Insect Cell Culture
Mammalian or insect cells are cultured in a sealed chamber referred to as a
bioreactor. Although mammalian or insect cell culture has superficial resem-
blance to fermentation, the process is, overall, quite distinct. The objective
in both systems is to produce a recombinant protein product that is either
stored within the cells or secreted into the medium. Both bioreactors and fer-
menters are closed and sealed systems with a high level of monitoring and
environmental control. Mammalian or insect cells typically demand more
complex substrates than bacteria and yeast. Hence, cell culture media used
in a bioreactor contains a complex mixture of nutrients and vitamins. When
animal products are used for biopharmaceutical production, in fermentation
or cell culture, they must be carefully tested and controlled, so as to ensure
Stationary
Lag
Growth
(exponential)
Decline or
death
Deceleration
Time
C
el
l n
um
be
r/
m
L
FIGURE 6.7
Phases of microbial growth in fermentation.

221Biomanufacture
that potential microbes and other impurities do not contaminate the cells or
the product. Typically, mammalian or insect cells must be grown as adherent
cells. This is because, unlike bacteria or yeast, these cells in nature exist in a
tissue or an organ, where cells are interconnected and held firmly to a base-
ment membrane or other connective tissue protein. Alternatively, hollow
fibers, convoluted vessel surfaces, and microcarrier beads, sometimes coated
with collagen or other connective tissue matrices, may be used to create opti-
mal microenvironments, facilitating cell anchoring or adaption to culture
conditions, yet confining cells to a small area in the absence of mechanical
stresses. Mammalian cells engineered to grow without anchorage are grown
in suspension, but agitation or stirring is exceptionally gentle, because insect
and mammalian cell membranes are fragile. Gentle air movement or wave
action is used in some systems to maintain the requisite movement of the cell
medium for suspension cultures. Mammalian cells are particularly suscep-
tible to reduced growth because of low oxygen tension, high carbon dioxide
tension, buildup of waste, changes in pH, and other metabolic-environmen-
tal influences. Cell bioreactors are closely monitored for temperature and
the addition of gases, buffers, and nutrients is highly controlled, both by the
operator and by microprocessors. The vessel environment of a cell bioreac-
tor is monitored using specialized probes and microprocessors by operators;
adjustments in gasses, buffers, and nutrients are efficiently achieved using
aseptic ports in the vessel.
Mammalian and insect cells demonstrate growth curves, which represent
cell growth as a logarithmic or semilogarithmic phase, followed by a pla-
teau phase, and finally a decline phase. Although the growth and protein
secretion of mammalian cells are typically slower than that seen in bacte-
rial or fungal cell fermentation, under well-controlled operating conditions,
the growth and protein secretion of mammalian cells may be sustained for
much longer periods. A cell culture is terminated at an exact point in the
growth and protein production cycle, so as to maximize protein production
and minimize contaminants. The result of successful cell culture is a bio-
reactor vessel filled with a slurry composed of cells, cell debris, expended
medium, and the intended recombinant protein product.
Upstream Process: Recovery
Immediately on stopping cell growth in a fermenter or bioreactor, the mate-
rial is harvested, chilled, and the cells and other large solids separated from
the liquid phase. This is done by moving, with pumps, the contents of the
chamber into a capture vessel. In the case of cells anchored to the substrate,
or when product is contained within the cells, it may be necessary to dis-
lodge the cells by mechanical or enzymatic means. If most of the product is
harbored in the cells, as would be the case with protein that is not secreted,
the cell paste is retained and the supernatant is discarded. Whole cells con-
taining product are then lysed using a single or a combination of methods,

222 Biotechnology Operations
which may include mechanical methods (e.g., sonication and homogeniza-
tion), chemical methods (e.g., lysozyme and detergent), and/or shock (e.g.,
freeze-thaw cycles and water). Soluble proteins are then harvested, again
traditionally, by using a combination of centrifugation and filtration meth-
ods. Primary clarification, to remove any remaining cells, cell debris, and
other large solids, is performed by centrifugation or tangential flow filtration
(Figure 6.8). The resultant filtrate or supernatant containing the recombinant
protein is then kept in a storage tank under controlled conditions, until it is
purified. The storage step is referred to as a hold.
Downstream Process: Purification
No matter what the source—fermentation, cell bioreactor, transgenic ani-
mals, or plants—recombinant proteins and other biological molecules must
be purified from typically a complex milieu of cellular debris, impurities,
and contaminants. By way of definition, impurities are undesirable materi-
als, both particulate solids and soluble molecules, that remain with product
after production. Common impurities derived from biological processing are
host cell proteins or DNA, endotoxin, or other microbial toxins; cellular debris
and organelles; and materials from the culture media. Contaminants are the
substances that enter the product stream, often during purification, and are
frequently shed or leached from the process materials or equipment. Small
particles from tubing, glass, or metal containers, chromatography gels, and
heavy metal ions, leached from metal containers, are the examples of contam-
inants. Impurities or contaminants may be debris, suspended particulate, or
soluble in nature. Both of them are considered necessary evils, because their
presence reflects the contents and environment of the culture that yielded the
product. However, levels of impurities and contaminants are greatly reduced
Pump
Membrane
cassette
PP
Pf
Pr
Permeate
Retentate
(product)
Feed
Permeate tank
Batch feed
FIGURE 6.8
Purification scheme for a recombinant molecule. This is a classic purification scheme, or down-
stream process, for a recombinant protein and includes precipitation, centrifugation, filtration,
and multiple chromatography steps, yielding bulk substance.

223Biomanufacture
during purification, and, in the end, biotechnology products are tested for
common impurities and contaminants (Chapter 7) to ensure that the levels
meet predefined specifications and the product is deemed to be safe.
Purification steps are designed to remove one or more impurities or con-
taminants, at least to the greatest extent possible, and yet retain the desired
recombinant protein, or other biologically active molecule, thus maximizing
the yield of product. Yield is especially important because a low yield of a
very pure product is as unacceptable as a high yield of the product with sig-
nificant levels of impurities. Hence, in-process testing (Chapter 7) is applied
throughout purification to ensure improvements in purity and maintenance
of yield at every step. The field of biomolecular purification has progressed
rapidly in recent decades and dozens of methods, some simple and others
quite complex, have been developed and have entered the market. We will
mention a few of the most commonly used methods.
To plan downstream processing, a purification scheme is produced
(Figure  6.9). To do this, it is first necessary to understand the biophysical
and biochemical properties of the recombinant molecule, because purifica-
tion methods take the advantage of those properties. This understanding is
based on experimental data, derived from the research laboratory, about the
product and the nature of that product as it enters purification. Knowledge
of the possible contaminants and impurities and their properties is also
needed. For example, it is critical to know the isoelectric point of the desired
molecule under given conditions, the pH or the salt concentration at which
the molecule precipitates from solution, the size and shape of the molecule,
the glycosylation profile, or any propensity to bind to other molecules or
inert substrates. Purification schemes, as shown in Figure 6.9, take advan-
tages of these characteristics or attributes.
Many technical methods, or purification tools, are available for down-
stream processing. Choice and application of a method are based on the
nature of the molecule and the knowledge that certain tools have been suc-
cessfully used in the past to purify similar molecules. Some purification
tools are quite simple and inexpensive, whereas others require significant
investment. The sequence in which methods are applied is as important as
the choice of the tools themselves, and the downstream plan must design
their use very carefully. It is often necessary to test each purification method
alone and the full sequence of methods, at small scale in the laboratory, in
an effort to derive the optimal sequence of events, before beginning opera-
tional purification. In-process tests are another critical component of a puri-
fication plan. These are developed to ensure that materials such as solutions
are of the correct composition, pH, or strength and can be effectively used
to measure the levels of the desired molecule and impurities throughout the
purification process. These assays, which can require expensive instrumen-
tation and extensive development efforts, must be available from the outset
of purification process development, because they are essential to the under-
standing of the purification outcomes.

224 Biotechnology Operations
Cell supernatant
or cell paste
Precipitation
Centrifugation
(clarification)
Tangential flow
filtration Depth filtration
Size exclusion
chromatography
Ion exchange
chromatography
Affinity
chromatography
Ultrafiltration
Bulk substance
Process
SDS-page
Total protein
SDS-page
SDS-page
activity
Total protein
HPLC
SDS-page
activity
Total protein
HPLC
SDS-page
activity
Total protein
HPLC
SDS-page
activity
Total protein
HPLC
BS panel of tests
Control testing
FIGURE 6.9
Tangential flow filtration. Material enters the filtration scheme from the batch feed, usually
a storage vessel (holding tank), and it is pumped under pressure (feed) across a membrane
cassette, where some material of the correct molecular weight passes through the membrane
cassette as permeate and is then held in a tank. Material that does not pass through the cas-
sette re-enters the batch feed tank and is again pumped across, and in some cases through,
the membrane cassette. Continuous flow across the membrane cassette deters clogging the
cassette selective filter.

225Biomanufacture
Early in downstream processing, and often at other stages in biomanu-
facture, the liquid fraction must be clarified, without significant loss of the
desired protein, using combinations of precipitation, centrifugation, and fil-
tration. Precipitation is a simple and inexpensive application; however, many
methods for precipitation, such as changing the pH or adding simple salts,
may either degrade the protein of interest or add contaminants to the prod-
uct. Precipitation is based on the knowledge that a desirable protein or an
undesirable impurity becomes insoluble under certain conditions such as
low pH or high salt concentrations. Once a precipitate is formed, it is sepa-
rated from the undesirable materials by centrifugation or filtration. Desirable
molecules in the precipitate are recovered by diluting the precipitate with
physiologic buffer. Undesirable proteins in the precipitate can then be dis-
carded. An example of precipitation is purification of an immunoglobulin
on addition of buffer with a high salt concentration. Immunoglobulin pre-
cipitates in this environment, leaving impurities in the supernatant. This is
centrifuged, and the pellet is recovered and diluted with normal saline to
again solubilize the immunoglobulin protein. Another example is the appli-
cation of a polycationic agent, such as polyethyleneimine, which precipitates
undesirable nucleic acids; however, the desired recombinant protein remains
in the solution. After centrifugation, the pellet with impurities is discarded
and the supernatant is retained or vice versa, depending on which fraction
holds the desired product.
Centrifugation, a relatively simple and often effective method, is employed
whenever possible and often constitutes the first step in a purification scheme.
It separates materials based on density, shape, and other physical properties
that impact their gravitational movement in a fluid. It can be used without
other treatments, such as in effective separation of large impurities (e.g., cell
walls and nuclei), from smaller particles and soluble proteins. Centrifugation
is also used in a step-wise manner to sequentially remove matter of different
density. It is often applied in conjunction with other applications such as pre-
cipitation. Centrifugation equipment is available in many designs and range
from small instruments to large, continuous-flow machines that can process
large volumes of product.
In addition, flow filtration or tangential flow filtration are the methods used
in purification schemes. Both methods remove debris and clarify a solution
in which the recombinant protein is suspended. Flow filtration involves pass-
ing the material through a selective membrane filter, a synthetic sheet that
has holes of a specific size. As solution is pushed against the filter, solids of
that size or less move through the membrane and larger particles are trapped
atop the membrane. However, a disadvantage of flow filtration is the buildup
of material on the membrane surface, which can clog and foul the filter. Some
filtration protocols, therefore, use a series of flow filtration filters. The fluid
stream is first fed through filters with larger membrane holes. Then, in a
series, it is fed through filters with smaller holes, thus distributing particles
over many filters and avoiding fouling and clogging of a single filter.

226 Biotechnology Operations
Tangential flow filtration (TFF) is a more expensive, but often more effec-
tive, method and is also a choice for processing larger volumes. In TFF, solute
passes over the filter in a horizontal stream, even as filtration is happening
in a vertical plane (Figure 6.10). This horizontal movement constantly sweeps
debris off the filter surface and prevents clogging. As it can be performed rap-
idly, TFF is often used to exchange solutions, such as one buffer for another
and to selectively remove low-molecular-weight impurities, all in a single step.
Although extremely useful, filtration must be applied judiciously, because
under some circumstances it may also destroy molecular integrity. Filtration
causes shearing forces as the fluids move under pressure across or against
a membrane and shear can destroy cells or molecules. (Each product has a
unique tolerance for shear.) The only way to fully realize the effects of shear
on a given molecule is through experimentation followed by characteriza-
tion of the desired molecule. Movement of fluids rich in proteins may create
foaming, an indicator of protein denaturation, which is an undesirable out-
come of any biomanufacturing process and must be avoided or countered.
A third cautionary note is avoidance of adsorption of the desired molecule
to surfaces of equipment, transfer tubes filtration membranes, and even to
Fraction collector
Filter/bubble trap
Pump A
Pump B
Load
Buffer A
Buffer B
Regen
Detector
Chromatography
column
Controller
FIGURE 6.10
Flow diagram for preparative chromatography. This scheme depicts the equipment and flow
for a chromatography system used as one step in the purification of a recombinant protein.
The product (load) and Buffer A are pumped into the column via pump A, where the gel
matrix of the column binds or otherwise slows the progress of the molecule of interest (e.g.,
through affinity binding, size exclusion, and ionic interaction). Other molecules pass through
the column and are detected and collected into fractions. Once this has been completed, Buffer
B is pumped onto the column, with the intention of releasing the bound molecule. Thus, the
desired product is detected as it comes off the column, and it flows into later fractions, where
it is collected.

227Biomanufacture
impurities. Again, any purification step must provide consistent and useful
yield, and adsorption can greatly reduce the amount of desirable cells or
proteins left in the product stream.
Chromatographic methods are used in most biomanufacturing purifica-
tion schemes that involve molecular purification. Chromatography is based
on various properties of proteins and other macromolecules: charge, size,
shape, or affinity to a substrate. Preparative chromatography is used to
purify significant amounts of materials, whereas analytical chromatography,
described in Chapter 7, is used to characterize macromolecules. Preparative
chromatography, the subject of this discussion, is performed using aque-
ous suspension of resins or gels packed into a vertical column (Figure 6.10).
Preparative chromatography columns come in various sizes and shapes
(Figure 6.11) to suite the intended purpose, with some exceeding the vol-
ume of household refrigerators. They are controlled with pumps, valves,
and microprocessors. Each column-and-resin chromatography system has
a unique set of properties that allow for the differential separation of mol-
ecules, based on the physical or chemical properties of the molecules to be
FIGURE 6.11
Equipment for preparative chromatography. Two preparative chromatography columns rest
on tripod supports. On the table top are controller units and pumps with tubing that lead
to the glass columns. This is performed within a chromatography suite of the biomanu-
facturing facility. (Courtesy of Waisman Clinical Biomanufacturing facility, http://www.
gmpbiomanufacturing.org.)

http://www.gmpbiomanufacturing.org

http://www.gmpbiomanufacturing.org

228 Biotechnology Operations
separated. In the simplest chromatography protocol, clarified supernatant
containing both product of interest and impurities is placed at the top of
the column. Then, using gravity or a pumped stream of buffer, the super-
natant is passed through the column. As this fluid passes over the column,
the molecules contact the resin and may or may not bind to the column. As it
passes out of the column, it is collected in a series of tubes, each fraction rep-
resenting a specific volume and time of collection. By eluting with various
buffers, a gradient is established on the column and desirable proteins leave
the column in one fraction, whereas undesirable proteins exit the column in
another fraction. This process is shown by simplified format in Figure 6.10.
Chromatography uses distinct molecular properties to separate one mol-
ecule from another. For example, ion-exchange chromatography uses the
charge properties of various molecules to separate desirable from undesir-
able proteins. Here, the resin has a known electrical charge at a given pH and
ionic strength (salt concentration). Elution buffers added to the chromatog-
raphy column may be changed by the operator over time. Each buffer has a
given salt concentration and pH. Taking advantage of the ionic properties of
both the gel matrix and the desired protein, the buffer strength and the pH
of the elution buffer are adjusted to ensure that the target protein binds, by
ionic interaction, to the ion-exchange resin. For example, at pH 7.0 and ionic
strength of 100  mM (Figure 6.10, Buffer and Pump A), a recombinant pro-
tein might bind to the resin; however, impurities pass through the column,
to be collected in the early factions. Next, a second buffer and Pump B in
Figure 6.10, of pH 6.9 and ionic strength of 150 mM, is added to the column
to release the recombinant protein, and so on. Eluate is collected in later
fractions, and some of these contain product, largely free of impurities.
Many other types of chromatography are available to the biomanufactur-
ing operator. Hydrophobic interaction chromatography takes advantage of
a molecule’s affinity for or, alternatively, rejection of water. To purify hydro-
phobic proteins, a gradient with high-to-low gradient of salt concentrations
are established in a column containing hydrophobic interaction resins. Size
exclusion chromatography takes advantage of the size and/or the shape of
a molecule. It is particularly useful to purify proteins of interest if they are
particularly large or small or have an unusual shape. Affinity chromatog-
raphy methods employ ligands, attached to chromatography resins, to cap-
ture the desired protein as it passes through the column in the presence of
physiological buffer. For example, Protein A resins are commonly used to
retain monoclonal antibodies to a column. The protein A molecule, derived
from bacteria, naturally sticks to antibody molecules. When Protein A is
immobilized on a resin and placed into a column, any antibody passing over
resin will, in physiological buffer, adhere to the Protein A while impurities
pass through the column. In the second step, a buffer solution, known to
force Protein A to release the antibody by molecular or ionic competition, is
passed over the resin. Now, monoclonal antibody, without impurities, elutes
into the subsequent fractions.

229Biomanufacture
Another purification method is the use of tags in affinity chromatography,
with polyhistidine tag being quite popular. Here, the protein of interest must,
in research or early development, be genetically engineered to have at the
C- or N-terminus a series of nucleotides that repeatedly code for histidine.
Polyhistidine tag chromatography is an affinity method in which the chroma-
tography resin immobilizes nickel ions. As the protein harboring the polyhi-
stidine tag passes over the column, it binds to nickel, while other impurities,
without the tag, pass through the column. The bound his-tagged protein
is then conveniently rinsed with several volumes of buffer. Subsequently, a
selective elution buffer is passed through the column, triggering the release of
protein from nickel. More sophisticated tags may include a cleavage sequence
to facilitate the release of the tag from a therapeutic protein. Although provid-
ing an efficient purification method, the use of tags is discouraged by regu-
latory agencies, as they introduce an added impurity with the capability of
influencing the protein structure and function or elicit an immune response
if used in the production of a therapeutic protein.
The pharmaceutical industry commonly employs the use of two indepen-
dent chromatographic methodologies in tandem (e.g., size exclusion followed
by ion exchange) as a common purification technique, which result in a rela-
tively pure protein product with minimal impurities. More chromatographic
methods are available and still others have been developed to purify spe-
cific biomolecules. For some molecules and even for living cells, in situations
where readily available purification methods are not useful, a new and very
product-specific chromatographic method such as affinity chromatography
is often developed out of necessity.
Purification is a lengthy process, as the tools are applied over days or even
weeks. Pauses, referred to as holds, in a series of events are commonly incor-
porated into process schemes to allow for in-process testing and operating
staff breaks in schedule. However, pauses require product storage, and stor-
age can result in product degradation. Therefore, pauses must be carefully
planned, controlled, and monitored. During a hold, the product may be
susceptible to degradation as a result of impurities such as proteases in the
material, to other influences of the hold environment, such as oxygen or pH,
or even to the container surface, which acts as a catalytic agent. In general,
greater lengths of storage time and higher storage temperatures accelerate
product degradation. To prevent degradation, it is important to understand
the contributing factors along with remedies to allow for planned and con-
trolled holds, in order to prevent degradation of the product. For example,
protease degradation is reduced by storage in various buffers or addition of
protease inhibitors, substances that are inherently safe and can later be sepa-
rated from the product. Oxygen tensions can be adjusted, antioxidants can
be added, or containers may be lined with inert materials to prevent product
breakdown. Planning each process and hold step is based on the knowledge
of the product, possible impurities and contaminants, and the product’s sta-
bility profile.

230 Biotechnology Operations
The end result of purification efforts is BS, a pure, potent, and stable prod-
uct within the proper bulk container. For a biopharmaceutical, this is referred
to as bulk (drug) substance.
In summary, purification processes are planned on the basis of the prop-
erties of the product and possible impurities or contaminants. Success at
purification frequently involves technical planning based on the product’s
characteristics and then experimentation and trial and error in the research
laboratory. Purification is first attempted at small or model scale, to better
understand the attributes of each application. The biomanufacturing opera-
tor does not expect to get it right the first time. Indeed, it may be shown that
a purification tool or a series of methods, intuitively good choices, negatively
impact the molecule of interest; or it may be discovered that product yields
are unacceptably low. If the negative impact is irreversible and the protein
cannot be recovered to the native form, then the operator might drop that
step and try another. Alternatively, the method can be modified. A third
possibility is the application of a recovery step that intends to return the mol-
ecule to its native or desired state. In reality, it may be necessary to apply sev-
eral tools and determine, experimentally and by trial and error, the impact of
each tool, before the correct process or formula is discovered. Again, success
in this endeavor is based on the knowledge of the protein and on the fact that
various tools are available to the operator.
In-Process Testing and Analysis of Bulk Substance
In-process testing is a hallmark of product purification. The operators need
to know, at each step, whether their purification scheme is achieving the
intended objectives of removing impurities and contaminants while enrich-
ing the desired product, without significant product loss. Hence, quality con-
trol (in-process testing) is applied to samples taken at the completion of each
step. More information is provided in Chapter 7 about the individual ana-
lytical tools commonly applied for in-process testing of biopharmaceuticals.
Examples are measurements of product, particles, contaminants, or impuri-
ties. At each step, the operator is interested to learn whether the product
remains in the stream and, if so, to identify its molecular integrity. Relatively
rapid methods, for example, examination and measurement of protein bands
after sodium dodecyl sulfate polyacrylamide gel electrophoresis of a sample
quickly provide information, both qualitative and quantitative, about the
yield of both the intended protein and the contaminants and impurities at
each step in the process. These tests must be readily available to a biomanu-
facturing operator.
Both quantity and quality of a BS matter greatly to the manufacturer.
Quality control test results of BS must demonstrate that product, at this stage,
possesses all intended attributes. In Chapter 7, we will discuss the tests used
to measure those attributes. Each test is classified under the attribute it mea-
sures: identity, safety, purity, and potency. Purity is of particular importance,

231Biomanufacture
because it is a key objective of downstream processing. However, in a general
sense, how do we define purity of a molecule such as a recombinant protein?
One guideline often applied to biopharmaceuticals is that more than 95% of
the BS is the intended and intact ingredient and less than 5% of the BS are the
known or unknown impurities and contaminants. Most biomanufacturing
operations strive for more than 98% or greater purity, certainly for commercial
manufacture. However, there are caveats to this purity guideline. First, the
balance of material in BS, the remaining 2% or 5% if you will, must be known,
indeed be characterized, for it cannot be toxic or allergenic or potentially toxic
to the user and it must consist of various materials without a predominant
molecular entity: impurity or contaminant. Second, it is not always possible
to reach the 95% purity level, and in such instances, it may be acceptable to
identify impurities and show that they cannot be harmful to the product or
the user.
Knowing what could be or what should not be in BS is helpful in making
these determinations. For example, a recombinant protein product derived
from bacterial host cells might be expected to have very small or trace
amounts of bacterial chromosomal or plasmid DNA, and it would be tested
for such impurities and measured against a specification for host cell DNA.
In addition, in-process testing focuses on the materials that are there and
those that could be there but not on those that are highly unlikely to be there.
For example, the bacterial product would not be expected to have mamma-
lian or yeast cell DNA, and so, the operator would not test for DNA from
eukaryotes. In-process testing will, in part, help the operator to understand
the makeup of material and to pinpoint the step at which it entered, or was
not fully eliminated from, the product stream. Understanding the potency
of product is another critical step in characterizing BS, and at this stage of
manufacture, there must be either an indirect or a direct measure of potency.
Recombinant proteins are not the only biotechnology products produced
by biomanufacturing technologies. The following paragraphs provide by
way of example an overview of possible manufacturing approaches for a
few of the many other biotechnology products.
Production of Bacterial Plasmid DNA
Bacterial plasmid DNA, used in DNA vaccines, genetic (DNA) therapeutics,
diagnostic tests, or as research laboratory chemical, is produced by bacterial
fermentation. Once purified, biomanufacturing processes may yield up to
1 g of plasmid DNA per 10 L fermentation. RNA can also be produced, albeit
in milligram quantities, by in vivo transcription in E. coli. To produce DNA,
a plasmid vector is constructed in the research laboratory and tested for the
intended biological effect. An appropriate cell line of E. coli is transfected
with the DNA plasmid and cell banks are produced from research seed.
Beginning with WCB, cells are grown in a fermentation vessel and then har-
vested and lysed to release supercoiled plasmid DNA, the intended product

232 Biotechnology Operations
for most purposes. The plasmid DNA is then purified using physical separa-
tion and chromatographic methods. Impurities, such as chromosomal DNA,
RNA, and bacterial host cell proteins must be considered during purifica-
tion. During all processing, the plasmid product must remain supercoiled,
that is, in the closed circular form that coils about itself. Quality control test-
ing focuses on identity, purity, and identification of minor impurities and
potency in a relevant biological assay for all DNA products. The form of
the plasmid (linear, circular, relaxed, or supercoiled) is also determined by
analytical testing.
Production of Live Recombinant Organisms: Bacteria and Virus
Live virus, bacteria, and even protozoa are used as biopharmaceuticals or
as diagnostic or laboratory reagents. Virus, for example, retrovirus, may be
used in gene therapies to transfer therapeutic genes to patients. Owing to
their efficient ability to infect cells, live viruses such as vaccinia, adenovirus,
and alphavirus are often employed as delivery vehicles for vaccines. Live
bacteria are used as investigational biopharmaceuticals, both therapeutically
and as vaccines. Even protozoa, such as attenuated malarial sporozoites,
serve as live vaccines.
There is considerable experience in bacterial and viral culture and puri-
fication, largely for vaccine production, yet there are potential issues with
some viral or bacterial constructs. One concern is the possible release of
live recombinant virus or bacteria into the environment or spread to close
contacts. Another concern is whether these microbes retain the capacity to
infect or cause diseases in humans, plants, or animals. To address these
issues, live organisms are carefully designed and endowed with redundant
systems engineered into their genome. Some systems ensure they cannot
survive outside the environment provided by cell culture or a living host
organism. Other systems delete the genes responsible for replication, infec-
tion, or pathogenicity, rendering them incapable of causing disease. Further,
safety issues related to exposure and release are considered in manufac-
turing plans and process controls. Extensive safety testing is performed to
measure the attributes of organism in research seeds, and stringent specifi-
cations are applied.
These recombinant organisms are constructed in the research laboratory by
using well-characterized, attenuated strains of virus or bacteria. Live bacteria
or viruses (Figure 6.12) are grown by fermentation and cell culture, respec-
tively. Since live virus is propagated in cultured cell lines, it is necessary to
identify a virus host cell, often of human origin, which yields large quanti-
ties of viral particles and is free of adventitious agents or other undesirable
traits. This requires significant resources, because the number of satisfactory
choices is limited, and the processes of adapting virus to the host, cell bank-
ing (both MCB and WCB), and testing for identity and purity are lengthy
and expensive. Cells are first grown to optimal cell density in a bioreactor

233Biomanufacture
Viral activity
Number particles
Surface protein
Certificate of analysis for
BDS
Certificate of analysis for
BS
Cell substrate
(Diploid cell and
culture collection)
Viral product
Virus research seed
Cell control
Characterization
Viability
Identity
Purity
Virus clonal selection
and expansion
Viral MCB and WCB
(virus seed)
Grow virus on cell
Substrate harvest cell/
supernatant
Cell lysis
Virus separation
(centrifugation and
filtration)
Whole virus
Purification
(centrifugation and
chromatography)
Killed or inactivated
whole virus product (BS)
Master cell and
working cell banks
(MCB and WCB)
Live purified virus (BS)
Viral subunit
Process
Viral activity
Number particles
Surface protein
Purity
Viral activity
Number particles
Surface protein
Viral activity
Number particles
Surface protein
Viral activity
Number particles
Surface protein
Chemical
inactivation
or kill
Viral
disruption
Viral activity
Number particles
Surface protein
Viral activity
Number particles
Surface protein
Certificate of analysis
for BS
Control testing
FIGURE 6.12
Production and preparation of virus (live, killed, or subparticle).

234 Biotechnology Operations
or in large flasks. Cell growth medium is both well defined and well char-
acterized to prevent contamination or cross-contamination of cultures with
adventitious agents. Raw materials from undefined or animal sources, such
as serum, are generally unacceptable as media supplements. Virus from WCB
is inoculated onto cells and, after a period of incubation and viral replication,
the cells are harvested, lysed, and then virus is separated from large debris
by centrifugation or filtration of the medium. It is then purified using density
gradient centrifugation or selective filtrations, to separate viral particles from
impurities such as cell debris and culture medium. Virus of high purity, BS,
results from the process, and this is extensively tested for identity, safety, ste-
rility, purity, and potency. Potency tests are developed to reflect the intended
use of each product. Virus may be examined for desirable traits, those that
enhance the intended mechanism of action. For example, if a vaccine must
first attach to epithelial cells to be immunogenic, testing might examine the
virus for the ability to bind that receptor. Hence, both potency and safety
tests are often complex assays, many immunological or molecular and others
performed in tissue culture or using live animals.
In contrast to virus, live bacteria are propagated from bacterial WCB as
recombinant biotechnology products. These are grown in defined medium,
preferably without animal-derived supplements and in the manner described
earlier for fermentation of recombinant proteins. Bacterial cell growth and
expression are monitored for amount and for desirable traits or attributes,
often identified in the research laboratory. The cells are harvested in a man-
ner that retains viability and are processed further to remove impurities,
such as dead cells, cell debris, and components of the medium. Purification
of live bacteria is based largely on physical separation with the help of cen-
trifugations, washes, and filtrations. Bulk substance consists of live bacterial
cells held in a physiological medium and perhaps with a cryopreservative,
because they will likely be stored in the frozen form. Testing protocols
are developed to ensure that traits are retained throughout the processes.
Identity, safety, purity, and potency testing are also completed. Potency test-
ing may include the ability to express an antigen or attach to a cell substrate.
Production of Products Composed of Mammalian
Somatic Cells or Tissues
With the advent of tissue and cell replacement therapies and the new era of
regenerative medicine, biomanufacturing operations developed methods to
expand somatic tissues and cells. While selected individual cell types, such
as those used in laboratory tissue culture, had been produced for decades,
the growth of somatic cells and tissues intended as replacement therapies in
human subjects present new manufacturing challenges. Today, autologous
tissue regeneration and replacement is a growing biotechnology industry
and a quality system suited for this technology, Good Tissue Practice, has
been developed as regulatory guidance.

235Biomanufacture
Replacement of knee joint cartilage provides one example. The objective is
to grow, in vitro, healthy autologous cartilage that can be used to replace dis-
eased cartilage in a joint. To begin, a piece of cartilage is surgically removed
from a healthy joint of the patient and the cells from this healthy tissue are
transferred to a biopharmaceutical production facility. Here, the cartilage cells
are expanded on an inert biological matrix to confluence and form a sheet of
cultured cartilage cells. This sheet of cells is returned to the surgeon, who then
implants it into the damaged joint. While this product does not utilize recom-
binant technology, it does apply the biotechnology practices of cell selection or
isolation, purification, cell culture expansion or growth production, and quality
control testing that accompanies a complex biomanufacturing process. Many
technical and quality hurdles have been overcome, largely through planning,
proper application of the existing technologies, and invention of new methods.
This approach has also been used to illustrate another example, growth
of autologous epidermis, new skin for patients subjected to severe burns. A
flow diagram for skin production is shown in Figure 6.13, in which major
Donor (patient) epidermal
cells
Dermal substrate
Adventitious agents
Viability
Bioburden
Quality of collagen
and fibroblasts
Mature epidermal tissue
(BS)
Certificate of
analysis for BS
Process
Viability
Purity
Identity
Biological function:
Viability
Histological analysis
Barrier function
Animal collagen
matrix
Downstream
control testing
Upstream
control testing
Epidermal cell
purification
Seed epidermal cells on
dermal substrate
Propagate keratinocytes
on dermal substrate
Stratified epidermal
tissue
Fibroblasts
FIGURE 6.13
Production of epidermal somatic cells and skin tissue.

236 Biotechnology Operations
attributes and production steps are highlighted. The seed tissue is taken
from an unaffected area of the skin of a burn patient. This critical raw mate-
rial must be of high quality. Culture methods encourage rapid and consis-
tent growth of skin tissues on a matrix or artificial membrane, to the stage
of confluence, thus achieving a sheet of tissue two to three cell layers thick.
Scrupulous aseptic technique is applied at every step to ensure a safe and
sterile product. Unique quality control methods focus on attributes of this
product. For example, measurements of skin tissue tensile strength ensure
that the tissue can be transported and then surgically sutured without tear-
ing. Identity testing confirms that donor skin sample matches exactly the
skin tissue yielded at the end of manufacturing. The outcome is a biomanu-
facturing system producing a high-quality skin or a tissue product able to
close horrific burn wounds, achieve homeostasis, and alleviate patient suf-
fering and provide new market opportunities.
Production of Cellular Products Derived from Pluripotent (Stem) Cells
The biomanufacturing community has rapidly evolved to produce prod-
ucts from pluripotent cells (i.e., stem cells); however, there remain many
unknowns regarding the clinical feasibility of this technology. The following
overview of technologies available for production and testing of differenti-
ated cells from pluripotent cells gives one an idea of how biomanufacturing
might be performed in the future. More times than not, groundbreaking sci-
ence and dire clinical need are what drive the rapid evolution of technology
to accommodate the applications of new cellular therapies from benchtop
to bedside. One fairly recent example of rapidly evolving technology is the
development of induced pluripotent stem cell (iPSC) methodology, which
was initially discovered in 2006. Induced pluripotent stem cells are spe-
cialized adult stem cells reprogrammed either chemically or genetically to
a more undifferentiated and stable stem-cell-like state. These specialized
cell populations are essentially coaxed into dedifferentiating from what
appeared to be a committed state into a less-defined developmental state,
which is better equipped to promote tissue repair or organ-specific regenera-
tion. For example, the seemingly most appropriate cellular therapy product
for a heart indication would be to use progenitor heart cells (iPSC-derived)
destined to become a cell type specific to the heart (e.g., cardiomyocytes)
rather than using stem cells isolated from bone marrow, which rely on micro-
environmental cues when delivered to the injured heart to facilitate heart
muscle regeneration.
Although iPSC technology is nearing evaluation in the clinic, many
issues have yet to be better understood or resolved to ensure that a reason-
ably safe product will be used in human clinical studies. One such issue
is the challenge of controlling and characterizing the differentiation state
of the cellular product; the desire is to have a homogenous cell popula-
tion, which has been difficult to achieve. Although much improvement

237Biomanufacture
has been made, this inefficient programmed cell differentiation is much
less than 100%, which raises concerns about the multiple cell populations
and the potential variability associated with every differentiation process.
The differentiation inefficiency results in a less characterized heteroge-
neous cell population, with undesirable carryover of cells that represent
an impure cell population with variable differentiation potential. This
differentiation inefficiency is apparent in vitro and as such raises safety
concerns in the regenerative medicine community of unpredictable cell
fate and unwanted differentiation potential after in vivo delivery of an
iPSC product. Technology developments will continue to strive to main-
tain control and confidence of the differentiation state regardless of the
manufacturing process scale and will be able to demonstrate that the cell
population can be safely administered to patients (e.g., maintain nontu-
morigenic potential). In vivo distribution and persistence in the human
body will remain an important safety consideration, until control of the
differentiation process is achieved, thus resulting in a homogenous cell
product that is predictable.
A biomanufacturing plan for products derived from stem cells is based on
a cell therapy indication and a product composed of differentiated, tissue-
specific, but viable growing cells of a particular lineage. As a new technology,
regulatory guidance—even general information from FDA—is important to
developing a compliant product. Fortunately, FDA has announced general
guidelines for development from pluripotent cell sources and established
helpful information on related products under Good Tissue Practices and
other regulatory guidelines. These recommendations, while still quite gen-
eral and largely unproven for pluripotent cell-derived products, provide a
foundation for production schemes.
However, many questions regarding application of these types of new
regenerative technologies to ensure production of a safe and effective prod-
uct have yet to be answered. First, do the proposed manufacture and control
methods result in a safe product and how exactly do we demonstrate the prod-
uct safety by using currently available scientific methods? Second, can any
biomanufacturing scheme actually generate differentiated cells and tissues
from pluripotent cells, and, as BS, FP, and after growing in the patient, will
these cells demonstrate the attributes of identity, purity, and potency? Third,
is it possible to apply to pluripotent cell-derived biomanufacturing protocols
those methods and quality criteria that are used for somatic cell and tissue
production, or will it be necessary to begin anew and develop unique schemes
for these cell types. In addition, when produced in great numbers, will dif-
ferentiated cells, derived from pluripotent cells, remain differentiated or will
they revert to an undifferentiated status or even to a malignant state of differ-
entiation? In addition, how do we ensure that pluripotent stem cells, derived
from an unknown source, do not carry adventitious agents? Do traditional
methods applied to somatic cells provide adequate safeguards for pluripotent
cell-derived products?

238 Biotechnology Operations
The biomanufacturing design and subsequent plan must address these
issues and answer questions by using novel manufacturing methods that
go hand in hand with appropriate quality control tests. A hypothetical
scheme for biomanufacture and control of a pluripotent cell-derived prod-
uct is proposed in Figure 6.14. Despite the novel technology and source of
the product, issues that confront the manufacturing team are very similar
to those experienced several decades ago by teams of biomanufacturing
scientists who intended to produce a recombinant protein in E. coli. Then
and now, application of precedent, good planning and careful and thorough
experimentation are keys to preparing a biomanufacturing plan, moving a
novel product through the manufacturing cycle, and bringing it to market.
Indeed, by application of good scientific and manufacturing practices, it is
conceivable that any biotechnology concept can be taken from the research
laboratory and be successfully produced and marketed for the benefit of
mankind.
Production of Biological Molecules by Transgenic Animals or Plants
A host of alternative production systems for recombinant biopharmaceutical
molecules are currently in development. Most purport to make a product
that is of equal or greater quality when compared to biomanufacture pro-
duced by traditional methods such as fermentation or cell bioreactor pro-
duction. Most animal and plant systems promise expediency, higher quality,
and lower cost. Although increasingly more popular, today only a few plant
or animal biomanufacturing concepts are technically mature or proven and
many have already fallen by the wayside, as they have proven difficult to
manage, give small yields, or produce an unacceptably impure or impotent
product. Nonetheless and based on some successes, notably production of
recombinant protein in transgenic goats, there are a host of new plant- and
animal-based biomanufacturing technologies in development. Biopharming
is a casual name given to the application of transgenic plants or animals to
produce biopharmaceuticals.
A transgenic plant or animal is a plant or animal that has been genetically
altered using recombinant DNA techniques to create a genetically unique
organism. The transgenic organism contains an exogenous gene or genes
that have been intentionally inserted into their genome. Once inserted, the
expression of the exogenous gene can express the protein of interest, often
a glycoprotein, and, in some cases, secrete this protein with tissue fluid. In
the case of a transgenic plant, the protein can then be extracted from bio-
mass, that is, stems or leaves, or from seed. In case of transgenic animals, the
protein is available from secretions, notably milk. Hence, the plant or ani-
mal functions as a bioreactor, producing appreciable amounts of the desired
protein as BS. As one would anticipate, production of a transgenic organism
capable of producing and secreting the perfect protein is highly technical and
requires significant experience and skill. As with any biopharmaceutical, the

239Biomanufacture
Process Control testing
Human stem cell
(Pluri/multipotent)
Clone: Derive cell line
Master and working cell banks
Expand culture from cell bank
Differentiation in culture flask to
smooth muscle
Harvest and pool
Purification: Centrifugation
selection methods
Selective adherence to substrate
Differentiation protocol for
smooth muscle
Collagen substrate (Urinary
Bladder Fibroblast)
Degree heterogeneity
Adventitious agents
Microbial burden
Phenotype and genotype
Differentiation
Heterogeneity
Phenotype
Genotype
Differentiation
Heterogeneity
Phenotype
Growth characteristics
Degree differentiation
Safety testing
Quality of raw materials
Heterogeneity
Phenotype
Growth characteristics
Degree differentiation
Safety testing
Heterogeneity
Phenotype
Growth characteristics
Degree differentiation
Safety testing
Expand smooth muscle to tissue
Mature smooth muscle to tissue
Induction factors
(Cytokine and Growth)
Certificate of analysis for BDS
FIGURE 6.14
Hypothetical scheme for biomanufacture and control of human cells and tissues (bladder
smooth muscle) derived from pluripotent cells.

240 Biotechnology Operations
protein product must be isolated and purified from other molecules and it
must possess posttranslational modifications and structure that allow full
biological function and should be without modifications that could make the
molecule allergenic or nonfunctional. This process, using a transgenic goat
secreting in milk, is provided in Figure 6.15.
Biomanufacture of proteins that demand complex glycosylation and post-
translational carboxylation has been successful using genetically modified
animals where it has failed using genetically engineered bacteria or yeast.
Recombinant whole animal systems, such as transgenic goats, have been
used to produce effective molecules of human blood proteins such as anti-
thrombin, fibrinogen, or alpha-1 antitrypsin. Further, to allow for convenient
collection and purification of the protein, the transgenic gene product must
be expressed and secreted into a harvestable body fluid. To achieve such
design objectives, human proteins have been transferred to dairy animals,
such as goats, for the purpose of gene expression in the mammary tissues
and protein secretion into the animal’s milk. Protein is then purified from
milk by using protocols that consider separation from cellular debris, other
milk proteins, and fat. This is accomplished using downstream purification
methods such as centrifugation, filtration, and chromatography, but under
the design plans that consider the unique impurities in mammalian milk.
Issues related to adventitious agents have been addressed. Additional infor-
mation on downstream protein purification methods is provided later in this
chapter. A specific example of how transgenic goat emphasizes the economic
advantages and the scale of production this technology can offer are pre-
sented in Box 6.4.
Rabbits have also been used as a reliable and efficient source of a clotting
factor for a rare blood disorder. In this case, a transgenic rabbit is created as
a bioreactor, and a C1 esterase inhibitor, produced in the milk of transgenic
rabbits, is licensed for the treatment of hereditary angioedema. A lactating
rabbit can produce 10–12  g of protein per liter of milk, whereas traditional
methods of protein expression from cell culture systems are less efficient,
with yields ranging from 0.2 to 1.0  g of protein per liter of culture media.
These are just a couple of examples where cutting-edge biotechnology has
demonstrated the proof of concept and then has been successfully developed
to reach the marketplace.
A second method, producing biopharmaceuticals in plants, has been tested
in a variety of plant species for over two decades. A scheme for production
of a recombinant protein by a transgenic higher plant species is depicted in
Figure  6.16. The ability to transform commercially useful plants, first with
genes of other plant species and then with genes of animal origin, has pro-
vided the foundation for this technology. Cultured plant cells of both higher-
and lower-order plants, for example, maize or tobacco and algae or mosses,
have been tested as biomanufacturing systems. Higher-order plants are usu-
ally used; however, systems using lower-order species are under develop-
ment. The first step is the production of a transgenic plant, a process that is

241Biomanufacture
Vector containing transgene
Injection of transgene into goat embryos
Transgenic goats
Offspring
Goat milk containing
biopharmaceutical protein
Embryo implantation
Vector
FIGURE 6.15
Transgenic goat producing biopharmaceutical protein in milk. The recombinant process
begins with constructing a vector containing the protein of interest, designed to target pro-
tein expression in the milk. The transgene is injected into goat embryos by using pronuclear
microinjection methods and then embryos are reimplanted into a host female goat. To iden-
tify germline transmission of the transgene, offspring are screened for transgene expression
in the milk. Milk is collected from the transgenic offspring and milk proteins are isolated.
Recombinant protein is easily purified from endogenous milk proteins and other impurities
that reside in goat milk.

242 Biotechnology Operations
facilitated by several novel gene delivery methods well suited for transfec-
tion. Once mature, samples of various plant tissues are tested for the desired
trait, such as expression of a mammalian protein. Next, these plants must be
cross-bred, by using methods applied to the development of hybrid plants;
this is followed by another round of selection. The ultimate source of the
recombinant molecule may be any plant tissue expressing the product, but
seed is a preferred choice, given its ability to store large amounts of pro-
tein in an environment with a low microbial bioburden. The plant tissue
must then be processed to release the desired recombinant protein from cells
and tissue matrix and bring it into solution. The solution is clarified of plant
debris and processed using purification methods described above for other
proteins in order to derive the recombinant molecule as BS.
BOX 6.4 THE TRANSGENIC GOAT AS A BIOREACTOR
• In 2009, FDA approved ATryn, a recombinant antithrombin
indicated for the prevention of thromboembolic events in a
rare human clotting disorder.
• Antithrombin can be isolated from human plasma; however, to
fulfill market requirements, 100 kg would take one year’s sup-
ply of plasma donations.
• However, the same amount of recombinant antithrombin can
be collected more efficiently and economically from 150 trans-
genic goats, animals expressing and secreting this protein in
milk. A lactating female goat produces up to 800 L of milk in
a year.
• The transgenic goat model as a bioreactor can in theory and
in practice yield, after protein purification from milk, approxi-
mately 4 kg of a recombinant protein.
• The cost comparison to produce the same amount of recom-
binant pharmaceutical-grade protein is quite staggering when
comparing traditional methods with biopharming.
• Purification is achieved by utilizing isoelectric precipitation,
affinity chromatography, and size exclusion chromatography.
These methodologies result in a 90% yield with greater than
99% purity.
• The production cost from a bioengineered domestic animal is
estimated to be approximately one-tenth the cost of manufac-
turing the same recombinant therapeutic protein in a commer-
cial cell culture facility by using a bioreactor.

243Biomanufacture
Choose transfection method
Molecular or Biological: DNA, agrobacterium
Mechanical: Gene gun, electroporation, and
optical laser
Other: Nucleofection and impalefection
Construct expression
gene
(selection and marker)
Donor seed Process
Transfection
Seedling+transfection
method+genetic construct
Genetic construct
Test in seedlings
Ensure high-rate
transfection
Select stable
Transformed plant
seed
Grow and cross-breed
Second and third
Generation plant and
seed
Centrifugation and
filtration
Evaluate recombinant
Protein trait in
plant tissue
Discard
Unproductive
Transfection
Test tissue for
Protein trait
First generation plants
Field growth
Harvest seed
Crush and solubilize
seed
Purifications and
chromatography
Protein
BS
BS panel tests and
certificate analysis
Activity
Total protein
SDS-PAGE
HPLC
Test tissue for
Protein trait
Test seed for
protein trait
Activity
Total protein
SDS-PAGE
HPLC
Test tissue for
Protein trait
Control testing
Establish gene
Hybrid seed
Plant seedling
FIGURE 6.16
Production and control of a recombinant protein by a transgenic higher plant species.

244 Biotechnology Operations
Several hurdles had confounded or currently confound the application
of biomanufacture with transgenic plants. Glycosylation is one example.
Plants have evolved unique methods to make posttranslational modifica-
tions to proteins. Glycosylation modifications of plants are unlike those
in animals, yet they are encoded in the plant genome. Sometimes, expres-
sion of a mammalian gene by a transgenic plant unpredictably results in
a molecule that has the expected protein backbone but possesses a totally
different and undesirable carbohydrate moiety. This is the result of glyco-
sylation by plant enzymes, a process that would not happen if the protein
molecule were manufactured by an animal cell. A case in point is a mono-
clonal antibody produced in maize cells and glycosylated with a series of
carbohydrates unique to plants. Such modifications change the proper-
ties, often the potency, of the recombinant molecule. Hence, careful plan-
ning, based on an understanding of the product and the transgenic host
system, is essential to successfully using transgenic plants or animals for
biomanufacture.
Another issue is the need to redefine cell banks to meet transgenic plant
technology. For example, corn, unlike bacteria or immortalized cells, relies
on cell banks composed of actual monocotyledon seeds. Owing to sexual
reproduction and other traits, higher plant seeds are quite heterogeneous in
nature and genetic makeup. A corn seed bank is not derived from a clone
and is not genetically pure or homogenous. This can cause problems with
variable field growth, sexual reproductive capacity, or protein expression.
In addition, the environment of field-grown plants is quite difficult to con-
trol. Weather in a corn field is highly variable, and higher plants grow, quite
literally, in dirty environments, certainly as compared to that of the fermen-
tation vessel in an aseptic biomanufacturing facility. Thus, plant-derived
recombinant molecules begin as septic entities, adding challenges to aseptic
purification processes. Finally, purification of biopharmaceuticals has also
presented new challenges, many unexpected, because extracts of plants
have impurities and contaminants that are not found in bacteria, yeast, or
mammalian cell systems. Novel approaches, such as use of unicellular and
asexual plant species (e.g., algal and moss species), growth of plants in con-
trolled environments, and derivation of recombinant proteins from selected
plant tissues, have overcome some, but certainly not all, of the issues facing
biopharming.
Yet, there are potential advantages, notably production of large quantities
of product at low cost, that are derived by biomanufacture from transgenic
plants or animals. Whether produced in domestic animals or plants, human
recombinant proteins intended for therapeutic use need to consider the real
potential of eliciting an unwanted immune response due to the xenogenic
nature of the protein. The FDA has provided guidance regarding the produc-
tion of therapeutic proteins by both plants and animal bioreactors. Further
recognizing the potential immunogenicity and associated safety con-
cerns surrounding xenogenic protein technology, FDA guidance discusses

245Biomanufacture
requirements to minimize the risks of immunogenicity and the likelihood of
generating an undesirable immune response.
Production of Biologically Active Lipids,
Glycolipids, and Complex Carbohydrates
Peptides, lipids, complex carbohydrates, and glycolipids are potentially
important biomolecules of economic and therapeutic value. This is perhaps
best demonstrated by complex carbohydrates and glycolipids, which have
been successfully used as vaccine antigens, and by lipid molecules, which
are then considered as vaccine adjuvants. Each has a biological derivation
and function in nature and can be manufactured by man in the laboratory.
Biomanufacture of these molecules in larger quantities is done through
one of the following two routes: (1) production by a live organism that is
not recombinant but naturally expresses the molecule in nature, followed
by isolation and purification of the macromolecule and (2) chemical syn-
thesis, using processes analogous to, but often more complex than, those
applied to production of small-molecule drugs. An example of a natural
product (platelet-rich plasma [PRP]) derived from bacterial cultures and
then purified using biomanufacturing methods and conjugated to a toxin
(T) to yield Haemophilus b conjugate (PRP-T) is outlined in Figure  6.17.
This complex carbohydrate is used as a vaccine against Haemophilus influ-
enzae type b.
The chemical synthesis of biologically active, nonproteinaceous macromol-
ecules is becoming more elegant and widely adopted. For example, instru-
ments are used to synthesize complex carbohydrates on lipid-like backbones,
a method that is dependable but still gives low yield.
The downstream or purification steps used for lipid, carbohydrate, or gly-
colipid biomolecules vary in some respects from those used to purify pro-
teins. This is understandable because lipid or carbohydrate molecules are
quite unique in chemical structure and composition and physical properties.
Manufacturing planning takes into consideration the molecular attributes of
these candidate products and matches them with technologies available for
purification. Many of the methods applied to a manufacturing step may be
borrowed from the research laboratory and adapted to biomanufacturing.
Production of Biologically Active Peptides
A peptide is a string of amino acids, 40 or less, by most definitions, in a given
sequence. Biologically active peptides are used or tested as biopharmaceu-
ticals, enzyme inhibitors, laboratory chemicals, and for other purposes. As
they are shorter than proteins, peptides typically have no posttranslational
modification and lack complex secondary structure. Various technologies
are available to manufacture peptides, and the choice of method is based on
the intended use and specifications, notably purity, the number and nature

246 Biotechnology Operations
Purified protein
carrier
(Diphtheria toxoid)
Conjugation
(Reductive
amination)
SDS-page
HPLC
Safety
Identity
Molecular weight
purity
Hib, master and
working cell
banks (Bacterial)
Growth in culture
Collection by
centrifugation
Centrifugation and
tangential flow
filtration
Kill bacteria
Remove
polysaccharide
Purify
polysaccharide
Chemical
modification
(activation) of
polysaccharide
Purification
Bulk conjugate
Certificate of analysis
(BDS)
Certificate of
analysis
Cell banks
Hib identity
Contamination
impurities
Purity
Identity
Molecular size
Moisture
Polysaccharides
Impurities
Molecular size
Degradation
purity
Growth Rate
yield
Polysaccharide
pH
Control
testing
Process Controltesting
PRP: Protein ratio
Residual conjugation
agents
Unbound PRP
Molecular sizes
FIGURE 6.17
Production of a polysaccharide-protein conjugate product (Haemophilus influenzae type b
vaccine).

247Biomanufacture
of amino acids in the string, the amount required, and the cost. Peptides are
often manufactured using automated equipment, the peptide synthesizer, to
build the string, one amino acid at a time, beginning at a solid matrix such
as a plastic bead and continuing to the end. Hence, most peptides are made
using tools common to synthesis of other organic molecules.
Impurities—truncated peptides, fragments, and free amino acids—
remain in solution with the peptide product after synthesis, so purification
is required. Further, peptides in their unprotected state, perhaps because
they represent incomplete or fragmented protein sequences, can be unstable.
Formulation, fill, and finish procedures must carefully consider postmanu-
facture hold and storage environments to ensure a potent and stable product.
Certain other biomolecules are peptide-based biomolecules, in that they
are peptides but with another large molecule bound to them. Examples
include glycopeptide and protein–peptide combinations. The production
of such molecules may require considerable planning and technical effort,
as there are no established manufacturing methods available for these
biopharmaceuticals.
Production of Combination Products: Biopharmaceutical
with a Drug or Medical Device
Biomolecules are frequently used with material from another source to
produce a combined effect. For example, a recombinant bacterium used to
clean oil spills may be applied to the spill along with a short-chain organic
molecule that disburses the oil and facilitates the metabolic activity of the
bacterium. In biopharmaceuticals, the pairing of a biological product with a
medical device, a drug, or both is considered a combination product by reg-
ulatory agencies (Chapter 3). A recombinant DNA vaccine that is delivered
exclusively with a special injector device is one example. Another example is
an engineered retrovirus that must be given with a specific drug to enhance
the potential therapeutic activity of the retrovirus. A case in point is the use
of a drug to facilitate insertion of a therapeutic gene into the genome of the
recipient. For the full intended therapeutic effect, both substances, drug and
biologic, must be pure and potent under a defined schedule of usage.
Biomanufacturing plans identify combination products and present a strat-
egy for producing both products to specifications that will yield the desired,
combined effect. Manufacturing specifications for combination products are
often challenging for the operator, because individual roles must be assigned
to attributes of each product and synergistic effect also needs to be consid-
ered. For the example of a recombinant DNA vaccine (a biopharmaceutical
composed of plasmid DNA), delivered by a needleless jet injector, a medical
device, the vaccine plays the dominant role, because it imparts the therapeu-
tic effect by stimulating the immune response. However, the device must
perform per established specifications, otherwise the DNA vaccine will not
be delivered correctly and might not then exert the intended pharmacological

248 Biotechnology Operations
effect, that is, vaccination. Early considerations for a manufacturing plan
takes into account this needed synergy in combination products. Specific
concerns in DNA-related examples might be as follows:
• DNA formulation must be compatible and stable with materials of
the device.
• DNA must be concentrated so that it can fit into the device chamber
during storage, but it must also be capable of rapidly exiting via the
device needle on actuation.
• Device must consistently deliver an exact amount of DNA.
• Device must be easily and correctly used by medical staff.
From this example, it is clear that design, actually a codesign of a biological
product and a medical device, is critical to success whenever a biotechnology
product is partnered with a drug, a device, or even another biopharmaceuti-
cal. Manufacturing plans and technologies consider complex performance
issues and ensure that these issues have been addressed early in develop-
ment. Experimentation is performed to test the effects of additional vari-
ables, as each product brings into play a new set of issues.
FP: Formulation, Fill, Finish, and Labeling
A product is used in clinical investigations or commercially only after it
has been properly formulated, tested, placed into a protective container or
a delivery device, and then labeled. Having reviewed the various types of
biotechnology products presented earlier in this chapter, one might well
imagine the need for a host of formulations, containers, and labels, as well
the procedures, to complete their FP. In this section, we review the technolo-
gies used to bring a biopharmaceutical from BS to the format intended for
the user, that is, the FP.
The biomanufacturing plan for an FP focuses on the indication and the
user. Biomanufacturing is a market- and user-driven process, and therefore,
the formulation, container, and label are designed for a specific purpose,
that is, the needs of the user. For biopharmaceuticals, the user is the patient
or a subject enrolled in a clinical investigation, but for many products, a
full definition of the user includes the medical professional prescribing or
administering the product. For a tissue product, such as the skin or cartilage,
mentioned in an earlier example, the surgeon is also a user of that product.
Indeed, the patient may never see the cartilage tissue product as provided by
the manufacturer, because the cartilage is transferred by the surgeon from
the package into the knee joint. In contrast, for the combination product of

249Biomanufacture
recombinant monoclonal antibody in an autoinjector, the patients, not the
medical professional, use the product, receiving it directly from the phar-
macy and then injecting it themselves. The medial professional is, however,
familiar with the product and its attributes before a prescription is written
and may train the patient on the proper use of the product. Most biophar-
maceutical FPs are used by more than one person and all of their needs are
considered in the FP design.
The FP design considers various product attributes. Formulation decisions
rest on the intended shelf life of a product, possible and desirable storage
conditions, and the proposed dose. The target dose also allows planning for
the amount of product that will be included in the final container and the
packaging and labeling that are provided with the container.
A scheme for FP production of a typical biotechnology product is shown
Figure 6.18. First step is to select or devise a formulation that suits the
nature of the product, intended use, route of delivery, and container. There
are many formulation choices available, and others can be developed for
a unique product. Common formulations for biotechnology products con-
sist of salt solutions, buffers, and a variety of excipients. Once a promising
formulation is identified, experimentation and trial and error are the bases
for selecting the one that is just right for that product; this means signifi-
cant testing must be performed for product attributes and for product sta-
bility. Since a product may need to be matched with several formulations
before the correct combination of ingredients is identified, the process can
be extensive.
Once a formulation has been selected, the actual formulation process for
a biopharmaceutical begins with final clarification and, for most products,
sterile filtration of the BS. After purification and until formulation, bio-
pharmaceuticals are held as BS under refrigerated or frozen conditions. If
the active ingredient in BS is stable, as a frozen recombinant protein might
be, it may be stored for several weeks or for even months. In contrast, a
tissue product, such as skin tissue, might withstand refrigerated storage
only for a few days or weeks. Hence, the time allowed between comple-
tion of bulk manufacturing and formulation, fill, finish, and labeling var-
ies greatly, depending on the product type. Product stability testing is
discussed in Chapter 7. No matter what the product, once the decision
is made to begin formulation, the process is completed as rapidly as pos-
sible to reduce hold time and thus minimize product exposure to the
environment. Since most biopharmaceuticals are sterile products, formu-
lation must be a strictly aseptic process applying stringent techniques and
environmental controls.
An example of formulation, fill, and finish is instructive and a recombinant
protein, such as a monoclonal antibody. After sterile filtration, the storage
buffer for a monoclonal antibody, an active ingredient in BS, is exchanged for
the FP buffer. In this example, 0.9% sodium chloride in water (normal saline)
for injection is exchanged with a small amount of detergent. After mixing,

250 Biotechnology Operations
Formulation
buffer
Excipients
Test final product
certificate of
analysis
Inspect label
Initial
formulation
Fill into vials,
syringes, injectors
final product
Label vials
Package and
package insert
In-process
testing
Inspect Package,
carton, and lablesOuter carton
Storage and
distribution
Test formulation
buffer
Hold and transfer
Process
Test excipients
Control
testing
Formulated
product
Product as bulk
substance (BS)
FIGURE 6.18
Manufacture of final product: Formulation, fill, and finish process.

251Biomanufacture
the formulated product is again sterile filtered and then transferred to the fill
area, where it is dispensed into vials.
In contrast to a recombinant protein, a more complex biopharmaceutical
product, such as skin tissue, may require more formulation effort. In this
case, it may be necessary to rinse the skin tissue with various buffers and
then place it into a nutrient solution enriched with oxygen and chemicals
that maintain tissue viability. Terminal sterilization, even by filtration, is not
possible with skin and other cellular (or many other biotechnology) prod-
ucts, and so, strict aseptic technique is practiced throughout the formulation
process. To meet stability profiles of some fastidious biotechnology products,
such as live bacterial or viral vaccines, some products have unique additional
steps in formulation. For example, dry powders may be prepared by spray
drying the product, once it is in the final salt solution. Freeze drying, referred
to as lyophilization, is also used to prepare powder from formulated liquid
product, after it has been aliquoted into vials.
Fill is the next step in the process. In this step, product is placed into a
final container with fill procedures tailored for each product-and-container
combination. For the example of skin tissue, product might be placed into
a pouch of some type. For the living tissue, the outer container might be
a special transportation apparatus replete with systems to provide sterile
nutrients and oxygen and an external heat source to maintain a temperature
suitable for this living product.
Returning to the example of recombinant protein, the monoclonal anti-
body product formulated in saline with a detergent, container and handling
requirements present a less complicated fill and finish procedure. Standard
pharmaceutical glass vials are chosen as the primary container. These vials
have butyl rubber caps, and aluminum crimps are used to seal the cap to
each vial. Vials and caps of the highest quality are scrupulously cleaned and
heated to remove any contaminants before use. Vials either are filled and
then capped and crimped (force a seal over the cap) manually by operators
(Figure 6.19) or are filled, capped, and crimped using a machine. Prefilled
syringes and injectors are becoming popular containers for biopharmaceuti-
cal products, and these are always filled using automated pieces of equip-
ment, which are fast, accurate, and less likely to break the aseptic nature of
this operation.
Once containers have been filled, capped, and crimped, a permanent label
is placed onto the container to provide the user with an abstracted descrip-
tion of the contents. Containers include a description of the product, dose,
volume, total number of doses per container, source (manufacturer), expira-
tion date, lot number, and special instructions or warnings. Imagine fitting
this much information onto a label, 2.5 × 1 cm, for a small vial!
Packaging is the next step in producing the FP. Containers are placed into a
protective inner package, for example, a light cardboard. A package insert is
added to this box before an adhesive label is attached to identify the contents
and then it is closed with a tamper-proof seal. The package insert, sometimes

252 Biotechnology Operations
referred to as labeling, provides product information to both the medical pro-
fessional and the patient or user. Multiple product containers in their outer
package are then placed into a larger carton, the outer container, and this too
is labeled.
By way of a final example of formulate, fill, and finish, we consider a biotech-
nology product that is not a biopharmaceutical but instead is a genetically
engineered plant. It is a strain of disease-resistant corn, grown in the field,
harvested, cleaned, and then placed into storage in 50-pound bags as BS.
The product will be planted in fields by farmers. Before packaging, the seed
is formulated by various treatments, perhaps by drying to an established
moisture level, followed by addition of powdered fungicide to prevent decay
during storage. The seed is then filled, placed in moisture-proof containers,
such as 10-gallon plastic buckets, and the buckets are sealed. These buck-
ets are then labeled on the outside, and several buckets, along with product
information (the package insert), are placed into an outer carton made of
cardboard, and this too labeled. The package may then be shipped to a dis-
tributor or the user. Although technically quite different from formulation–
fill–finish production methods used for a biopharmaceutical, the processes
for this and many other biotechnology products follow the same general
steps in production, protecting them from harmful environments and mak-
ing them ready for the user.
FIGURE 6.19
Fill and finish of final product in a clean room. Two operators, working under a Class 100 hood,
manually fill and finish vials of a biopharmaceutical. They are carefully gowned and covered
to prevent any possibility of contaminating the FP. The operator on the left is filling the vials,
held in a box at the center of the hood, with a dispensing pipette, whereas the operator on the
right adds a cap to each vial. In the next step, an aluminum seal will be crimped to tightly close
each vial and a label will be added. (Courtesy of Waisman Clinical Biomanufacturing facility,
http://www.gmpbiomanufacturing.org.)

http://www.gmpbiomanufacturing.org

253Biomanufacture
Biomanufacturing Facilities, Utilities, and Equipment
Facility Design Considerations
Biotechnology products, notably biopharmaceuticals, are manufactured in
very special facilities. Biomanufacturing facilities are not only unique to our
industry but are also custom designed for each class of product and even for
a specific product. All the activities we have mentioned in this chapter, with
the possible exception of upstream production in biopharming, must be per-
formed under a roof, above a floor, and between four walls. Biomanufacturing
facilities are complex and expensive to both build and operate. Staff are pro-
fessional specialists and perform everything, from emptying the trash and
cleaning surfaces and equipment to aseptically filling the final containers
follows and writing procedures and results in permanent records. As each
biotechnology product and every biomanufacturing process for a product
are unique, facilities are custom designed and have been built and equipped
in every imaginable way. Still, there are similarities in facility designs for dif-
ferent products, and our discussion of biomanufacturing facilities, utilities,
and equipment will provide basic principles of facility design and operation.
Biotechnology firms skilled and lucky enough to find themselves in devel-
opment are faced with the need to manufacture their product. After this
realization, the first question to be posed by management to the product
development team is as follows: When, where, and how will this be accom-
plished and at what cost? The answer must be in the manufacturing plan;
therefore, options must be considered and choices must be made. A team
composed of individuals with business, financial, management, biomanu-
facturing, and facility and process engineering experience is chartered to
reach a decision on manufacturing options. There are at least three possible
ways to meet a biomanufacturing requirement, and these are as follows:
(1) do not manufacture the product (and hence, do not develop it further);
(2) manufacture the product in-house; and (3) manufacture the product at
a CMO. A fourth possibility is to split manufacture, that is, do some pro-
cesses in-house and have others completed at a CMO. Not surprisingly, few
biotechnology firms select the first option and the rest seem evenly divided
between the choices numbered two, three, and four. Many factors beyond
the technical need or a desire to have an in-house operation often influence
the final decision; these are resources, location, and business and exit plans.
A biomanufacturing facility is designed to produce a specific class or type
of product. One would not expect to see a recombinant seed corn operation
co-located with a formulation, –fill, and finish operation, and it would be
unusual to find, in the same biomanufacturing building, both epithelial cell
culture and bacterial cell fermentation. However, some facilities can process
quite a number of different products, that is, manufacture on a campaign
basis, but even then, they are limited in scope. When considering the building,

254 Biotechnology Operations
one must choose between erecting a new building or remodeling an exist-
ing structure. Many excellent biomanufacturing operations have been built
within an empty warehouse, albeit one of high quality in a good location
and with adequate services. Actual facility design is best left to professional
engineers and architects with experience in biomanufacturing process engi-
neering and building, equipping, and maintaining facilities. Early drawings
are produced by engineers and architects. These should be carefully exam-
ined by process engineers, the individuals with an understanding of the pro-
cess intended for the facility. General descriptions of utility and equipment
requirements are added to the facility plan. After discussions and one or two
more rounds of review, a final facility plan is established. Cost estimates are
made by professional biomanufacturing facility engineers.
The Facility and Utilities: A Controlled Environment
Most biopharmaceuticals are sterile products of exceptionally high qual-
ity and reliability. Biopharmaceutical production requires special controls,
because products are intended for use as medicines by large numbers
of people and many of these medicines are given by the parenteral route.
Biopharmaceutical production and the facility producing the biopharmaceu-
tical are highly regulated, and for marketed product, the facilities must have
a government license, so as to ensure that only safe, pure, and potent prod-
ucts reach users. Regulations and guidelines make clear the exact standards
and specifications for biopharmaceutical- manufacturing facility design and
operation. Further, good business practices also demand a quality manufac-
turing facility for biotechnology products. Not a month goes by without a
news story covering an inadequate and substandard drug, biologic, or medi-
cal device facility that produced and continued to provide substandard, even
unsafe, product to consumers. These incidents result in expensive product
recalls, consumer illness or even death, and regulatory actions against the
sponsor (Chapter 4). Such cases lead to negative publicity for the firm and
loss of product sales, even for the product not made in that facility. Indeed, a
single incident at a manufacturing facility has many times led to the loss of a
product line or even to the financial demise of a biopharmaceutical firm. The
combined lesson is that a manufacturing facility must be first-rate from the
ground up and in all respects.
A biomanufacturing facility is, from the outside, a building that looks like
any other commercial structure; however, from the inside, it is established
and equipped exclusively for the production of biopharmaceutical product.
How then is the proper environment for biomanufacture established under
the general facility plan? First, that building is designed to house a particular
process or several similar processes. Second, the design considers the need
for production by aseptic processes, so as to reduce the incidence and spread
of microbes and other potential contaminants through the use of segregated,
clean work areas. The facility is also planned to house adequate utilities and

255Biomanufacture
equipment, which are again properly designed. Biomanufacturing requires
sufficient space for operators, various processes, utilities, and equipment;
further, regulations require and common sense suggests separation of vari-
ous activities to prevent mix-ups, contamination, or cross-contamination of
product or spread of microbes. The design must consider utilities, reliable
sources of water, electricity, natural gas, and heat, ventilation, and air condi-
tioning (HVAC). Finally, a facility must be well managed by highly trained
and experienced professionals, and, just as with the manufacturing process,
it must be run by strict and compliant written procedures and records. Does
this sound rather expensive? Indeed, it is.
Once a decision is reached to build a biomanufacturing facility according
to a design, additional planning is in order. Detailed product-manufactur-
ing plans allow the facility planner to impose on the facility design a pro-
cess map, a schematic in which the manufacturing process is drawn, stage
by stage and step by step onto a general facility design. This allows one to
determine whether or not all the pieces—processes, equipment, utilities,
and workflow—will work in harmony. Consulting engineers and architects
and biopharmaceutical process engineers who have experience in pharma-
ceutical manufacturing are retained to examine rough plans and refine the
drawings to ensure compliance with local and state regulations. Plans and
drawings are revised, discussed, changed, and finally finished. The facility
plan now supports business functions such as accepting bids from building
contractors and utility and equipment manufacturers. Now, upper manage-
ment can be given a firm estimate of the cost.
Operation of Clean Work Areas for Biomanufacture
Controlled processes and aseptic processing are crucial to biomanufacture.
At the heart of aseptic processing is the need to keep viable particulate mat-
ter, specifically bacteria and yeast, from contaminating a product. The pri-
mary source of microbes is humans (e.g., skin and hair) and materials that
enter into a clean area. Introduction and spread of microbes are controlled by
facility, utility and equipment design, environmental awareness regarding
microbial burdens (Chapter 7), and sound aseptic operational procedures.
The facility floor plan is critical to maintaining a clean environment, as it
allows for segregation and the logical flow of activities and products. Entry of
people and raw materials and exit of waste are carefully controlled to reduce
the entry of contaminants into a clean work area. Before entering, individu-
als don special gowns, footwear, and masks that cover much of the body,
thus reducing the microbial burden shed by them into the clean room envi-
ronment. Materials are sterilized or disinfected immediately before these
are brought in a clean area. Before entry, water that is to be used in a clean
area is filtered to remove any viable particles. As microbes and contaminated
dirt particles move freely in air, the HVAC system is designed to constantly
filter air through high-efficiency particulate air filters. Moreover, the air is

256 Biotechnology Operations
circulated rapidly and in great volume to ensure that any particles generated
by process activities are swept from the room and filtered to prevent product
contamination. Doors and pass-through openings control airflow between
rooms, moving air by an established pattern from the cleanest to less clean
rooms, thereby further limiting particulate and microbial movement. Since
microbes adhere to and multiply on surfaces, all ceilings, walls, and floors
are finished with highly resistant epoxy surfaces to discourage microbial col-
onization and to withstand repeated scrubbing and disinfection. All equip-
ment surfaces are designed to tolerate harsh antimicrobial treatments.
Movement of everything—people, raw materials, trash, and product—
within a facility must flow in a predetermined direction. Highly controlled
aseptic operations, such as fill and finish, are performed in highly classi-
fied areas segregated from other manufacturing and nonproduction areas,
so as to prevent contamination of the FP. During and after biomanufacturing
operations, measurements of air quality are taken, and each room must meet
an air quality standard or classification. There are two manners of classify-
ing air within a clean area; both of them are shown in Box 6.5. Everything
in a clean area, including air, water, surfaces, and the gloved hands of every
staff member, are sampled or swabbed to test for microbial contamination.
Operations not requiring a clean environment are kept apart from clean
areas dedicated to aseptic processing. For example, packaging and labeling
are generally relegated to an area that is not highly controlled or classified. In
addition, quality control laboratories, offices, and meeting rooms are estab-
lished outside the clean area. In summary, a facility design facilitates the
BOX 6.5 MEASUREMENT OF PARTICLES IN A
BIOMANUFACTURING CLEAN ROOM OR AREA
Room Classifications
Published Specifications
ISO 14644–1 FED STD 209E (U.S.)
ISO 3 Class 1
ISO 4 Class 10
ISO 5 Class 100
ISO 6 Class 1000
ISO 7 Class 10,000
ISO 8 Class 100,000
Note: The particle counter instrument is used to measure particles in air. An air sample
is taken into the instrument; particles of a specific size (e.g., >0.5 µm) are counted
and total air volume is measured. Particle counts are then given as number of
particles per cubic foot or particles per cubic meter (ISO 14644–1; International and
European). A manufacturing facility might have air classified as 100,000/ISO 8 in
general preparation or laboratory areas, as 1,000/ISO 6 in clean work areas, or as
100/ISO 5 in areas for performing aseptic operations, such as filling vials.

257Biomanufacture
prevention of mix-ups, provides a flow from clean to dirty, allows segrega-
tion, and isolates critical steps in an effort to ensure pure, potent, and safe
product.
A biopharmaceutical operation also considers the utilities in support
of manufacturing operations. Temperature is always controlled by heat-
ing air-conditioning and ventilation equipment. The production of some
products also requires humidity control. Gases to incubators, steam to
sterilization equipment, and water to make solutions must enter the clean
area. Water for injection is the purest grade and may be produced by the
manufacturer in-house. However, towing to the complexity and expense
of producing WFI, many biotechnology firms simply purchase it in bottles
as a United States Pharmacopeia (USP) reagent (Chapter 7). Steam is used
in most biomanufacturing facilities to sterilize raw materials or equipment
and to clean and disinfect surfaces. In such cases, the steam is produced
as clean steam, generated with special equipment from WFI. Mechanical
equipment, such as HVAC and water purification systems, are always
monitored and alarmed.
Biomanufacturing Equipment
Many pieces of equipment, including biological safety cabinets, centrifuges,
filtration apparatus, fermenters, bioreactors, chromatography apparatus,
controllers, microprocessors, and incubators, to name a few, are operated in a
clean area. Special or unique pieces of manufacturing equipment—and there
are many to manufacture biopharmaceuticals—must be of the highest qual-
ity and some are specially designed for the biomanufacture of one product.
Equipment specifications ensure proper performance, and all equipment
are periodically calibrated, controlled, and monitored during operation.
Rigorous cleaning is necessary. Cleaning protocols are often complex pro-
cedures, because all residual product and cleaning agent and microbes must
be removed after use. In addition, many pieces of equipment are sterilized
before use or reuse. Once the facility has been built, facility, utilities, and
equipment are commissioned and validated, along with the process itself.
In addition to product-specific requirements, there are also basic quality
requirements for cGMP operations.
Contract Manufacturing Options
It is no wonder that many biotechnology firms select a CMO to produce their
product. At first, many resist this option because they believe that the firm
will lose some control of product manufacture, especially if the CMO is some
distance away. However, renting capacity can be economically attractive and

258 Biotechnology Operations
there are many ways to establish a partnership in which the sponsor retains
adequate control of the biomanufacturing program after it is placed at a
CMO. Indeed, procedures, reflected in Box 6.6, are recommended for selec-
tion of a CMO.
Even if a CMO is retained to perform critical biomanufacturing stages,
many firms elect to perform some processes in-house. Using outside ser-
vices to formulate, fill, finish, and label is quite common in the biotech-
nology industry. An added benefit of contracting these types of services
is that this approach necessitates the technology to be mature enough and
BOX 6.6 CONSIDERATIONS FOR RETAINING
A CONTRACT MANUFACTURING OPERATION
Plans List your needs from a contract manufacturing operation (CMO),
regarding type of product, phase of development, and
biomanufacturing by stage. Is this a clinical Phase 1, 2, or 3 study?
What exactly must be done by the CMO: early development; cell
banking; upstream processing; downstream processing; formulation,
fill, and finish; quality control testing; or more than one of these
functions? What are the deliverables: BS; final product; amount of
product; or number of containers, reports, or records?
Competencies Identify core competencies of CMO and match to your requirements:
history; size; management philosophy; experience and willingness to
work with small, medium, or large biotechnology firms; experience
by type of product, phase, or capacity; dedicated or shared facility;
references; location (region and country); profile in the CMO
community; and possibility of strategic partnership.
Equipment Will it be necessary to purchase or lease specialized utilities or pieces
of equipment, or is everything already in-house at this CMO? What
is the cleaning and change-over process in case of dedicated
equipment or shared equipment?
Microbiology If necessary, can the CMO work with live organisms or material that
requires stringent or unusual aseptic technique or environment? Are
antibiotics used or allowed in the facility?
Design Consider responsibilities for manufacturing design, planning, and risk
analysis and mitigation.
Quality Note quality responsibilities and willingness to enter into a Quality
Agreement and Technology Transfer Agreement. If it is a shared
facility, evaluate change-over procedures; staffing of multiple projects
in facility; and systems to prevent potential for mix-ups,
contamination, and cross-contamination.
Schedule Examine scheduling possibilities, typical slack and busy periods,
opportunities on calendar, and flexibility going forward. Plan for
onsite observations and audits as necessary.
Cost What are the projected costs and unusual expenses associated with the
CMO, and will it be cost-effective, resource-effective, and time-
effective to retain this CMO?

259Biomanufacture
be amenable to technology transfer from in-house to an outside contrac-
tor for this to be successful. At some stage in development, technology
transfer is likely going to be imminent, either to support later stages of
clinical development or to meet commercial demands requiring large-
scale production. Therefore, an early lesson in technology transfer, iden-
tifying subtle but important vulnerable processes in the manufacturing
process, may add value during the early stages of product development
and be less costly in the end. Recall that it is much easier and generally
more acceptable to tweak the production process in the early stages of
development than in the later, more codified stages. Preferably, there are
multiple CMOs capable of doing the chosen biomanufacture. Selection of
a CMO requires considerable diligence by the sponsor. A history of previ-
ous projects provides assurance that a CMO can work with this product
and is capable of performing the processes properly. Precontract site vis-
its, audits, frequent communications, and a detailed contract are the best
means of choosing the right CMO. However, a sponsor’s efforts do not
end with the award of a contract, because there must be frequent visits,
communication, and coordination between the sponsor and the contrac-
tor throughout the life of the contract. Including contractor representa-
tives in the project management team is an especially effective method of
managing a contract.
Validation of Biomanufacturing Facilities,
Utilities, Equipment, and Processes
Validation is an expensive and time-consuming, but very necessary, process
that is completed during late-phase development and only after a manu-
facturing facility, with all equipment, utilities, and staff, and the exact pro-
cess have been established and commissioned. It is considered a regulatory
requirement and a good business practice. Marketing approval is not possi-
ble unless a manufacturing plant and process are fully validated. Validation,
which is defined in a dictionary as to make sound, defensible in common prac-
tice, carries a more complex definition in biopharmaceutical development.
For biotechnology operations of any type, validation is a formal process of
establishing documented evidence that a specific process (or test, equip-
ment, facility, or utility) consistently performs and will, with a high degree of
assurance, continue to perform within determined specifications and qual-
ity criteria. Quite a mouthful, but once broken down, this definition makes
sense in light of the complexities of biomanufacturing and the importance of
quality in the endeavor.
All manufacturing systems have inherent variations, much of it being accept-
able and some being undesirable. Validation is based on an understanding of

260 Biotechnology Operations
the nature of that variation, its impact on the process, and the ability of pro-
cess controls to keep that variation within manageable levels. Hence, valida-
tion is an experimental endeavor based on deep knowledge of the process
and under which evidence is generated under a protocol. Perhaps, this is one
reason that validation is undertaken only in the later stages of manufactur-
ing development. Careful planning is an absolute requirement, and valida-
tion efforts are always included in a manufacturing plan.
A validation master plan is prepared, usually in late-phase development,
for any major validation effort. The scope of the plan is broad and the level
of detail is great. The overall philosophy and approach taken to validate the
facility and process are provided in the validation plan. The master plan
outlines the validation activities related to a facility, including the physi-
cal plant, environment and utilities, equipment—both installed and mov-
able—computer systems, software and hardware, critical raw materials,
biomanufacturing processes, with all stages and key steps, including aseptic,
cleaning, and monitoring processes or procedures. Many analytical tests are
also validated at this time (Chapter 7).
From the validation master plan, validation protocols are written to exper-
imentally examine each critical step of a manufacturing process. Validation
of a facility might involve dozens or even hundreds of protocols. A protocol
breaks down a system into simple parts and describes each critical parameter,
quality attribute, and operational specification. It ensures that attributes are
measurable and testable and that each measurement is scientifically sound.
Specifications are developed for results derived from each test or measure-
ment. Validation activities are spelled out in detail, usually with standard
operating procedures, production records, and testing instructions, and
these are identified in the protocol. The work is done by scientists and engi-
neers, working closely with quality assurance professionals, and it is normal
to employ consultants and contractors to assist employees with these her-
culean efforts. Good statistical practices are also utilized in most protocols.
Validation is fully documented, from the master plan to the final validation
reports. Validation has a pass-versus-fail outcome; either one meets all the
predefined specifications outlined in the validation or the validation fails.
Validations are labor intensive, time consuming, which make them costly.
With this in mind, the prudent biomanufacturer ensures that all systems are
performing as expected before executing validation protocols.
Technical steps are involved in the validation process. First, each piece of
equipment, utility, and facility component must undergo installation quali-
fication, a process to confirm that each item was correctly designed, built,
and installed. Limits for usage are also confirmed in installation qualifica-
tion. These are simply operational limits that could not be exceeded during
normal operations. For example, instructions and specifications limit the use
of a 10 L fermentation vessel to 5–8 L media volume. The next step is opera-
tional qualification, a verification process in which the operating ranges of
each item are confirmed under operational conditions. Items may be stressed

261Biomanufacture
to the farthest ranges of operational performance. For the fermentation ves-
sel, it might be tested, against specifications, three  times, with 5, 6.5, and
8 L media volume. In operational qualification, calibration is completed on
mechanical or electrical systems, and control systems are shown to work as
designed. Process qualification is the third step, in which the manufacturing
process is performed, typically three or more times, so as to demonstrate
control, consistency, and achievement of specifications. In process qualifica-
tion, critical systems are stressed to ensure that they function properly at
the limits of operating ranges. Two or three successful repetitions are the
norms under validation protocols, and each one must meet specifications.
Validation is not a one-time endeavor, and critical systems must undergo the
process of revalidation at periodic intervals, after market approval. In addi-
tion, any significant manufacturing change must be validated.
A primary outcome from validation efforts is the confidence that the
product will be successfully manufactured within specifications over a long
period of time. Validation also ensures that product quality, safety, and effi-
cacy are designed into the product as confirmed in early manufacturing
endeavors and that ongoing process monitoring will be a part of the product
manufacturing life cycle.
Summary of Biomanufacture
Biomanufacturing activities typically entail three major components: pro-
duction, purification and final fill/finish. It is a phased process, improved
throughout the life cycle of product development, aiming to ultimately yield
a sustainable process of high quality and adequate scale of production and
purification. The amount and quality of the biological substance must meet
clinical study and marketing requirements. Further, there is a diversity of
biotechnology products, and so, the manufacture of a given product is based
on a custom design, which is based on a carefully crafted biomanufacturing
plan. There exist precedence for manufacturing most types of biotechnol-
ogy products, but for any new product, no matter the amount of precedence,
actual production always requires careful planning and some trial and error.
Therefore, biomanufacturing requires a skillful and experienced team, and
patience and resources from the management. The result is a process that
consistently yields a pure and potent biopharmaceutical product.
Successful biomanufacture requires a well-designed product with product
specifications, because the design will change during the course of develop-
ment, it also requires design control and carefully stated technical consid-
erations. Further, the development process requires the use of appropriate
raw materials, a step-wise effort to increase the amount produced, scale-up,
quality control, and consideration of GMP. Early stages of biomanufacturing

262 Biotechnology Operations
include production of the construct, that is, the active ingredient, a molecule,
or a cell line. This is followed by production of cell banks and reference stan-
dards. Quality control assays and process controls are developed along with
the biomanufacturing process. Upstream processing, that is, the production
of the active ingredient in bulk, often impure form; downstream process-
ing, that is, purification; formulation; and filling of the FP require multiple
steps, each composed of a particular subprocess. There are as many subpro-
cesses as there are products. Product yield, activity, stability, and purity can
be drastically different based on the nature of the product and the selection
of the processes. Some examples of product classes are bacteria, viruses, pro-
teins, glycoproteins, DNA, RNA, cells or stem cells, transgenic plants or ani-
mals, and peptides, and they may be derived from biomanufacture involving
prokaryotic or eukaryotic cell lines, biological fermentation or culture, and
chemical synthesis.
Each manufacturing process occurs within an environment and environ-
mental controls that surround the manufacturing process. Most early phase
manufacturing is performed in small facilities, following the basic guide-
lines for aseptic manufacture and a proper environment. However, as the
scale of manufacture increases, the quality criteria also become stringent,
until at the later stages, where both the manufacturing process and the facil-
ity are validated. Appropriate facilities, utilities, and equipment are selected
to provide an acceptable environment, one that meets regulatory require-
ments. Production of the FP involves formulation, fill, and finish, a process
that is scrupulously aseptic.
References
Hamburg MA. U.S. Food and Drug Administration/FDA Strategic Priorities 2014–2018,
September 2015.
Woodcock J. 2005. American Association of Pharmaceutical Sciences-FDA-ISPE Workshop,
October 5.

263
7
Quality Control
Quality Control Overview
Quality Control (QC) is a laboratory endeavor aimed at ensuring that the
highest-quality biotechnology products are manufactured and released to
users. QC tests do not just happen; they are designed to meet certain objec-
tives and quality criteria. Indeed, their design follows the principles of
Quality by design (Chapter 5), which promulgates the concepts of superior
quality testing programs and is based on an understanding of the product
and the analytical tools used to test it. This chapter reviews the principles
of QC planning, describes the life cycle of QC test and product specification
development, identifies analytical methods most often applied to biophar-
maceutical development, discusses the qualification and validation of these
methods, and mentions the application of QC tests for product release and
stability.
In contrast to the management and administrative nature of Quality
Assurance (QA) (Chapter 5), QC is a technical or laboratory endeavor, which
uses analytical methods to achieve specific objectives in a biotechnology
operation. In the past, the terms Quality Assurance and Quality Control were
used interchangeably, particularly by regulatory agencies, and this was con-
fusing. Today, FDA still defines the term Quality Control as a largely adminis-
trative function, which is not the same as that applied in most biotechnology
operations. In this book, we define Quality Control as a laboratory or testing
function and Quality Assurance as a quality management and administrative
function (Chapter 5). Further, and as with any endeavor in biotechnology,
QC has developed a trade or technical jargon, so commonly used terms are
defined in this chapter and in the glossary.
The technical objective of QC is to apply laboratory testing to measure the
quality of materials, be they raw or in-process materials and bulk substance
(BS) or final product (FP), and whether the purpose of testing is at release or
over time for stability evaluation. Quality control planning involves several
steps, outlined in Chapter 1 and described in detail throughout this chapter.
Quality control planning leads to important documents, the Certificate of
Analysis (CoA) and the stability protocol. These are data-reporting formats

264 Biotechnology Operations
that identify product attributes, test methods, specifications, and, after the
completion of testing, the results.
There is a second dimension to QC planning, that is, consideration of the
biopharmaceutical QC development cycle, as shown in Figure 7.1. The cycle
identifies various QC functions in which processes are progressed, sequen-
tially, in many small steps, or generations, and in close coordination with bio-
manufacturing, clinical studies, and nonclinical testing. Each step in this cycle
is considered one generation in the life cycle of a given test method and involves
the test, the specification, and the product that is tested. Presumably, each gen-
eration of test and specification is a slight improvement over the past, and with
each step, there is a greater understanding of how test, specification, and over-
all product quality relate to each other for a given biotechnology product.
Quality control planning is complex in part because a biopharmaceutical
product requires many QC tests, each measuring an attribute. It is typical for an
investigational product to have 10 or more QC tests and for a marketed product
to have more than 20 tests. Together, the tests developed to support one product
are complementary, that is, one test adds to knowledge gained from the others.
Second, ongoing nonclinical and clinical studies often validate the usefulness
Begin: product
attribute
Analytical
requirement
Test design
Hypothetical
specification (S-1)
Final test and
specifications
Product
(P-1)
Final
specification
Refine specification
(S-2)
Refine specification
(S-3)
Develop test
(T-1)
Product
(P-3)
Redevelop test
(T-2)
Redevelop
test (T-3)
Product
(P-2)
Validate tests against
specifications
Test results
(R-2)
Test results
(R-3)
Test results
(R-1)
Final test procedure
Phase 2 clinical
Nonclinical
and phase 1 clinical
Phase 3 clinical
Phase 3 Clinical
FIGURE 7.1
Quality control cycle. The quality control test life cycle begins with an understanding of a
product’s attributes and the analytical requirement. An appropriate test is then chosen or
designed, and the development cycle then applies specifications (S), test development (T), input
of manufactured product (P), bulk substance or final product on which to perform the assay,
and results (R) from tests at that development step. It is a multistep process (e.g., S-1, S-2, S-3,
and final specification).

265Quality Control
or validity of a test by demonstrating biological activity of that product in ani-
mals or man. At some point in QC development, perhaps in the mid phase,
some tests are qualified, and later in development, all of them are validated.
These processes add considerably to confidence in using the assays.
Finally, QC testing is required for samples of BS and FP at release, for prod-
uct on stability protocol, and at several points during manufacture (in-pro-
cess testing). Multiply the number of QC tests with the number of times each
QC test is performed and again with the number of samples per test, and it is
clear why QC is a critical yet time-consuming function in biopharmaceutical
product development.
Quality control test development begins very early in biopharmaceutical
development, because all operations, especially biomanufacturing, require
excellent analytical support. Failure to have available analytical methods
often delays other efforts such as clinical and nonclinical studies and bio-
manufacturing process development. Time and again, this point is proven,
as biotechnology firms ignore the need to plan and develop QC technologies
and soon find that they are unable to evaluate product quality, and subse-
quently, the development program stalls.
We now describe each step in developing a panel of tests, using QC of BS
as the first example and then reviewing this process as it is applies to FP and
to stability testing.
Definition of Product Attributes
The QC development cycle begins with a Quality Control Plan, drafted only
after there is an understanding of the intended product, a treatment indica-
tion, and at least an early or research version of the candidate biopharma-
ceutical (Chapter 1). In addition, some experimental work must precede QC
planning, as it is not possible to develop a test for a product that has not been
at least slightly characterized in the research laboratory. For example, if it
is a protein, the primary, secondary, and tertiary structure, as well as the
molecular, cellular, or immunological basis for its activity, should be known.
Quality control planning begins once one understands how the product will
eventually be used, the intended treatment indication, and the mechanism
of action the product must have to achieve a desired endpoint.
An example product will be applied throughout this chapter to illus-
trate the principles of biotechnology QC. Consider a recombinant protein
(r-protein) product, a biopharmaceutical that functions in man by neutral-
izing an undesirable molecule, perhaps a complex carbohydrate, located
within a diseased cell. The therapeutic r-protein has the potential to ame-
liorate a disease. However, this r-protein must first enter the cell via a
receptor on the cell surface. Further, it is known that r-protein binding to
the cell receptor must be of that magnitude that triggers the cell to inter-
nalize the r-protein. In addition, the r-protein must survive within the cell
and bind to the target molecule, an undesirable carbohydrate associated

266 Biotechnology Operations
with cell death and disease. Binding leads to functional elimination of
the carbohydrate molecules from the cell. Further, to enter the cell, the
r-protein must be an intact molecule with proper primary and secondary
structure, that is, it should not be degraded or unfolded.
With such knowledge of a product and it’s mechanism of action, the QC
planner begins by developing a list of attributes for the product. An attribute
is simply a desirable or necessary characteristic that a must possess to be safe
or effective. In biopharmaceutical development, the most commonly applied
attributes are appearance, identity, strength, purity, and potency. Returning
to our example, several attributes of the r-protein—binding to a cell recep-
tor, internalization into the cell, survival within the cell, and binding to the
target molecule—are easily identified. Each of these attributes is listed in a
typical CoA, and some appear several times, as they are measured with dif-
ferent tests. The CoA, to be described later, is a formal document that lists
each attribute, QC test, specification, and the test result.
Further to our example, the active ingredient in the example product is the
r- protein. It is also referred to in testing parlance as the analyte or test sub-
strate, the material we wish to measure in whatever way. The r-protein also
possesses attributes. When in solution, it has a distinct appearance. Our
r-protein has an identity, just like every human has a distinct fingerprint.
In a given configuration, such as a vial of FP, the r-protein has a particular
strength or level of active ingredient (e.g., a concentration). It also has purity,
and, as with most products, impurities accompany the r-protein, even if pres-
ent in trace amounts. Finally, like all biopharmaceuticals, the r-protein has a
biological potency, in that it affects a biological system. A biopharmaceutical
may possess other attributes, but this list is adequate to begin planning QC
tests for most biotechnology products.
Analytical Methods to Measure Attributes
The heart of QC testing is developing and applying tests that measure a
product’s attributes. Quality control scientists map a strategy, matching tests
to particular attributes and sometimes applying multiple tests for each attri-
bute. Further, they determine whether an attribute requires, or deserves, a
qualitative or quantitative measurement. This necessitates an understanding
of what makes an assay a good means of measuring an attribute.
Nonetheless, a perfect match between a method and an attribute is often not
possible. In such cases, QC scientists adapt a given analytical method to suit
the exact nature and intended use or indication of their product. Fortunately,
most analytical tools are quite adaptable. Even then, some attributes for a
given product cannot be measured with off-the-shelf analytical methods or
even with available but slightly modified tests. Here, QC scientists must be
creative and design a new or unique analytical tool, right from the begin-
ning… This is often the case for potency tests, which measure complex bio-
logical functions such as an immune or cellular physiological response.

267Quality Control
Traits of Analytical Methods
Each assay has one or more unique traits, somewhat like a facial feature, that
distinguish the assay from other analytical tests and make it attractive for a
particular application. Some tests have traits of qualitative analysis, others
of quantitative analysis, and a few possess both. Tests are selected for their
traits, a description of what they can really do for us. The QC scientist must
have a pool of analytical methods available, at least in theory, once he or she
begins to match a test to measurement of each attribute. Fortunately, a num-
ber of analytical tools, each with its own peculiar trait or traits, are available
for testing common attributes of many biopharmaceuticals.
One trait of any test is system suitability, which means that the chosen ana-
lytical method must be appropriate in all ways for the intended purpose and
measurement. Specificity is the second trait, and it means that a test has the
ability to measure the intended product and nothing else that might be in the
test material. Precision is the closeness of agreement between several mea-
surements, much like precision in shooting an arrow means coming close to
one point on the target with several arrows. The trait of linearity is applied
when the assay must generate a linear curve. Linearity means that the results
are directly proportional to the concentration of the analyte. Range, closely
related to linearity, is the interval between the upper and lower concentration
of analyte in the linear part of the curve. Limit of detection (LOD) is to under-
stand how little of the analyte can be reliably detected in a sample. Limit of
quantitation (LOQ) defines the lowest amount of analyte that can be quanti-
tatively measured and not just simply detected. The trait of robustness car-
ries many related meanings, but overall, it means that a test is reliable with
respect to normal or expected variations in the analytical or testing environ-
ment. For example, if three operators obtain the same result after each of
them performs the test on three different dates, then an assay is robust and,
one would think, reliable. Each trait will be discussed further in this chap-
ter. Analytical tools are chosen to measure an attribute only if they possess
traits that allow them to complete a stated measurement. Hence, possession
of proper traits is a criterion for choosing the proper analytical tool. To para-
phrase a saying of automobile mechanics, you must select the right analytical
tool for each job.
Drafting a Certificate of Analysis (Bulk Substance)
As attributes and methods are identified, they are listed in tabular form on
a draft document referred to as the CoA, as shown in Table 7.1 for BS and
in Table 7.2 for FP. Each attribute, further defined below, are listed in the
first column of the CoA. More than one test may be applied to an attribute,
as each test measures a different parameter of that attribute. This discus-
sion and the accompanying tables use examples of commonly applied QC
or test methods, described later in this chapter, and reflect the analysis of
our r-protein example product.

268 Biotechnology Operations
TABLE 7.1
Certificate of Analysis for a Biopharmaceutical Bulk Substance (r-Protein Example)
Attribute Analytical Method
Reference to
Method Specification Result
Appearance Visual inspection of BDS
in clear glass tube
SOP# QC01 Liquid, opaque, off-white
to straw color, no
particulates or aggregates
Safety Microbial enumeration
test
USP <61> <1 cfu/mL Identity N-terminal sequence SOP# H411B Confirm known sequence Identity SDS-PAGE SOP# QC-02 Single band at 41 kDa Safety Bacterial endotoxin (gel- clot LAL test) USP <85> <1.0 EU/mg protein Purity SDS-PAGE SOP# QC-03 Single band, comparable to reference standard Purity HPLC SOP# QC-04 Single peak integrated ≥98% of material in sample Purity Aggregates by size exclusion chromatography SOP# QC-05 ≤2% of material is aggregate Purity Peptide map SOP# QC-06 Map equivalent to reference standard Purity Host cell protein, ELISA SOP# QC-118 <0.1 mg host cell protein/ mg total protein Purity Host cell DNA, fluorescence probe SOP# QC-120 <10 µg host cell DNA/1 mg total protein Strength Total protein by BCA SOP# QC-07 1.0 ± 0.1 mL containing 2.0 ± 0.1 mg/mL protein Purity Silicon lubricant by atomic absorption Contract laboratory SOP# X147–1 <10 ng silicon/mg total protein Purity Aggregated protein Aggregated protein by light scatter SOP# QC-08 <7 µg aggregated protein/1 mg total protein Potency Receptor binding SOP# QC-111 0.6–1.05 µg r-protein/1.0 µg receptor Potency Viability of cultured cells at 1, 2, 4, 6, and 12 h SOP# QC-09 and SOP# QC-10 >70% viability versus time
0 at each time point
Potency Accumulation of
molecule in cultured
cells at 24 h
SOP QC# 11
and SOP#
QC-12
<10% accumulation over baseline, time 0 Prepared by/Date:___________ Approved by Quality Control/Date:__________ Approved by Quality Assurance /Date:______________ 269Quality Control TABLE 7.2 Certificate of Analysis for a Biopharmaceutical Final Product (r-Protein Example) Attribute Analytical Method Reference to Method Specification Result Appearance Visual inspection of FDP in final container SOP# QC-21 Clear, colorless liquid without particulates or aggregates Safety Endotoxin gel-clot LAL USP <85> ≤10 EU/1 mL dose
Safety Sterility, compendial USP <71>
21 CFR 610.12
Sterile
Safety Osmolality by
osmometer
SOP# QC-26 200 ± 10 mOs/kg
Safety pH by pH meter
microprobe
SOP# QC-27 7.1 ± 0.2
Identity N-terminal sequence SOP# H411 Confirm known
sequence
Identity SDS-PAGE SOP# QC-22 Single band at 41 kDa
Purity SDS-PAGE SOP# QC-23 Single band at
30 kDa, comparable
to reference
standard
Purity HPLC SOP# QC-24 Single peak
integrated >98%
material in sample
Purity Excipient glycerol
atomic absorption
SOP# 11–4422C 1 ± 0.1 μg/mL
Purity Excipient, human
serum albumin by
ELISA
SOP# 11–2244C
contractor
200 ± 20 μg/mL
Strength Total protein by BCA SOP# QC-25 1.0 ± 0.1 mg/1 mL
dose and per vial
Purity Aggregated protein
by light scatter
SOP# QC-28 <1 µg aggregated protein/1 mg total protein Potency Viability of cultured cells at 1, 2, 4, 6, and 12 h SOP# QC-09 and SOP# QC-10 >70% receptors
versus time 0 at
each time point
Potency Accumulation of
molecule in cultured
cells at 24 h
SOP# QC-11 and
SOP# QC-12
≤10% accumulation
over baseline time 0
Potency Reduction of disease;
transgenic mouse
model
SOP# QC-23,
SOP# QC-24,
and SOP# QC-25
>50% reduction as
compared to control
Prepared by/Date:___________
Approved by Quality Control/Date__________
Approved by Quality Assurance /Date:______________

270 Biotechnology Operations
• Appearance: Most products have a distinct appearance to the eye. Bad
product often looks bad. Under appearance, traits may be further
defined as color, clarity or opaqueness, or presence or absence of
particulates or aggregates.
• Identity: This trait simply ensures that the product is what we believe
it to be, and what we have labeled it as, and not something else. Each
biotechnology product is unique and possesses fingerprints that can
be analyzed.
• Safety: A safety test cannot by itself tell us whether a product is safe
or not, but it can provide some assurance that it is not overtly toxic or
otherwise lethal or overtly harmful to the user. In addition, multiple
safety tests, each examining a specific aspect of the attribute, can
additively increase the chances that a product is, in fact, safe.
• Purity and impurities: All biotechnology products are purported to
be pure, that is, to have only those molecules or cells or other active
ingredients they are supposed to contain. Purity is a measure of that
product claim, and impurity testing informs us as to the nature and
amount of anything else in the product vial.
• Strength: It is important that each product have enough of the active
ingredient, so that it has the potential to cause the intended effect. If
we say that there is 25 mg/mL of product in 1 mL per vial, then there
should be 25 mg/mL and 1 mL in each vial.
• Potency: This is the most challenging trait to measure. It tells us that
the product, in fact, has the intended biological effect.
This is the first step in drafting a CoA. The next step, as shown in column 2
of  Tables 7.1 and 7.2, is to identify analytical methods to measure each of
these attributes.
Selection of Analytical Methods
This section provides information on selecting a test to measure each attri-
bute; by way of example, it identifies a few assays commonly applied to
recombinant protein biopharmaceuticals in BS (column 2, Table 7.1). Selection
of tests for FP is discussed in a later section, and a list of assays, with techni-
cal descriptions, is presented there and in Table 7.2.
Laboratory tests used in QC, also referred to as analytical methods, or
just methods, can be classified in several ways. Tests described in a compen-
dium (e.g., a pharmacopeia) are nationally or internationally recognized
and are performed in a very standard manner, no matter what the product.
Tests are matched to attributes in the example CoA, and in column 3 of
Tables 7.1 and 7.2, reference is made to the specific manner in which each
test is performed on BS.

271Quality Control
A compendial test, such as that for sterility, microbial limits, or endo-
toxin, is applied to a wide range of biological and pharmaceutical products.
Examples of compendia, technical handbooks, or encyclopedia are given in
Box 7.1. An outline and contents of the United States Pharmacopeia (USP 2015),
a commonly used compendium and QC reference, are given in Box  7.2,
BOX 7.1 EXAMPLES OF COMPENDIA AND REFERENCE
TEXTS FOR QUALITY CONTROL
• United States Pharmacopeia (USP): The official pharmacopeia
for the United States, is published by the U.S. Pharmacopeial
Convention (http://www.usp.org). The USP, along with a sister
publication The National Formulary, or USP-NF provides test
methods, standards, and references for analysis of medicines,
reagents, and other materials. If an analytical method and a
standard are applicable to a biopharmaceutical and they are
approved and published in the USP, then it is very likely that
FDA will demand that product testing meets this standard.
Specifications may or may not be recommended by USP.
• European Pharmacopoeia (EP): The official pharmacopeia for the
European Union, developed by the European Directorate for
the Quality of Medicines (http://www.edqm.eu).
• British Pharmacopoeia (2016): The official pharmacopeia for
Great Britain (http://www.pharmacopoeia.com).
• Merck Index (2011): This encyclopedia provides precise and
comprehensive information on chemicals, drugs, and biologi-
cals written as monographs and carefully indexed. It is used
in biopharmaceutical development for materials and products
that are well characterized and may be predicate or compara-
tors for a novel compound in early development.
• Merck Manual (of Diagnosis and Therapy) (2011): A medical ency-
clopedia, organized by disorders or diseases of various sys-
tems or organs. It explains the symptoms or diseases, their
diagnosis, and their treatment. It is a leading medical reference.
• Martindale’s: The Complete Drug Reference (2015): A very com-
plete reference book that provides monographs, albeit brief,
on thousands of drugs and biologicals with reference citations
and manufacturers. It is carefully indexed.
• Physician’s Desk Reference (2015): A collection of the product
labeling of the most commonly used drugs and biologicals in
the United States. It has information on drug indications, dos-
ages, side effects, and detailed instructions for use.

http://www.pharmacopoeia.com

http://www.edqm.eu

http://www.usp.org

272 Biotechnology Operations
BOX 7.2 AN OUTLINE OF THE UNITED STATES
PHARMACOPEIA AND EXAMPLES OF ITS SECTIONS
RELATED TO BIOPHARMACEUTICALS OR DRUGS
• General Information: Provides guidance on a variety of product
classes spanning the spectrum of drug and biopharmaceuti-
cal development. Examples are general guidelines for oph-
thalmic preparations and water for pharmaceutical purposes.
Section <1231> identifies classes (qualities) of waters and their
standards.
• Reagents: All types of reagents and their quality standards are
described in this section. Examples are acetic acid (diluted),
used in various test procedures, and pancreatic digest of
casein, used in culture media.
• National Formulary (NF): Here are the recipes for making a host
of products that are used in or as drugs, including many over-
the- counter preparations. An example is the procedure for pre-
paring the peppermint solution added to certain oral drugs.
• Official Monographs: In this section of the USP are monographs
on commonly used drugs; many drugs described here are long-
standing and generic. An example is aspirin delayed-release
tablets. Others are products used largely in medical treatment
facilities, such as lactated ringers and dextrose solution.
• General Tests: Many tests that are used in drug and biopharma-
ceutical quality control are found under this heading. Certain
tests used for biotechnology products are described in great
detail. Examples are as follows:
• <621> Chromatography: Gas, Paper and Column
• <85> Bacterial Endotoxin Tests
• <71> Sterility Tests
• <61> Microbial Limits Tests
• General Tests also covers information that provides guidance
and procedures for biopharmaceutical testing and assay devel-
opment, in general. Examples are as follows:
• <111> Design and Analysis of Biological Assays
• <1041> Biologics
• <1045> Biotechnology-Derived Articles
• <1046> Cell and Gene Therapy Products
(Continued)

273Quality Control
along with the titles of some compendial tests commonly used to measure
attributes of biopharmaceuticals. Methods of compendial tests cannot be
easily modified, but sometimes they are adapted for novel applications.
Another group of analytical methods, often not found in a pharmaco-
peia and referred to here as generic tests, is used to measure the attributes
of identity, purity, strength, and, sometimes, potency of biotechnology
products. Although they are not compendial, there may be industry or
regulatory precedence, procedures, or even quality standards for their per-
formance. This depends on the nature and history of the product. These
QC tests can be established in most laboratories, or if they require expen-
sive instrumentation, they can be performed by a contract research orga-
nization (CRO). Most are readily adaptable to a variety of products, and
in many cases, their methods can be changed to suit specific purposes. A
biologics sterility test, described in FDA regulations (21  Code of Federal
Regulations [CFR] 610.12), or visual appearance of a product would be con-
sidered generic tests. It is worth noting that even some commonly used
tests (e.g., high-pressure liquid chromatography [HPLC] or polyacrylamide
gel electrophoresis [PAGE]) are performed in a very specific manner for
each analyte, and, at most, published guidelines, such as in USP or by FDA,
are general in nature.
A third group of tests includes those developed for one product or a few
closely related products. These methods (some examples are given later in this
chapter) often originate in a research laboratory and are further developed,
adapted, and refined by the QC laboratory for use as a QC test to measure an
attribute of a specific biological product. Biological potency assays often fall
under this group of tests.
Quality control assays are also classified in yet another way, that is, by
their intended use or application. These include tests for raw materials, for
BOX 7.2 (Continued) AN OUTLINE OF THE UNITED STATES
PHARMACOPEIA AND EXAMPLES OF ITS SECTIONS
RELATED TO BIOPHARMACEUTICALS OR DRUGS
• <1047> Biotechnology-Derived Articles—Tests
• <1048> Quality of Biotechnological Products: Analysis of
the Expression Construct in Cells Used for Production of
r-DNA-Derived Protein Products
• <1049> Quality of Biotechnological Products: Stability
Testing of Biotechnological/Biological Products
• <1050> Viral Safety Evaluation of Biotechnology Products
Derived from Cell Lines of Human or Animal Origin.

274 Biotechnology Operations
in-process testing, for drug substance, for drug product, or for stability test-
ing of any material, substance, or product. Hence, a given method might be
applied at one or more points in the product manufacturing cycle.
As noted earlier, another means of classifying QC tests is by their intended
outcome or application. Examples of test applications are appearance or
description, identity, purity, impurities, potency, quality, and special tests.
It is not unusual for a very adaptable test method to be used to measure two
attributes. For example, one test may be used to measure both identity and
purity. A test is also classified according to the method’s enabling technol-
ogy, such as identification of bacteria and yeast, pH measurement, HPLC
chromatography, peptide mapping, or receptor binding.
The information entered into the third column of the CoA (Figure 7.1),
references by name the test, or procedure used. For compendial tests, ref-
erence is made to a section of a monograph to describe the sterility test
(e.g., USP <71>). For noncompendial tests, standard operating procedures
(SOPs) are identified in this column, citing the number of the SOP. If a CRO
performs a test, then that laboratory and the SOP used by it are identified
under the test method column of the CoA.
To summarize, a key element of QC planning and performance is match-
ing the proper test to an attribute, and this requires knowing which tests
are available to the analyst and understanding how the attribute relates to
each available test. Only then can a meaningful panel of tests be selected
for the product. We return to our example in an attempt to better explain
the matching process of attribute-to-test method. First is the identity test.
Identity tests reveal whether or not a product is, in fact, the intended mate-
rial, that is, the fingerprint of a biopharmaceutical. For the r-protein exam-
ple, we know from research that it is a globular protein of known molecular
weight and a defined sequence. How should we ensure that the material we
manufactured is, in fact, that r-protein? A common approach is to sequence,
by amino acid determination, from the N-terminus of the r-protein until
about the tenth amino acid. It is highly unlikely that another protein would
have the same 10 amino acids in that order at the N-terminus. One might
also identify the isoelectric point of a protein by performing electropho-
resis at various conditions, for example, various pH and ionic strengths.
Another approach to demonstrating identity is to measure the molecular
weight of the molecule under reducing or nonreducing conditions by gel
electrophoresis. Although not as definitive as N-terminus sequencing, this
test differentiates the analyte from many other proteins. Gel electrophore-
sis is made more powerful as an identity test if, after the electrophoresis
step, the protein is blotted to an inert but absorbent membrane and then
probed with an antibody specific for the product. This is known as Western
blot test.
A sample of reference standard, that is, a protein known to be the desired
protein, is always tested in parallel with a test sample. If results of the test
sample match theoretical or expected values and the results obtained from

275Quality Control
testing match the reference standard, then there is a high probability that the
test sample is the intended molecule.
The strength of a preparation is a general measure of how much of the
desired active ingredient is in the product. For the r-protein, strength
might be reflected in the total amount of protein, as long as the vast major-
ity of protein in the sample is, in fact, the r-protein. How do we ensure
this is the case? First, we perform identity testing on that sample to know
whether the molecule in the sample is the intended r-protein. Second, and
as described below, we measure the purity of the molecule in a sample
of product. Back to the concept of strength, for a recombinant protein,
this may be measured by a total protein assay, such as bicinchoninic acid
(BCA) assay, or by ultraviolet absorbance at a specific wavelength in a
spectrophotometer. Then, this measurement, given in milligrams per mil-
liliter, is multiplied by the percentage purity and total volume in millili-
ters to give the total amount, in milligrams, of the desired protein. This
paradigm demonstrates the interdependence of various QC tests and the
need to interpret the result any one assay may give in relation to the result
from another assay.
Several test methods may be applied to measure the purity of any product.
Further, major impurities are characterized. Methods are developed to mea-
sure the level of product purity, that is, the percentage or the actual amount
of the stated product and the percentage or the amount of all impurities
and contaminants. Other methods are then used to identify specific major
impurities or contaminants to include even tiny amounts of potentially
toxic or otherwise undesirable substances that might have entered into the
production stream and remained in the product. The development of test-
ing schemes and the selection of tests is based on an in-depth understand-
ing of the raw materials, equipment, and processes used in manufacturing,
as well as of the scope of possible impurities or contaminants. Impurities
and contaminants are further discussed in relation to biomanufacturing
(Chapter 6).
Returning to our example of r-protein, we consider the purity of that mol-
ecule as well as impurities that might exist with the r-protein as BS. Here,
r-protein purity is measured by sodium dodecyl sulfate PAGE (SDS-PAGE),
an assay also used as an identity test. However, this assay is, at best, only
semiquantitative in that it cannot accurately measure the amount of the r-pro-
tein or the amount of impurities in the sample of BS. It may, however, give
a reasonable estimate of purity. The SDS-PAGE test is often used in-process
to follow the progress as molecules are purified across multiple bioprocess-
ing steps (Chapter 6). A more exact method is applied to the measurement
of r-protein purity by using an analytical chromatographic technique; HPLC
is a common choice. Here, the example r-protein should appear on the chro-
matogram as an independent, major peak, whereas impurities might appear
as smaller side peaks or shoulders of the major peak. Further, we might
use size exclusion chromatography (SEC) to show that the molecule in our

276 Biotechnology Operations
preparations is not aggregated. This test demonstrates that the r-protein has
maintained a native form and that it has not otherwise distorted through
clumping. Yet another purity test, peptide mapping, may reveal that molecu-
lar integrity is still present. Protein aggregates are measured by light disper-
sion, if their presence is suspected. Special tests such as mass spectroscopy,
nuclear magnetic resonance, and capillary electrophoresis may be consid-
ered. Carbohydrate analysis may be applied if there is a need to examine cer-
tain posttranslational modifications. Today, many new methods are applied
to molecular characterization of certain products and some are noted later
in this chapter. Since purity is critically important to molecular integrity and
function and because impurities must be characterized and measured to
prevent them from causing undesirable reactions in the consumer and from
increasing in amount with later production, several purity and impurity tests
are typically applied to a product, both as BS and as FP.
Detection of contaminants presents a different challenge because these
can enter the stream from so many sources, especially if a failure goes
undetected during processing. For example, most proteins are filtered at
some point during purification and filters may fracture and release fibers,
particles, or fragments into the product stream, thus contaminating the
product. Contaminants, particulate and soluble, may enter the product
stream from raw materials or virtually any substance that contacts the
product stream. Given that one cannot test for every possible material that
might contaminate a biotechnology manufacturing process, what should
the QC scientist consider as possible contaminants for any given product?
Anything that might be toxic or otherwise dangerous to the user in small
amounts and is a part of the process comes first to mind. For example, if
there is a possibility of bacterial growth, then endotoxin testing, described
below, is critical. If there is a piece of biomanufacturing equipment that is
essential but is known to sometimes shed particulates of silicon lubricant
into the product stream, then it might be wise to test for silicon lubricant.
Both endotoxin and silicon lubricant are considered in the example CoA
for BS, as shown in Table 7.1. Clearly, an effective yet affordable contami-
nant testing program involves discussion between manufacturing and
control staff, with decisions based on full understanding of the production
processes and the intended use.
Potency assays are critical to a QC testing scheme because they are used
to predict whether the product will function as it was designed to func-
tion. It would be futile to produce any biopharmaceutical product and test
it for purity, identity, and safety and still not know if it could function
as intended. Unfortunately, this is far too often the case with biopharma-
ceutical development programs. For testing BS, potency assays are often a
surrogate assay, meaning that it does not directly measures the biological
function in a complex system such as a whole animal but instead measures
a physiological attribute of the product in an in vitro or a cell-based assay.
Surrogates are used in all aspects of biotechnology development, but any

277Quality Control
surrogate measure must be appropriate, well designed, and, eventually,
validated against the intended use. The QC scientist developing a surro-
gate assay must be knowledgeable about both the product and the thera-
peutic indication, particularly the mechanism of action and the biology
and molecular biology involved in the product’s therapeutic effect. Using
this knowledge, scientists become inventive, even crafty, in finding analyti-
cal methods that predict potency (or lack thereof) while keeping those tests
as simple, inexpensive, and practical as possible. The best source of new
potency tests is the research laboratory.
Returning to the example of the r-protein and examining Table 7.1 further,
we see that two potency assays were developed for this BS. One measures
specific binding of the r-protein to its cell-surface receptor. The rationale is
that receptor binding is a critical step in the pathway to molecular activity and
a biologically active r-protein must bind to that receptor. For this example,
the receptor was identified and the gene was cloned in a research laboratory,
so that it is now produced in small amounts (enough for testing purposes).
Using analytical instruments, QC scientists next develop an in vitro assay
that measures the amount of r-protein that binds to a given amount of the
receptor. For example, between 0.6 µg and 1.0 µg of the recombinant prod-
uct binds to 1.0  µg of the receptor. On repeatedly testing three batches of
r-protein with the newly developed assay, scientists determine that between
0.75 µg and 0.92 µg of the product binds to the receptor in this test. This test
is chosen as one of the two potency assays for r-protein in the BS.
For an attribute as important as potency, one always considers two or
more complementary assays. This is because a single assay measures only
one aspect of a product’s potency attribute. In the r-protein example, the QC
scientist chooses a second assay, also developed in the research laboratory,
to measure the degree to which r-protein inhibits the buildup of the unde-
sirable molecule within the target cell. This is a relevant surrogate to the
intended biological response because it measures the ultimate activity asso-
ciated with therapeutic value, at least at the molecular and cellular levels.
Shown in Table 7.1 as the last assay on the CoA, the measurement is an in
vitro assay, which is likely rapid and inexpensive but hopefully very precise
and sensitive to product activity.
This section of provided an overview of the early test selection, basing
each assay on a product attribute. After this process has completed, the next
step in the planning process is consideration of specifications. Later in this
chapter, we discuss in greater detail analytical tests and their application to
the QC of biopharmaceutical products.
Development of Specifications
Identifying a test to measure each attribute is important, but it is also critical
to know whether each batch or lot passed or failed. This decision is based on
the results obtained when the test is applied to a particular batch of product.

278 Biotechnology Operations
The word specification carries great meaning to both QC scientists and bio-
pharmaceutical development projects. A specification is a descriptor, numer-
ical or verbal, that a product must achieve to be considered suitable for use.
It also serves as a requirement or condition, the basis upon which a product
is accepted or rejected. Specifications may be established by regulation, by
precedence and proven value and capability, by outside guidance, or by a
product development team. Often, a specification is quantitative, stated as a
range of values (e.g., ≤12.5 units or 1.0–3.0 mg/mL or 2.0 ± 1.0 mg/mL), but
it may be qualitative, a term that compares the specification to a reference,
such as comparable to values of reference standard #0017, or it can be purely a
descriptor such as clear, colorless solution free of particulate matter. Examples of
specifications for BS are shown in the fourth column of Table 7.1.
During the development process, that is, before submitting a marketing
application, many specifications may be considered interim or temporary.
However, specifications codified (e.g., sterility test in 21 CFR) or established
by precedence or compendium (e.g., sterility test in USP) are less flexible and
are typically inviolable during the development life cycle. Specifications are
taken quite seriously by both regulatory authorities and the sponsor; indeed,
final or ultimate specifications established through the validation process in
Phase 3 guide the release of a marketed biotechnology product in the years
to come. Specifications for marketed products may be changed, but this is
done only with scientific evidence to support the adjustment and follows
strict change control rules. Hence, there is a great need to carefully choose
and then fully develop a specification during the development process, bas-
ing interim and final decisions on experimental data generated by testing
multiple lots of the manufactured product.
Advances in analytical technologies for biopharmaceutical products have
increased the number of tests used on any product. Regulatory authorities
are quick to suggest yet another test that might ensure safety or better pre-
dict efficacy of a product. In addition, specifications themselves have become
more complex, quantitative, and sensitive. Indeed, the role and importance
of the QC function to biotechnology development has grown considerably
over the past 30 years.
Establishment of specifications for purity or impurities is challenging for
biopharmaceutical product development teams, as the questions raised have
no simple answers, especially because the data are limited or do not exist at
the time. What quantitative limits are acceptable for purity and impurities,
and what is allowed and how much? The correct answer must be based on
highly regarded existing scientific data, and, for a unique product, it must be
experimentally determined for each product. Thus, a final purity specifica-
tion is not finally established until late-phase development, after much data
have been generated. For many products, there are at the outset of testing
no existing guidelines for purity specifications or impurity characteristics.
At first, the sponsor must assume that a product will not be 100% pure and
that impurities will exist in both the BS and the FP. Upstream manufacturing

279Quality Control
generates, and downstream processing concentrates, certain impurities and
contaminants; no product is expected to reach 100% purity by using current
purification technologies. Upstream production is a dirty process, whereas
downstream processing, notably chromatography, may introduce and con-
centrate novel, yet undesirable, impurities and contaminants, even while
removing other impurities or contaminants and concentrating the product.
Contaminants enter the stream out of necessity, because they are inherent
to a required process, a necessary evil. Examples of contaminants are endo-
toxin, in some systems shed by the very recombinant bacteria making the
product; chromatography gels or matrices; particles from vessels and tubing;
and chemicals leached into the product stream from various contacts and
surfaces. Impurities are the molecules that are derived from the ingredients
used to make the product but are not wanted in the BS or FP. These include
cellular debris and molecules derived from the cells in which the recom-
binant protein or tissue was produced and the components of the nutrient
medium that fed those cells. Impurities are also seen as breakdown prod-
ucts of the desirable biomolecules and include improperly folded protein,
shortened versions of the protein, posttranslational product variants, and
fragments or aggregates of the protein.
As noted before, a rule of thumb for establishing purity and impurity
specifications in biotechnology is that the BS or FP specification should be
that the product is at least 95% pure. However, this rule does not apply to
every biopharmaceutical, and specifications for purity must be developed
based on the intended use and attributes of the product and the nature of
the impurities. For example, a protein that will be used to enrich cattle feed
might be fine at 75% purity, as long as none of the impurities were toxic to
cattle or man and consisted largely of protein fragments and aggregates. A
biopharmaceutical intended for injection at large doses in human patients
with serious disease might need to be well more than 99% pure and com-
pletely free of any toxin or foreign or aggregated protein. Here again,
careful planning is required to ensure that analytical methods and speci-
fications developed for purity and impurities match exactly the intended
use and other attributes, for example, safety, of a product. Impurities such
as a virus or lethal toxin are simply not allowed in a biopharmaceutical.
However, if such materials could possibly have been introduced, meth-
ods must be designed to ensure their removal and highly sensitive and
specific tests should be introduced to ensure that product is free of such
substances. Here specifications are very stringent. The guidelines for set-
ting specifications for other impurities or contaminants are established
based on prior experience, on the probability that they do in fact exist,
on the availability of tests to identify or measure them, and, mostly, on
common sense and good scientific practice. As noted in Chapter 6, speci-
fications for levels of impurities or contaminants are, in the end, often
negotiated between the sponsor and the regulatory agency. This brings
up a final point on the subject of setting specifications for impurities, both

280 Biotechnology Operations
qualitative and quantitative. The views of national populations and their
regulatory agencies vary greatly on the perceived risk of certain impuri-
ties to the user, and the biotechnology firm should consider all market-
places and national or international guidance, not just the United States,
when establishing specifications for a product.
There are other outcomes to the processes of establishing and applying test
methods and specifications in this cycle. Sometimes, a well-considered ana-
lytical method fails miserably and is not predictive of the attribute or is oth-
erwise unable to predict product quality. There is no need to consider further
refinement of the test or of the specification. In other instances, the hypotheti-
cal value established by QC scientists is not at all close to the experimental
values. In such cases, the assay may be further studied or it may be reworked
to either explain the differences or to optimize the method. More often than
not, however, a well-considered analytical method is meaningful to measur-
ing product quality, with only the need to adjust the specification, and thus
ensures consistent quality for future batches or lots of product.
Some QC tests have, in the eyes of regulatory authorities, absolute specifi-
cation requirements. As noted before, sterility tests are performed by com-
pendial methods and they must meet standards published in a compendium
or by regulatory agencies. There is, in the eyes of regulators and the market-
place, but one definition of sterility, and adjustment of the sterility specifica-
tion is simply not acceptable for a biopharmaceutical such as the r-protein
mentioned in the earlier example. Other historical tests, even certain com-
pendial methods, may allow specification variance, but this depends on the
nature of the product and the risk to the user associated with a change in
specification. Such changes must be negotiated with regulatory authorities.
For example, the appearance and description for a parenteral biopharmaceu-
tical, formulated as protein in a buffer, is expected to read clear solution with-
out particulates. However, for some proteins at high concentrations in a buffer
solution, it may be normal for the product to be cloudy or opaque. Hence,
a highly concentrated protein solution may be allowed to deviate from the
standard specification for most protein solutions and be considered accept-
able if it has a specification for appearance of cloudy colorless solution without
particulates or foreign matter.
Establishing specifications for contaminants and impurities is a chal-
lenging task, because it is impossible to know in early development how an
impurity might impact the safety or efficacy of a product. How does one
evaluate the impact of minute amounts of a given contaminant, unless it is
a known toxin? Establishing purity and impurity guidelines has led to long
discussions within the international biopharmaceutical community. These
discussions are based on the risk posed by certain impurities found in some
products, formulation, dose, or patient population. An example relevant to
biopharmaceutical protein preparations is the impact that protein aggregates
might have on parental products. Years ago, it was felt that they had little
impact on product safety or potency, unless they were present in significant

281Quality Control
amounts. Even then, a definition of significance was elusive. Recent evi-
dence suggests but does not prove that protein aggregates, even in small
amounts, may be immunogenic and potentially elicit an antibody response
in a patient to a recombinant protein. If this is the case, the impact is prob-
ably quite variable, depending on the route of delivery, amount given over
the lifetime of the patient, exact nature of the protein and the aggregate, and
the patient. Then how does one establish a specification for maximum allow-
able amounts of aggregate for any given protein product? Panels of experts
may be called to address such questions, but even then, recommendations
tend to be fuzzy. Hence, challenges to planning product attributes, tests, and
specifications continue through the life cycle of product development and
also into marketing phases.
As with other attributes, specifications for potency of product in BS are
established early in development, even when little experimental data are
available. Potency tests, identified in the planning process, are created in
a laboratory and then used to analyze some early batches or lots of BS or
FP, respectively. Referring back to the development of the r-protein receptor-
binding potency assay (Table 7.1), we know that in theory, 1.0 µg of the recom-
binant product should bind to 1.0 µg receptor and also that laboratory testing
revealed a range of binding activity, that is between 0.75 µg and 0.92 µg of
pure r-protein binds to 1.0 µg receptor. At this time, the QC scientist must
establish a specification for this potency assay based on both theoretical and
derived experimental data but with the knowledge that this data might,
by chance, reflect an incorrectly high or low estimate of the actual binding
activity. The early or hypothetical specification, referred to as S-1 in the QC
cycle drawing (Figure 7.1), is the following specification in this example:
Range of protein binding, 0.60–1.05 µg of product per 1.0 µg of receptor (Table 7.1).
After testing additional and subsequent batches of BS, the sponsor might
discover that the range of values is too broad and, based on the data, narrow
the range of acceptable values in the specification, perhaps to 0.80–0.90  µg
protein per 1.0 µg of receptor (e.g., the refined specification, S-2, in Figure 7.1).
Alternatively, with additional data, the original or S-1 specification could be
a firm estimate, holding up to experimental results and remaining the same
throughout the product development life cycle. This then is the accepted
process, experimentally intensive but proven effective to establishing mean-
ingful tests, test results, and specifications.
The other potency assay for BS used in our example (Table 7.1) measures
another trait important for measuring the activity of the r-protein, the ability
to halt buildup of a molecule inside cells. The specification was established
in the same manner.
In summary, the early establishment and then later adjustment of a specifi-
cation is normal part of QC testing in the overall product cycle. In early devel-
opment, the process is a mix of scientific, iterative, and intuitive approaches
and later, it becomes heavily scientific, driven by data from clinical, nonclini-
cal, and laboratory studies.

282 Biotechnology Operations
Entering Test Results
Results are added to the fifth and final column of the CoA (Table 7.1), after test-
ing has been completed. Results are given in the same format as specifications
to allow for comparison. For example, if the specification for range of protein
binding is 0.60–1.05 μg recombinant protein bound per 1.0 µg of receptor, then
the result should only be given as milligrams of protein bound per 1.0 µg of
receptor. If the result was 0.73  μg, then this batch of BS would Pass by this
standard. However, if the result was 0.55 μg, then it would Fail. The subject
of handling a failure to meet a specification is discussed later in this chapter.
Certificate of Analysis for Drug Product
The process of planning QC test methods and specifications is also applied
to developing testing plans for FP. Indeed, the process of potency test devel-
opment is often more challenging with the final formulated product than it
is with the BS.
Once BS has been tested and released, it is formulated, filled into a final
container, such as a vial or syringe, and finally labeled and packaged
(Chapter 6). Once finished, testing begins and results are included in a CoA
for FP, a sample of which is shown in Table 7.2. This certificate, once signed
and reviewed, becomes one of the several documents that support the release
of FP to the user or, if product fails, the destruction of this material. The
contents of the CoA for a lot of FP are important, and so, great care is taken
during QC planning to choose the correct analytical tools and specifications.
Again, the choices are based on the nature and attributes of, and the indica-
tion for, the FP.
Quality control tests for FP may be more stringent than those for BS, and
they are always focused on an attribute that is important to the intended use
and the well-being of the user. In designing the tests that apply to a biophar-
maceutical FP, we consider many aspects of quality. The appearance test is
performed on a representative sample of FP when it is in the final container,
filled and finished. The specification for appearance is designed to ensure
that the QC examiner inspects a representative number of vials for certain
attributes and to note the absence of undesirable and visible impurities or
contaminants. For the r-protein example, we expect the FP to appear color-
less and to not contain any aggregates or particulate matter. The appearance
test illustrates an important point of QC testing: the SOP must be written in
such a way that the operator or inspector examines for these attributes and
properly identifies substandard FP. This general procedure, use of trained
operators or technicians, adequate equipment and SOPs, and exact report-
ing are applied to every test that is performed in a QC laboratory and to the
results reported in a CoA.

283Quality Control
FP is subjected to several safety tests, because this is the most important
attribute of any biopharmaceutical. Most biopharmaceuticals are given
parentally, and hence, they must be sterile. They must also be free of or have
very low levels of endotoxin or other toxic substances. Tests for specific types
of undesirable contaminants or impurities are defined in a compendium or
in regulations and are described elsewhere in this chapter. In addition, the
general safety test is performed in the United States on most biopharmaceu-
tical FPs to detect any highly toxic properties that the product might have.
The CoA in Table 7.2 identifies one test for appearance and four methods for
safety.
The active ingredient in FP must be exactly what it purports to be on the
label and nothing else. Hence, identity testing is performed on FP. A variety
of generic tests, such as SDS-PAGE, perhaps in conjunction with Western blot
using a specific monoclonal antibody, HPLC, N-terminal sequencing, tryptic
digest mapping, and cell karyotyping or phenotyping, may be employed in
a panel of identity tests. In Table 7.2, the chosen identity tests for an FP, our
example r-protein, are N-terminal sequencing and SDS-PAGE.
Despite the fact that purity has already been demonstrated and impurities
identified at the BS stage, it is important to test FP for purity and impuri-
ties. This is because impurities or contaminants might have been generated
or introduced during formulation and fill, the processing steps from BS to
FP. For example, some r-protein might degrade during processing or micro-
scopic contaminants, such as endotoxin, might enter the FP if, for example,
the containers were not scrupulously clean. In the CoA for FP (Table 7.2),
two purity tests, SDS-PAGE and HPLC, are used to detect and identify (or
measure) macromolecular impurities in FP.
Formation of aggregates in FP is a problem with formulations of certain
biopharmaceuticals. They are considered impurities but can also impact the
safety and potency of FP. Hence, an additional purity step for measuring
protein aggregates is added to the CoA for the r-protein (Table 7.2).
The FP is also tested for concentrations of any excipients, materials that
are added during processing and should exist in the FP. In the example
(Table 7.2), both glycerol and human serum albumin were added to the for-
mulation and the amounts of each are measured to ensure that they meet the
specified concentrations.
Strength of FP is determined by an assay that measures the total amount
of active substance. In the case of a recombinant protein, this might be a BCA
assay to measure total protein (Table 7.2). A variety of analytical methods
are available to measure all macromolecules in FP or count and measure the
amount of cells or tissues in FP.
Other tests performed on FP measure attributes of the formulation that are
important to product purity, potency, and stability. In the example (Table 7.2),
osmolality and pH were measured to ensure that the salt concentration was
correct in the formulation buffer. Maintaining pH is also important to main-
tain the stability of most biopharmaceutical products.

284 Biotechnology Operations
Development of relevant potency tests for FP challenges the design and
subsequent execution of any QC plan; this requires considerable abstract
thinking, laboratory testing, and interaction with scientific colleagues. FP
potency tests must be meaningful and practical. A potency test that mea-
sures a noncritical potency criterion is not very helpful, and any test that
takes more than 60  days to complete and report is impractical. Consider
also that a potency assay must stand as surrogate for the ultimate potency
test: performance in many human users. There, probably, never was, and
never will be, a single perfect potency assay, one that stands alone to pre-
dict the biological efficacy of a FP. Therefore, sponsors seldom rely on a
single potency assay but instead apply three or more potency assays, each
of which may be imperfect. This does not always happen from the start of
product development, but it should begin in the early phase, so that mul-
tiple FP potency assays are available in mid- and late-phase development.
Another objective of potency testing is to learn whether FP possess attri-
butes that result in optimal performance for the end user. This is difficult
to achieve, because we often do not understand every biological factor that
leads to optimal and consistent efficacy in all users. For biopharmaceutical
development, this means, in theory, if not always in practice, that the potency
test applied to FP mirrors exactly the potency when it is used in man. A
single potency test seldom, if ever, achieves this objective, but application of
multiple potency assays may support such conclusions.
Biological responses and biological molecules or cellular systems are com-
plex. Application of a biopharmaceutical to a biological system-cultured cells,
animal or human, is an attempt to disrupt or bring back a biological system
to equilibrium. Indeed, biopharmaceutical treatment may further disrupt or
complicate a biological system already out of control. As compared to small
drug molecules, many biopharmaceuticals are complex biological entities.
Given this information, consider how difficult it is to measure a product’s
potency in a complex system. Hence, multiple potency assays provide a
greater chance of ensuring product efficacy than does a single potency test,
because several potency tests evaluate the impact of the product at multiple
points in complex biological pathways. Although the use of a complete liv-
ing organism (e.g., a whole animal) for FP potency testing brings into play
all biological influences on the product and allows measurement of product
potency, it is often difficult to develop and validate an appropriate animal
model that mimics the human situation. Often, however, it is worth consider-
ing animal models for potency testing over in vitro models.
Again, consider the example of our r-protein, indicated to treat a disease,
based on buildup of an undesirable molecule inside certain cells. To reach
the desired biological endpoint, the r-protein must function properly at sev-
eral cellular locations and in a number of ways. Unlike the situation of test-
ing for potency in a simple and highly defined in vitro laboratory model,
there are other, extracellular influences that impact this molecule, as it
exists in a human. Hence, a well-designed panel of potency assays for this

285Quality Control
biopharmaceutical takes into consideration the functions at the cellular level.
For the r-protein, the initial QC plan considers three potency assays: one that
examines potency in a living animal; another that focuses on the r-protein
entering the target cell; and a third that involves the measurement of a spe-
cific desired activity within the cell. This plan applies a commonly used and
practical approach to ensure a potent biotechnology product in every lot of
FP and performance of product under multiple potency tests, each of which
measures a different aspect of the potency attribute. Could an animal model
possibly be used to measure the potency of r-protein? Perhaps. As noted ear-
lier, the best potency tests are developed in or adapted from the sponsor’s
research laboratory, where the technology might have already been applied
for investigational purposes.
In-Process Testing
The concept of in-process testing during product manufacture was intro-
duced in Chapter 6, and several examples for various products were provided
therein. Analysis of any product is important to establish effective manufac-
turing processes and to maintain quality. First, timely feedback regarding
product, impurities, or contaminants in the product flow allows production
scientists and staff to make adjustments and resolve the issues. Stopping a
process midstream and then reworking a particular step is usually a much
less costly solution than uncovering a problem at the end of manufacture and
then having to repeat the entire process. In-process testing also gives a level of
assurance that product will be pure and potent once it is tested at the end of
production. This too can save time and resources.
Although in-process testing is included in manufacturing protocols and
samples are taken by manufacturing staff, typically the QC scientist will
develop in-process assays and test samples supplied to the laboratory by
manufacturing staff. In-process test results appear on manufacturing docu-
ments such as batch production records (Chapters 5 and 6). Most in-process
tests characterize products by using chemical or simple biological measure-
ments, examining strength, measuring the amount of product, or testing
either for product purity or for specific impurities and contaminants. Many
in-process tests are developed for release testing of BS or FP and others
are modifications of tests found on those CoAs. Whatever the test, it must
be designed to generate results in a short period of time, usually within
hours or a few days. This means that many tests, such as complex biologi-
cal potency assays and those methods performed by CRO laboratories dis-
tant from the manufacturing facility, are unlikely candidates for in-process
use. In addition, in-process tests are simple to perform and do not require
expensive, dedicated instrumentation or staff with special analytical skills.

286 Biotechnology Operations
Despite these disclaimers, a number of tests mentioned in our discussions
on release of BS and FP can be adapted, and many more are available from
the methods listed later in this chapter. Some excellent in-process tests are
commercially available and used to rapidly measure protein concentration,
counting cells, or assess their viability. Most are adaptable to measure var-
ious products for two or more attributes; examples are simple analytical
chromatography methods, such as HPLC, and various types of electropho-
resis, notably rapid procedures like SDS-PAGE.
Analytical Methods
Having introduced the QC test development and specification development
cycle (Figure 7.1), we now examine the technical aspects and advantages and
limitations of several analytical tests commonly applied to biotechnology prod-
uct’s QC. Analytical methods, such as specifications, are adapted from many
sources by QC scientists. Some methods, such as sterility testing, are com-
pendia and are performed only using very specific recipes and reagents and
according to industry standard specifications. Other methods are traditional
or generic in basic design but adopted for use on a specific product or group of
related biotechnology products. Generic methods provide some flexibility in
the method of performance, and the specifications are product specific. There
are also novel tests and analytical methods developed for a special measure-
ment of one product. Methods are classified in other ways: by analytical instru-
ment, degree of difficulty, foundation technology, type of product, or level of
product manufacture or development. Presented below, but not classified or
listed in any special manner, are certain tests commonly applied to biophar-
maceutical development. Potency assays are given little attention, because, as
mentioned before, they are often home-made and relevant to one or a few prod-
ucts. Potency assays for a variety of biopharmaceuticals are, however, listed in
the manufacturing descriptions and figures (flowcharts) of Chapter 6.
Quality control tests are performed in a dedicated laboratory (Figure 7.2), or
samples may be submitted to contract laboratories for analysis. The small- or
medium-sized biopharmaceutical firm is well advised to use contract labo-
ratories for specialized tests (e.g., sterility), for methods that require complex
or expensive pieces of equipment (e.g., mass spectroscopy), and whenever
special scientific expertise is required (e.g., tests for posttranslational modi-
fications). Since regulatory requirements for QC testing are extensive, such
testing is rarely performed in a research laboratory.
• Sterility test: Sterility testing is required by regulatory agencies
under guidelines (e.g., FDA 21  CFR 610.12). Further, sterility test-
ing methods described in great detail in pharmacopeias sterility test,

287Quality Control
if passed, ensure that a biopharmaceutical is at the sterility assur-
ance level required for a parenteral product. The USP sterility test
provides 95% assurance that no more than one vial of product in
one million vials will have a bacterium or fungus. Considering it is
impossible to test 1 million vials in each lot of a product, the assur-
ance level is a statistical relationship to the actual number tested,
which can be surprisingly low. The test is performed with great care,
to exact procedures and with many controls. Sampling protocols are
carefully designed to ensure that representative product is selected
for testing. A specification for sterility test, USP <71>, might read:
Sterile or no growth, as growth of organisms is the measurement made
on a sample.
• Microbial limits test (MLT) or microbial enumeration test: For in- process
materials and often for BS, another compendial method, the MLT,
is used in place of the sterility test to determine the microbial load
or bioburden. The MLT, USP <61>, is designed to enumerate the total
bioburden in a sample and to identify a few select and highly unde-
sirable bacteria and fungi, should they grow from that sample. The
specification for the MLT is expressed as colony-forming units per
sample (dose or milliliter). A result might read: <2 Colony-forming units per milliliter of sample and no pathogens detected. Both MLT and sterility testing are very specialized and highly regulated endeav- ors; hence, biopharmaceutical firms may contract this work to spe- cialty laboratories. FIGURE 7.2 Quality control laboratory. This area of the quality control laboratory is dedicated to microbiol- ogy testing. 288 Biotechnology Operations • Endotoxin test: This test measures the amount of a toxic mol- ecule, endotoxin, that is produced and shed by many species of gram- negative bacteria. A gel clot, lymph amebocyte lysis (LAL) assay, is commonly used, but there are other accepted methods. It is an important test performed on most biotechnology products because the endotoxin molecule can result in adverse events, such as inflammation and even shock and death, in humans. Thus, endotoxin serves as a sentinel for past or present bacterial con- tamination of a product. Some expression vectors themselves pro- duce endotoxin, shedding it into the product stream. Endotoxin is sticky, adhering to surfaces or other molecules, and it persists, so absence or low levels of endotoxin signals good production and purification techniques for a biopharmaceutical. As is the case for MLT and sterility testing, national regulatory agencies or interna- tional advisory groups have established specifications for endo- toxin, that is, maximum acceptable levels that cannot be exceeded in certain classes of products intended for a specific use or route of administration. A result might read: 4.3 EU/mL endotoxin by gel- clot LAL. • Appearance: Appearance tests measure attributes such as color, pres- ence or absence of visible particulates or aggregates, and clarity. The appearance test is often performed visually by trained operators, who inspect a representative number of containers, selected at ran- dom at various times during the fill operation. Inspection of syringes or vials is typically done before an indirect bright light and a dark/ light background. Through training and carefully written SOPs, the examiner becomes proficient at identifying certain undesirable traits such as coloration, opaqueness, aggregates, or particulates. Instructions for reporting results are critical to success of appear- ance testing. Instrumentation is also used by some biopharmaceuti- cal QC laboratories to scan vials of FP or samples of BS to measure appearance. A specification might read Clear, colorless solution, free of visible particulates or aggregates. • General safety test: A general safety test, historically required by the Center for Biologics Evaluation and Research, USFDA, for release of most biopharmaceutical FP, is exactly described in 21 CFR 610.11. It can detect general toxicity of a biopharmaceutical. However, it is no longer routinely required by FDA for most products. • Osmolality: Virtually, all biopharmaceuticals are formulated in buffered salt solutions and kept at a particular ionic strength and pH. The ionic strength of FP, and sometimes of BS, is determined using an osmometer. Ionic strength is important to the stability of many products and reflects proper formulation. A result might read: 200 ± 10 mOs/kg. 289Quality Control • pH: A basic but important measurement is pH, because biopharma- ceuticals are often unstable and macromolecules can degrade at low or high pH values. Formulations are often designed to maintain a narrow range of pH for each product. pH measurements are made using a pH meter with a microprobe. A result might read: pH 7.25. • N-terminal sequencing: Used as an identity assay and described ear- lier, this inexpensive method establishes a unique identifier for a protein. It is performed by sequencing, from the N-terminus, the first 10 amino acids in a recombinant protein. Example of a result is: KQENMEVRLL versus known and reference standard KQENMEVRLL. • Polyacrylamide gel electrophoresis, native or reduced molecule (PAGE or SDS-PAGE): This assay determines the molecular weight of a mole- cule and it can disclose impurities in a preparation. It is typically used with proteins and glycoproteins. The sample is subjected to an elec- tric field, electrophoresed in a polyacrylamide gel matrix, and the gel is stained with a vital dye to disclose bands of proteins, distributed by molecular weight. An example of PAGE is shown in Figure  7.3. 41 kDa 175 80 58 46 30 25 kDa M ark er La ne 1 La ne 2 La ne 3 La ne 4 FIGURE 7.3 Polyacrylamide gel electrophoretogram. A polyacrylamide gel was stained for protein with Coomassie Brilliant Blue as a quality control test for identity of r-protein (molecular weight of 41  kDa). The gel is divided into five vertical lanes, with one standard or test sample applied to the top of each lane, followed by electrophoresis to separate proteins by molecular weight. Lane Marker receives standard protein sample containing six proteins of known molecular weight, 25, 30, 46, 58, 80, and 175  kDa. A negative control sample of r-protein, first digested with proteolytic enzyme, is applied to Lane #1. Lane #2 is a reference standard of r-protein, a positive control. Samples in Lanes #3 and #4 are from BS and FP, respectively, of a lot of r-protein manufactured by fermentation of recombinant bacteria with purification. Results of the samples from BS and FP show a single band at 41 kDa, identical to the reference standard. This electrophoretogram suggests that within the sensitivity of this assay, there is little or no other protein in the samples. 290 Biotechnology Operations It is not generally considered a quantitative test, but an estimate of purity can be calculated if comparisons are made on the same gel of test material versus qualified reference standards of known purity, for example, 100%, 95%, 90%, 80%, and 60%. Polyacrylamide gel elec- trophoresis, when used under various conditions, such as reducing or nonreducing, rapidly provide semiquantitative information and other valuable insights into product identity, structure (secondary, tertiary, and quaternary), and purity. When testing unreduced or native proteins, shape or change isoforms can be found, even in small amounts, and can be compared to reference standard. A high load of sample can reveal oligomeric or aggregate species at the top of the loaded lane. A typical test result might read: Native (nonreduced): Single dominant band at MW 41 kDa and two faint bands at approximately 20  and 10  kDa. Minimal amount of material at the top of the load lane. Reduced (SDS): Dominant bands at MW 20 and 36 kDa and faint bands at MW 10, 15, and 26 kDa. • Electrophoretic methods: Other electrophoretic methods may be used. Each method, aimed at identifying a unique attribute, uses a different matrix or format to retain macromolecules while they are subjected to an electric field, two or even three dimensional. Immunoelectrophoresis is one example. Most electrophoretic meth- ods are commercially available and easily adaptable to suitable QC testing protocols. Further, some electrophoretic methods can be immediately followed by immunological assays on the sample to further identify each type of molecule (e.g., PAGE and Western blot testing). The adaptive possibilities are many and varied. • Western blot (of PAGE or SDS-PAGE): Sometimes applied as an iden- tity test, but adapted to also detect certain impurities, a molecular profile by Western blot analysis is performed by transferring mol- ecules from a PAGE gel to a membrane and then treating that mem- brane with polyvalent antiserum, or with a monoclonal antibody, specifically reactive against the test protein product or an epitope of that test protein. The reaction is developed by immunohistochemi- cal methods to demonstrate colored band(s), which should fall at the molecular weight location of the protein and be comparable to the immunoblot bands of reference material. Oligomeric or aggregated species may also be detected at the top of the lanes. Using antisera specific for known impurities, Western blot may also identify those materials. Protein reference standards and negative control antisera or monoclonal antibodies are used. Western blots are not quantita- tive. A result might read: r-protein, major band at 41 kDa recognized as major band at 41 kDa by polyclonal rabbit serum to Protein-r and by mono- clonal antibodies 3D7, 8F8, and 4D2, specific for epitopes Ala3, Leu29, and Try54 of r-protein. No other bands detected. 291Quality Control • Host cell protein: After molecules are produced in a cell-based sys- tem, they are associated with impurities and contaminants, that is, a variety of host cell substances and process materials (Chapter 6). These are often proteins: enzymes or structural molecules. Host cell proteins can be identified by a variety of methods. Popular ones are antibody-based assays, such as an enzyme-linked immunoas- say. This measurement uses a polyvalent animal antiserum raised against proteins of the host cell to measure impurities or contami- nants. As it is not possible to identify or measure every host cell contaminant, specific or marker proteins may be measured. A result might read: <0.10 mg host cell protein per 100 mg recombinant protein in the solution. • Host cell DNA: DNA is an impurity, unless the product itself is com- posed of DNA (e.g., bacterial plasmid), in which case anything other than the desired DNA molecule is an impurity (e.g., chromosomal DNA). DNA is measured by a variety of methods, many commer- cially available, with great accuracy and specificity. Assays include threshold measurement with DNA-binding proteins, hybridization assays for specific DNA of defined origin, and quantitative poly- merase chain reaction probe methods, which are highly specific. A result might read: <10 µg DNA/1.0 mg recombinant protein. • Host cell RNA: Some host cell production systems can yield a consid- erable amount of undesirable host cell RNA. Commercial test kits are used to measure this molecular impurity. A result might read: <10 µg RNA/1.0 mg recombinant protein. • Carbohydrate: Some biopharmaceuticals must be posttranslationally modified to show activity. Indeed, glycosylation and a unique pat- tern (e.g., location on protein, composition, and structure or pattern of glycosylation of each carbohydrate side chain) can be important to potency. A variety of methods, adapted from classical carbohydrate chemistry and some now semiautomated, are applied as surrogate measures of potency to demonstrate identity of some biomolecules. • Light scatter for aggregates: Subvisible molecular mini-particles and aggregates can be disclosed and measured using instruments that measure the scatter of light as it passes through a solution of prod- uct. A result might read: <0.1% scatter at wavelength 300 nm. • Protein measurement: A variety of tests, some commercial and others developed in or adopted by the QC laboratory, are on the market to measure the amount of protein in solution. Each has advantages and limitations, so the QC scientist picks a test carefully to meet a partic- ular need and qualifies it for use with a given protein. The Bradford test or a BCA reagent-based test is commonly used in biotechnology. A result might read: Total protein, 1.06 ± 0.1 mg/mL. 292 Biotechnology Operations • Peptide mapping: This is an identity test. A protein in solution is digested with an enzyme, for example, trypsin, and the fragments are subjected to an electric current (electrophoresis) in a matrix and then stained. The pattern of fragments is characteristic to a given protein. The result would be obtained from the peptide map and might be given as: Matches predicted and reference maps. • Size exclusion chromatography: The SEC method examines a sample of protein for purity and impurities by using a chromatography gel that distributes proteins on the basis of size. Aggregates of protein are detected by SEC. The results provide a measure of purity and identify impurities based on molecular size. A result might read: Dominant peaks at MW 20 and 36 kDa and faint peaks at MW 10, 15, and 26 kDa. • Isoform characterization: The isoelectric focus assay, a high-resolution method that allows the separation of proteins based on their iso- electric point, evaluates the charge characteristics of a protein and can demonstrate isoforms, major variants, of the protein. Isoelectric focus gels are scanned and bands can be measured and identified by pI. Results are not quantitative, but estimates may be made from scans. Example of an isoelectric focus result: Major sample band lies between pI markers of 5.20 and 5.85 and is comparable to reference stan- dard. Minor variants constitute under 10% of total protein. • Amino acid composition: This method may be used as an identity test. An analytical instrument determines the amount of each amino acid and then calculates the ratio. This ratio is compared to the expected ratio and to that measured for a reference standard. The ratio rela- tive to a reference amino acid, say L-leucine, may also be determined and compared to theoretical and reference standard. To perform the test, a sample of protein is hydrolyzed with acid and the amino acid composition is determined by an automated method. Typical result might read: Correct amino acid composition ±10%. • Chromatography: Many analytical variations and instruments are applied to this methodology, which separates chemicals in a com- plex sample. Molecular characteristics, such as charge and molecular weight, are the basis for their separation. Sample may be entrained in a semisolid matrix (thin-layer chromatography) or in a gas stream (gas chromatography), held within a narrow column. A detector, scanner or in-line, measures molecules as they exit from the long tube. A chromatogram, to include quantitative data when reference standards are applied, is produced from the detector. Gas chroma- tography is especially useful for detecting residual solvents in a product. A result might read: Residual isobutane <10 ng/mL. • High-pressure liquid chromatography: This has been applied to mea- sure several attributes of many biological molecules. As the name 293Quality Control suggests, HPLC is distinguished by applying very high pressures to the enclosed column. It is especially useful with macromole- cules and shows excellent resolving power, with clear separations on the chromatographs. In addition, it is relatively fast and inex- pensive and quite adaptable. Sample is separated into components that appear on the output as distinct peaks or even bumps or shoul- ders on a major peak. An HPLC apparatus and sample chromato- gram are shown in Figure 7.4. It also has the ability to measure the amount of material under each of those peaks and, for reference purposes, to spike known molecules, such as impurities or contami- nants, into a sample of highly purified reference product. Indeed, this is how experiments are designed and initial results are seen (a) FIGURE 7.4 High-pressure liquid chromatography. Panel (a) shows a high-pressure liquid chromatography instrument, stacked modules on left, and dedicated computer containing analytical software on the lower right. (Continued) 294 Biotechnology Operations using several other modern chromatography and spectroscopy methods. Typical result might read: Major peak at 15.8″ comprising 97.68%. Two minor peaks at 22″ (0.88%) and at 18″ (0.27%), with slight shoulders at leading and trailing edge of major peak. • Electrospray ionization-mass spectrometry: This instrument-based test is used to measure mass of a molecule and may be applied to study protein folding. The results are compared to both reference standard and the theoretical mass of the molecule of interest. Controls might include proteins of known masses, especially those in the molecular weight range of the test material. Results are reported in kilodal- ton (kDa) unit of protein mass. Typical result might read: 41.111  kDa (versus theoretical 41.005 kDa). • N-terminal and C-terminal analysis by liquid chromatography-mass spectrom- etry: This method uses physical separation by liquid chromatography and mass analysis by mass spectrometry. It is highly sensitive and spe- cific and can be applied to characterize a variety of proteins. It confirms both the N-terminal and the C-terminal sequences. Typical result might read: N-terminal, KQEN; C-terminal, EIGGY; comparable to the reference standard. −3 .7 3 (b) −4 .3 3 −4 .4 6 −4 .8 5 −5 .1 9 −5 .4 9 FIGURE 7.4 (Continued) High-pressure liquid chromatography. Panel (b) shows a chromatograph of r-protein bulk substance. Here, the large single peak to the right, at 4.85 min, is r-protein (determined by comparison to a reference standard), whereas the peak on the left, at 4.46 min, represents a significant amount of a contaminant protein, which has a shoulder peak at 4.33 min, perhaps a second but closely related contaminant protein. 295Quality Control • Protein folding and unfolding by intrinsic fluorescence: Protein folding, often important to biological activity of a macromolecule, may be compromised because of incorrect posttranslational folding or mod- ification. Properly folded protein may denature during processing or storage. Hence, it is sometimes important to demonstrate correct folding in a protein product. The amino acids tryptophan and tyro- sine in proteins fluoresce under specific wavelengths of light. As proteins in the native or correctly folded state demonstrate a unique fluorescence signal and because denaturation of a protein results in a shift (e.g., red-shift) of the fluorescence emission barycentric mean value (which can be derived from an analytical instrument), protein folding can be measured. A specification for batch-to-batch variance can be established. An example is barycentric mean Lambdanm < 358 for fluorescence measured between 300 nm and 400  nm. A result might read: Lambdanm 340 for fluorescence measured at 380 nm. • Mass spectrometry-time of flight: This method relies on a complex piece of equipment and represents one of the several new and promising analytical tools that may be used to characterize proteins and other biological molecules. It is useful in describing protein folding. Additional Analytical Tools and Concepts The tests identified above are used to test the attributes of biotechnology products that exist as molecular entities, but what about testing for appear- ance, safety, identity, purity, and strength of living biopharmaceutical prod- ucts, such as attenuated organisms used in vaccines, retroviral vectors, and somatic or pluripotent-derived cell and tissue products, as introduced in Chapter 6? Here, we review, with very general descriptions, a few of the many methods used in the QC of various biopharmaceuticals, notably live materials. • Cell karyotyping: A karyotype represents the appearance and num- ber of chromosomes in the nucleus of a eukaryotic cell. Cytogenetic analysis of a cell’s karyotype is used as an identity or a purity test to demonstrate quality of a cell line, especially when that line was used as a somatic or pluripotent cell-derived therapeutic or to pro- duce macromolecules. Karyotyping is performed in specialty labo- ratories, and reference cell lines are required. The number of cells with an abnormal karyotype is measured. The species origin of the cells is also confirmed. The result would be presented to identify the cell line, as compared to a reference cell line, and might also attest to its purity. 296 Biotechnology Operations • Cell phenotyping: Any cell trait or characteristic distinctive of that cell line is considered phenotypic. Cell phenotyping, used when a cell line or tissue is derived from somatic or pluripotent cell sources, measures one or more molecular parameters to demonstrate that cells are identical to those intended and that the cells have neither differentiated or dedifferentiated and they are not contaminated. The result would be presented to identify the cell line and attest to its purity. • Microbial identification: Bacteria, fungi, yeast, and viruses, includ- ing retrovirus and bacteriophage, are biotechnology products. They are derived from many sources and manufactured in various ways, both with and without cell-based systems. An identity test demon- strates, in various ways, that the microbe is as purported. Bacteria and yeast are identified by traditional methods such as culture on selective media and metabolic properties. Viruses are identified by growth characteristics on selective cell lines. All microbes may be further identified using species-specific antibodies to agglutinate or label with fluorescent dyes, to kill them in the presence of comple- ment, or to neutralize their activity. Describing their morphology or ultrastructure is also an effective means. Polymerase chain reaction is increasingly used to identity live organisms or DNA molecular products. • Monoxenic nature of microbial product: The purity of a microbial prod- uct may be demonstrated to show that all organisms in a product have the same trait. Cell phenotyping or karyotyping or microbial identification methods, described above, can be applied for this purpose. Other methods use growth characteristics or a panel of chemical or immunological reagents to selectively identify possible contaminants. An example is the use of selective media that support the growth of most bacteria but not of the strain or species compris- ing the product. Polymerase chain reaction, using probes against DNA from a variety of possible contaminants, is another test used to reveal the purity of a culture. • Attenuation of microbial product: Many products, notably those intended for genetic therapy or vaccines, are attenuated, so that they do not produce disease. Quality control tests for safety focus on ensuring markers of attenuation, such as inability to grow on cer- tain substrates or in specific cell lines. These traits, or lack thereof, may also be evident by using antibody or molecular probes, such as immunofluorescence assays. Polymerase chain reaction and other genetic probes identify a gene of interest in any host. • Expression of a molecule by a vector or host cell: Other biotechnology products are engineered to express a molecule, which, in turn, exerts an immunological or therapeutic effect on the user. Quality 297Quality Control control tests are based on methods mentioned above, but instead of searching for the absence of a trait, they focus on confirming that an attribute is present and active or functioning. Immunological and molecular probes are used to ensure that an expression product is expressed and exists at the expected location, such as on the cell surface. • Adventitious agent testing: Adventitious agents may be found in a variety of cell products and cell banks. Considerable effort is put in examining samples for microbial agents, for example, mycoplasma and retrovirus. Some testing is performed to identify, through cul- ture or immunological methods, the agents themselves. More often, indirect measures of adventitious agents, such as electron micros- copy to detect viral or viral-like particles or polymerase chain reac- tion, are used to locate and, sometimes, identify undesirable microbes or the DNA fingerprint they leave behind. If infectious agents are not obvious at the outset, they may be induced by applying a vari- ety of chemical or biological stimulants to the cell line. For example, endogenous retrovirus can be induced with nucleotide analogues and then detected by electron microscopy as viral particles. Viruses can be detected by injecting cell lysate into newborn animals and then examining the animals months later. For some biotechnology and blood-derived or supplemented products, such tests are critical to ensuring safety. Quality Control of Cell Banks As noted in Chapter 6, the identity, purity, and viability of cell banks (micro- bial, mammalian, or insect cells) are very important attributes to the overall manufacturing quality and success. Every cell bank is tested both at the time of release and at specific intervals. The attributes considered in most test protocols are identity, sterility, purity, and viability (potency). Following are the tests applied to the QC of cell banks, both master and working cell banks, as well as the progeny of cell banks that are used in extended manufacturing campaigns. • Identity of cells: The identity of cells in a bank is established by applying tests to ensure that they are the intended species and strain. Particular methods are described above, under the headings Microbial identification, Cell karyotyping, and Cell phenotyping. • Identity of insert: If a cell line has been genetically engineered to express a product, then the process of expression and the expression product 298 Biotechnology Operations are tested to ensure that banked cells possess these intended capacities. Methods described in the earlier sections of this chapter are applied, as appropriate, to detect the nature and function of the molecule or other attribute. • Potency: This is defined as the capacity for cells in a cell bank to divide after removal from storage. This function is clearly basic to the intended use, and so, quantitative potency testing is an annual QC physical examination for any cell line. • Purity: To ensure that a cell line has not been contaminated with another product, and this does happen, then the monoxenic nature of the cell line must be established using the methods described above, notably adventitious agent testing. Samples and Sampling Consideration of sampling methods, assay controls, and reference standards is an important aspect of QC planning because it is critical for each test sam- ple to represent the whole of that batch or lot of BS or FP. Thus, sampling must follow standard procedures, with methods tailored to the intended scope and nature of each test and heed statistical considerations such as sample size or random selection. Indeed, papers and books are written about sampling methods for QC of various consumer products, including pharmaceuticals. Beyond the number of samples taken, there must be a plan for sampling per- formance. For example, if one wished to sample 100 glass vials containing a biopharmaceutical from a total lot of 10,000 vials, it would be important to take vials periodically, perhaps 10 vials at each of 10 time points, throughout the fill, as opposed to grabbing the first or last 100 vials in the fill line. Also, when seeking a representative sample from a single container it is necessary to take the sample in the proper manner. For example, if one is taking sample for aggregate testing from a small vial containing a recombi- nant protein, the container is first gently stirred and then the sample is taken, ensuring aggregates, that tend to settle to the bottom of a vial over time, are fairly represented throughout the sample. Sampling raw materials provided by a vendor in large containers drives the need to ensure that containers are selected in a representative yet random manner and, for each selected container, to randomly take material from within that container. The gen- eral concepts of sampling are included in a QC plan, and specific sampling methods for each assay are written into the test protocol or SOP. If not every sample is subsequently assayed, then it is important to ensure that tested samples are indeed representative of all samples taken. Consultation with a statistician is often helpful throughout the sampling process. 299Quality Control Analytical Controls and Reference Standards To ensure that each QC test is reliable and to maintain consistency in test- ing, control samples, reagents with known analytic values, are always tested in parallel with unknown samples. While every assay requires one or more controls, a full set of control materials is too often missing from an otherwise adequate test procedure. In establishing a QC test, a panel of control samples is carefully chosen and then applied correctly within the test scheme. Each con- trol reagent is certified for a particular purpose, and during assay qualification or validation, it is shown to produce the intended outcome. Positive and nega- tive control materials are generated through biomanufacturing or purchase from vendors, before beginning the analytical work. Controls are stored in a manner that retain all desired attributes. Controls for quantitative assays, such as those requiring generation of a standard curve, require particular attention. In contrast to control reagents, a reference standard is a test sample with known analytical values. It has a pedigree. A reference standard consistently produces, by repeated testing with a standard assay and over a long period of time, the same result. Reference standards are by definition used with every assay or panel of assays. In some instances, more than one reference standard is required for an assay, such as when a standard curve is produced by limit- ing dilution of the standard. A reference standard also is the same or nearly the same as the actual test material, the product or analyte. For example, if actual samples are substance or product that contains excipients and buffers, the reference standard is for- mulated in the same way. Hence, the best source of each reference standard is a batch of substance or a lot of product manufactured and controlled in the same manner as is currently used. However, in those instances when a recom- binant protein cannot serve as a reference standard due to issues such as insta- bility, it may become necessary to purify and store small amounts of the native molecules to avoid the issue, that is are stable formulations. Producing a refer- ence standard can be a very resource-intensive project. How does one obtain a reference standard? One option is to store BS or FP in aliquots and then use one aliquot, taken from the first batch or lot of product, as the first reference standard. Over time and as manufacture and control expertise improves, this first reference standard is replaced with material from new batches of BS or lots of FP. This is also the case when tests are redeveloped, as shown in Figure 7.1 as T-1, T-2, and T-3. It is not unusual for five or more individual, reference standard lots to be used over the development cycle of a single assay. Cross- over studies are performed to compare, in great detail, an old to a new refer- ence standard, that is, to ensure that the pedigree is maintained. To further complicate the matter, a reference standard, like a control, is used and relied upon over a long period of time and thus each is kept in extended storage. Early in development, and often for years into the development cycle, there is limited information on stability profiles of controls and reference standards. 300 Biotechnology Operations This creates unknowns regarding their reliability when used to test product over that same period or into the future. A  plan to meet each of these chal- lenges becomes a part of the QC plan and this is typically done for each assay. Test Failures, Out-of-Specification Results, and Retesting As one might expect, failures are experienced in the QC laboratory just as they are in other aspects of biotechnology operations. When the QC test result on a given batch or lot does not meet the specification it deviates or is out of specification. In some biotechnology operations, such as biomanufacturing, a failed process can be repeated, at least if there is an explanation and time and money allow. Failure in testing, specifically the failure for a test to meet a specification, can be difficult to resolve. There are strict controls on managing deviations in a regulated environment, and biopharmaceutical QC follows guidelines and regulations promulgated by FDA. Further, some highly pub- licized judicial actions taken by FDA originated from improper retesting and reporting of QC test results. Deviations in QC testing demand, by regulation, an internal investigation. Investigations involve a complex process, having four major components that are described in greater detail in Chapter 5. A case study of an out of specification for a potency test is reviewed in Box 7.3. An investigation uses established methods, a root cause analysis, a corrective and preventive action plan, and approval of the outcomes and recommendations by supervisors, QC, and QA. Further, findings of the investigation may lead to a recommen- dation of significant actions, such as qualification or validation even replac- ing an assay before it can be used to further test a product. Other resolutions, closely monitoring the performance of the assay, replacing key components such as a reference standard, and establishing formal audits or documenta- tion systems, may be recommended by the QA unit. Since management is ultimately responsible, the failure may be raised to that level. It is important to prevent test failures and this is achieved in several ways. First, full QC planning prevents many failures and sticking to the accepted plan avoids other. Another approach is to follow a QC development cycle (Figure 7.1), that is by developing each assay one step at a time and not skip- ping a step or a critical experiment. The most frequently cited reason for fail- ures in QC testing is, in this author’s experience, setting unrealistic (meaning too stringent) specifications in early and middle phases of the development cycle. In effect this means that the development team establishes a speci- fication before adequate data was available to support that specification. Following the hallmarks of quality and abiding by a quality system, current Good Manufacturing Practices for most biopharmaceutical QC operations is another way to ensure success with QC endeavors. 301Quality Control BOX 7.3 CASE STUDY: QUALITY CONTROL TEST FAILURE AND INVESTIGATION The r-protein was formulated and filled into single-dose vials for use in a Phase 3 clinical trial. Three final lots, #1, #2, and #3, each having 8000 vials at a cost of $25 per vial, were manufactured and then tested by QC. For Lot #3, a FP potency test (Table 7.2), Accumulation of Molecule in Cultured Cells at 24 h, failed to meet the specification of lesser than 10% accumulation over baseline, time 0 (of the test assay). The result for Lot #3 was 12%. QC and Quality Assurance (QA) would not release the lot, despite the fact that it was needed for patients in the trial and represented an investment of more than $20,000. QA called for an investigation by an ad hoc committee composed of supervisors from research, manufacture, QC, and QA. Careful review of all documents failed to reveal evidence of errors to properly per- form the test or maintain all records. It did reveal that the standard operating procedure (SOP) for preparing the cultured cells, a critical biological component of this potency assay, allowed a wide range in the age of the cells that could be used as cellular substrate in the assay. Specifically, cells could be used from 2 to 8  days. Research pointed out that older cells, those older than 5  days, could, at times, be in asynchronous cycles; indeed, some cells even experienced death at 7–8 days. Further investigation showed that Lot #3 was tested using 7-day-old cells, within limits stated by the current SOP but perhaps not advisable. Lots #1 and #2 and other lots tested previously used cells of 3–5 days of age. Laboratory investigation in a well-designed and -controlled study tested Lots #1, #2, and #3 using cells of various ages, 2–8  days old. Findings consistently demonstrated that older cells yielded higher val- ues, often greater than 10, of accumulation of molecule in cells. A second laboratory investigation tested Lot #3 repeatedly with cells of 3–5 days of age and all results were lesser than 10. There was a sci- entific basis to this finding as well, given the mechanism of action for r-protein. The committee recommended release of Lot #3, changes to the SOP, allowing only cells of 3–5 days of age, and careful and complete docu- mentation of the investigation and all laboratory results. 302 Biotechnology Operations Testing for Product Stability All BS and FP are placed in storage and transported for defined periods and at established environmental conditions. BS is usually stored for shorter periods, days or weeks, as compared to FP, for months or years. There are significant advantages, both economic and operational, to establishing a long shelf life for BS or FP. Substance and product stability profiles are established over the product development cycle by using many storage conditions, each exact, and by testing with multiple assays at many time points. It must be demonstrated that BS and FP remain pure, potent and safe during storage and transport. Because some products are likely to be subjected to shaking, inversion, humidity, and temperature incursions, cool and hot, during stor- age stability testing is performed. Stability protocols include subjecting both BS and FP to undesirable but possible conditions. Establishing a stability profile is a long, tedious and expensive endeavor but it is necessary and usu- ally well worth the effort. Experimental stability protocols are designed by QC and manufacturing staff to evaluate the attributes and desired traits of BS and FP, as might be expected under conditions of storage, handling and shipment. Elements of a stability plan are outlined in Figure 7.5. Most biotechnology products today are kept refrigerated or frozen, but all products are subject to fluctuations in temperature and humidity, or they face exposure to light. Environmental conditions certainly change as the product is moved: from manufacturer to truck; then to wholesale warehouse; again to truck or aircraft; then to phar- macy, mailbox, or automobile; and finally to the consumer. Also, if a prod- uct is to be shipped, stored, and used in regions with a warm moist climate where it is difficult to always maintain a cold chain, or in very cold regions where it might be exposed to freezing temperatures, if just for 1 h, then stabil- ity studies need to be especially rigorous. Stability data provides a sponsor with very useful information because it aids in developing proper product formulation and approaches to marketing, transport, and storage. Regulatory authorities demand that storage conditions be explicit and highly visible in labeling. In addition, because one cannot sell degraded product, stability test results impact both business and marketing plans. There are significant eco- nomic advantages for a product with a long shelf life at ambient temperature, and there are marketing hurdles for the sponsor of a product that must be kept frozen, especially if the product must be kept at −80°C or cannot reach room temperature even for 1 h. Consider also the difficulties of manufactur- ing, stocking, and rotating a supply of a product with a shelf life of just 1 year and one can imagine the advantages of a 3-year shelf-life. While experimental in nature, the design of stability protocols is driven by quality, business, and market interests, because the information derived from stability studies in part ensures the sales of that product. Hence, stability testing plans are an important aspect of the overall QC plan. 303Quality Control Stability testing, typically a QC function, is a formal process under which the actual shelf life of a biopharmaceutical is identified through experimentation. A  key milestone is early development of a written sta- bility plan, developed by a multifunctional team before stability testing begins. The  team begins by reviewing what is known about the product Elements of a stability study plan Study protocol Pull samples at predefined intervals for analysis Results Interim and final study reports Analytical testing Stability chambers Refrigerate Humidity Incubate Light/dark Cycle samples Warm → Cool Freeze → �aw Light → Dark Sample prep Degas or gas overlay Adjust volume Mix, shake, and invert FIGURE 7.5 Elements of a stability plan. The stability plan considers the physical environment (test cham- bers) for samples, a sampling and preparation scheme, cycling of some samples, selection of (pulling) samples to test, analytical testing, and interpretation of results. 304 Biotechnology Operations and similar products or molecules. First, it is important to know if a prod- uct could be stored at room temperature or in the refrigerator, as opposed to in a freezer, as simpler or ambient storage reduces cost and complica- tions of shipping and storing the product. Second, the shelf life is deter- mined empirically for each proposed storage condition. How can this be approached? Third, we often attempt to improve the shelf life or simplify the storage conditions for a product by experimenting with different for- mulations. For a given product, we know in general that certain chemicals preserve cells or proteins better than do others. What might these be and which might be experimentally evaluated? Finally, we seek an understand- ing of how long product can withstand excursions, such as high or low temperatures, high humidity, shaking, inversion, and so on, before it loses potency. These conditions must be carefully chosen, since resources limit the number of variables that may be tested. Stability testing is an experimental endeavor, designed as a matrix experi- ment and guided by a written protocol. It applies not one but a panel of assays, each capable of measuring a stability-indicating attribute for the product. Further, to provide enough material for testing, many samples of product, both BS and FP, are stored in a variety of configurations. For example, storage at three or four temperatures and, for FP, in two positions (upright and inverted). With testing by three to four assays required at each of the seven different time points, sample requirements may reach hundreds or thousands of vials or syringes of FP or samples of BS for every lot and batch, respectively. During stability testing, storage conditions must be care- fully controlled and documented. Thus, there is a need for many samples and a laboratory infrastructure able to accommodate various environmental conditions and perform many tests. Stability testing is very labor intensive and expensive, but it is absolutely necessary from both regulatory and busi- ness standpoints. Stability-indicating assays, if not available as QC release assays, are modi- fied from existing research or development assays or they are developed by QC scientists to fit this purpose. They are selected by first identifying those product attributes providing meaningful information about the shelf life of that product. Since any assay used to measure a product attribute is, in the- ory, a stability-indicating assay, the CoAs for the release of BS (Table 7.1) and FP (Table 7.2) provide a foundation for developing stability-testing protocols, as shown in Table 7.3 for BS and Table 7.4 for FP. However, a CoA intended for product release lists far more assays than are necessary or could ever be performed on the variety of stability test samples and at every time point. To downselect the choices to perhaps three to six assays per stability protocol, the QC scientist must first determine which attributes of the product are most likely to be the stability-indicating attributes. Next, they choose those attributes and experimentally determine if in fact they really are stability indicating. 305Quality Control What are the reasons for choosing an attribute and assay for inclusion in a stability protocol? First, certain types of assays are, by tradition, stability indicating for specific classes of biopharmaceuticals. Examples include peri- odic sterility testing and appearance of all products and tests to detect the degradation of protein in storage or cell viability for products with living cells. Second, it is good to have a balanced assay portfolio, choosing one or two methods for the attribute of purity or impurities, one for potency, and one or two for safety. The nature of the product gives a clue to what con- stitutes a good stability-indicating assay, as does the understanding of the mechanism of action. For example, if a protein product tends to aggregate at the formulated concentration, then a quantitative test for aggregates is in order. Further, each test must be sensitive, so that loss of product integrity or activity is, in fact, detected early and long before the product is completely unsafe, degraded, impure, or impotent. For some products and tests, this selection is challenging. For the r-protein in our example, instability might be reflected in denaturation or breakdown of the molecule. This would result TABLE 7.3 Tabular Synopsis of a Stability Protocol for Biopharmaceutical Bulk Substance (Example of Formulated r-Protein in Vial) Test/Specification Post Manufacture (Months) 1 5 12 36 60 Appearance Clear, straw-colored liquid without particulates or aggregates X X X X X Safety Microprobe pH 7.1 ± 0.2 X X X X X Purity SDS PAGE: Single band at 41 kDa, comparable to reference standard X X X X X HPLC: Single peak integrated >98%
material in sample
X X X X X
Potency Blocking assay: cultured cells: >60%
inhibition of secretion as compared to
reference standard
X X X X X
Safety Bioburden: USP <61> <5 cfu/mL and no evidence of pathogenic organisms X X X Note: X  =  This test will be performed at this time point. Accelerated stability testing only to 12-month time point. This protocol is performed on samples of BS that have been stored at static conditions in the upright position at the following temperatures (one set per temperature in static conditions): at all time points at -70 ± 10°C (recommended storage temperature); at Day 1 and Day 5 when kept at 21 ± 2°C (room temperature); and at Day 1 and Day 5 when kept at 37 ± 1°C (accelerated temperature). Another set of BS samples is tested after static storage at –70 ± 10°C but after having been subjected to three freeze/4-h thaw cycles before testing. 306 Biotechnology Operations in an inability to perform the key biological functions and reach the desired endpoint. Referring again to the r-protein, and focusing only on FP, initial consideration is given to several key assays for early stability experiments. The tests such as SDS-PAGE and HPLC are selected for purity and impuri- ties, and the potency assay is chosen for blocking accumulated molecules by cultured cells. These are chosen in part because both are meaningful to this protein and its biological activity and because each test is simple, accurate, well controlled, and can be performed on large numbers of samples, in a relatively easy and inexpensive manner. Since vials are opened to test the product anyway, we might add tests to record the appearance and measure TABLE 7.4 Tabular Synopsis of a Stability Protocol for Biopharmaceutical Final Product (Example of Formulated r-Protein in Vial) Test/Specification Recommended Storage Temperature 4 ± 2°C Post Manufacture (Months) 1 2 3 6 12 24 36 48 60 Appearance Clear, colorless liquid without particulates or aggregates X X X X X X X X X pH: 7.1 ± 0.2 X X X X X X X X X Purity SDS PAGE: Single band at 41 kDa, comparable to reference standard X X X X X X X X X HPLC: Single peak integrated >98%
material in sample
X X X X X X X X X
Potency Blocking assay:
Cultured cells: >60%
inhibition of secretion
as compared to
reference standard
X X X X X X X X X
Safety Sterility: USP <71>
Sterile
X X X X X X
Note: X  =  This test will be performed at this time point. Accelerated stability testing only to
12-month time point.
This protocol is performed on FP that has been stored at static conditions in the
upright position at the following temperatures (one set per temperature in static condi-
tions): 6 ± 2°C (recommended storage temperature); 21 ± 2°C (room temperature); and
during the first 12 months, 37 ± 1°C (accelerated temperature).
In addition, one set is tested after storage at 6 ± 2°C, with vials kept in both upright
and inverted positions.
Another set of vials with FP is tested after static storage at 6  ±  2°C, with vials in
upright position, but only after sample vial has been subjected to five freeze-thaw cycles
before testing.
Yet another set is tested at all time points and at the 6 ± 2°C temperature storage con-
dition in an upright position but only after sample vial has been subjected to shaking at
30 oscillations per minute for 6 h immediately before testing.

307Quality Control
the pH at every time point, because changes in pH are often related to pro-
tein instability and because degradation often discolors or adds precipitate
to a vial of product. In addition, using a single vial of product, both tests,
that is, the appearance in the unopened vial and the pH, can be performed
on a single vial, conserving the expensive product. Regulatory authorities
ask that sterility be examined once each year, because sterility test is a test
of container (vial) integrity. Returning to the example, SDS-PAGE provides
an indication of whether or not the r-protein was breaking down under
stressful conditions. In addition, HPLC, a much more sensitive test, confirms
and extends any observations of protein breakdown, perhaps detecting the
changes earlier than SDS-PAGE. Besides, HPLC analysis of r-protein might
provide, on the chromatogram trace, a clue as to the breakdown products
and impurities that accumulate over time. A stability-indicating assay is also
chosen for the attribute of potency, based on the requirement that r-protein
inhibits the buildup of intracellular carbohydrate molecules. The assay uses
cultured cells, providing a well-characterized and much used method that
should reflect biological activity, or loss thereof, in a relatively simple and
reproducible format. Now, this stability-testing concept is designed into a
written FP stability testing protocol, and the test scheme is summarized in
tabular outline, as shown in Table 7.4.
Consider also that controls are included with each assay to demonstrate
that a stability assay detects unstable product. These controls, partially
and fully degraded or inactivated product, are tailored for use with a par-
ticular assay. To develop a control, it is critical to first understand the most
likely routes of degradation and then mimic this degradation in a mean-
ingful way. For the r-protein example, it is not adequate to simply boil the
r-protein or completely digest it with trypsin; this would not mimic possible
environments in actual storage or transport. Instead, it is necessary to care-
fully devise accelerated degradation by using conditions that mimic possibly
real environments. These might include using room temperature found in
temperate and tropical environments or one or more cycles of freeze-thaw.
Producing controls is itself challenging and requires time and knowledge of
possible degradation pathways.
The same process is followed for developing a stability protocol for BS, but
some different assays may be chosen. It is seldom necessary to store BS for
long periods, that is, months or years, because BS is formulated and filled
to FP within days or weeks of manufacture. In addition, it is much easier
to faithfully keep bulk containers of molecules at extreme conditions, for
example, −80°C, as compared to FP. An example of a BS stability protocol is
shown in Table 7.3.
Stability testing is performed on FP and BS kept not only at recommended
(labeled) conditions, say refrigerated or frozen, but also at several subopti-
mal conditions that might be experienced in a real situation. For the r-protein
example, the intended storage conditions are refrigerated, but for stability
testing, the QC scientist also tests the samples kept at room temperature

308 Biotechnology Operations
and at a temperature above room temperature. Such protocols are referred
to as accelerated stability, when they often represent accelerated decline,
degradation, or decay. As noted earlier, vial handling is an important vari-
able. Initially, the vials are kept upright during stability test storage, but in
later stages of development, vials are placed in other configurations of stor-
age, such as inverted or horizontal. Shaking and exposure to humidity or
light might also be included in last-phase stability studies. Finally, several
time points must be tested at each of the chosen conditions, because it is not
known exactly how long the product will remain stable in any given environ-
ment. The end result is a set of protocols that provide a stability test matrix.
Although stability testing adds considerable effort and expense to a prod-
uct’s QC program, it results in extremely valuable information and becomes
the basis for ensuring proper handling, shipment, and storage of the FP. In
addition, of course, it ensures that a high-quality product—pure, potent, and
safe—is sold to the user. This, in turn, enhances marketing opportunities
and prevents future recalls or complaints.
Quality Control Testing of Raw Materials
The quality of BS or FP reflects not only the manufacturing process and
release testing, but also the quality of each raw material that goes into
making that product. Therefore, raw materials are carefully selected and
controlled, with involvement of staff from manufacturing, QC, and QA.
Manufacturing staff have primary responsibility for selecting the finest
materials to use in each process. Quality Assurance professionals approve
those selections and are further involved by later signing-off on use of
each lot of raw material, once it has arrived at the manufacturing facility.
Quality control staff consider the technical quality of raw materials, either
by reviewing the results of the tests performed by the vendor or by testing
or retesting the material at the sponsor’s laboratory.
Almost any conceivable material, live or nonliving and chemical or bio-
logical, is represented in the brief history of biotechnology. Microscopic
particles of pure gold, live invertebrates, radioisotopes with short half-life,
toxic or oncogenic chemicals, liable cells, and recombinant microbes are a
few examples, and for every manufacturing protocol, there are salts, organic
chemicals, and just plain water. Quality attributes of raw materials differ
based on their intended use and integration into the product. Items that
do not contact the product, such as a detergent used to clean the floors in
a manufacturing facility, receive the least attention. For example, QC at a
biopharmaceutical firm would review the CoA provided by the detergent’s
supplier to ensure that this material is generally recognized as safe, that it
has been tested by the manufacturer or distributor, and that it meets the

309Quality Control
specifications; the biopharmaceutical firm might not retest this detergent to
verify this CoA. In this manner, the manufacturer’s CoA attests to the safe,
pure, and potent nature of that raw material. Certain other raw materials,
such as culture media or salts provided to the biopharmaceutical manufac-
turer by an established and reputable vendor and carrying both a USP cer-
tification on the label and a CoA, might not be routinely retested. The QA
department might audit these vendors periodically (Chapter 5), but unless
there are potential issues with the vendor or the raw material, laboratory
testing might not be repeated, at least not often. The third and the greatest
level of attention is given to the raw materials that become part of the prod-
uct or directly contact the product and do not carry a recognized certification
(e.g., USP), those that are notoriously difficult to control, or those that have
even a remote possibility of harboring adventitious agents or toxic materials.
These raw materials, examples of which are given below, demand retesting
in the biomanufacturer’s QC laboratory, or they should receive other special
consideration to ensure that they are, in fact, safe, pure, and potent. General
testing requirements for a few classes of raw materials commonly used in
biomanufacturing are given below.
• Solid containers and process equipment: Containers, such as hold ves-
sels, and process equipment, such as plastic tubing and filters, are
known to shed particles or to allow chemicals to dissolve (leachates)
into the product stream. However, biomanufacturers take great care
in choosing the process materials that release particles or chemicals.
In steps such as product holds, in which this might happen even
with the highest-quality materials, the QC laboratory may be called
upon to test for those possible contaminants in the raw material, in-
process samples, or the product. Standard assays are available to the
biopharmaceutical industry for detecting particles and many leach-
able chemicals. Adventitious agent testing was described earlier in
this chapter.
• Water: As noted in Chapter 6, large amounts of water are used in bio-
manufacturing, and this must be of the highest purity and without
any microbial contamination or undesirable dissolved chemicals.
Water is purchased as purified or as water for injection by some oper-
ations, but biomanufacturers often purify water themselves, begin-
ning with an excellent source of tap or well water. Since it is used in
large volumes, impurities or microbes that enter at upstream steps
in biomanufacturing may be carried through or even concentrated
during the process and thus end up in the product. Hence, water of
all grades must be tested for traces of chemicals, such as total dis-
solved carbon; particles; endotoxin; yeast; and bacteria. Compendia
(e.g., USP <1231> Water for Pharmaceutical Purposes) describe the
various levels of water quality and the tests used by QC laboratories
to ensure that this critical reagent remains pure and safe.

310 Biotechnology Operations
• Inorganic and organic chemicals: Large amounts of salts or saline
solutions are purchased or prepared during biomanufactur-
ing. Whenever possible, compendial grade or otherwise certified
reagents are purchased from a reputable vendor and the CoA is
carefully reviewed before acceptance. Retesting may or may not be
called for, depending on the material source, the previous test pro-
tocols or certifications or lack thereof, and the risk profile. When a
USP-grade material is not available, the QC laboratory may test a
raw material to ensure that it meets the established specifications.
• Culture media and supplements: These raw materials, most of which
have a CoA but no USP designation, are critical to most biotechnol-
ogy operations, because recombinant molecules, cells, and tissues
must be grown in basal media, typically enriched with a variety of
natural or synthetic supplements (e.g., animal or human albumin
and vitamins). Although manufacturing operations attempt to use
only well- characterized or chemically simple materials, this is not
always possible. In addition, it is sometimes necessary to use plant-
or animal-derived materials to produce a biopharmaceutical. As
with inorganic and organic chemicals, it is important to understand
the nature and origin of each product used to grow and maintain
cells or tissues. Quality control plays an important role in analyzing
the nature of each raw material and ensuring that it is purchased
and inspected carefully. Again, vendor-supplied CoA are scrutinized
and, for some items, are tested again. Although synthetic and plant-
derived natural supplements, such as vitamins or growth factors, are
of moderate concern, animal-derived products are of great concern
to both sponsors and regulators. This is because animal products can
carry microbial toxins, animal viruses, or prions, and if present in
a supplement, these agents could be transferred via the product to
humans. Certificates of analysis are scrutinized, vendors are asked to
certify the origin and microbial purity of such products, and vendor
audits are commonly performed. In rare instances, such as with a
special animal serum, it may be necessary to further process and test
the product at the sponsor’s laboratory, or if a risk of disease trans-
mission even exists, it may be necessary to simply find an alternative
source or another supplement.
Quality Control and the Manufacturing Environment
As noted in Chapter 6, there is a great need to ensure a consistent and high-
quality environment in the areas of a facility dedicated to aseptic manu-
facture of a sterile product. One means of demonstrating compliance with

311Quality Control
environmental standards is to test swabs taken from personnel, equipment,
or facility surfaces and also from samples of air and water. The QC laboratory
is often responsible for this environmental sampling and testing. Samples
are taken during periods without manufacturing activity (static environ-
ments) and also during actual manufacturing operations (active environ-
ments). Instruments are used to sample air or water, to count the number
of nonviable particles, and to culture and count the colonies representing
viable particles, that is, bacteria and yeast. Samples are also taken by swab-
bing the uniforms and gloves of operators, as well as work surfaces, walls,
and floors. This information is used to better maintain a clean environment
and to alert or alarm the manufacturer whenever the aseptic or low-particle
nature is detected or a work area has been compromised. Data are then plot-
ted as a trend analysis, so that the staff may visualize the level of bioburden
as it changes over time. This facilitates early action in the face of a developing
problem. A trend analysis curve for environmental monitoring is shown in
Figure 7.6. The QC laboratory may also be responsible for additional testing
of environments in biomanufacturing, such as testing for residual product
or cleaning agents, and using analytical methods to identify trace amounts
of product or undesirable chemicals on work areas and process equipment.
10
9/2
4/2
00
2
10
/24
/20
02
11
/24
/20
02
12
/24
/20
02
1/2
4/2
00
3
2/2
4/2
00
3
3/2
4/2
00
3
4/2
4/2
00
3
5/2
4/2
00
3
6/2
4/2
00
3
7/2
4/2
00
3
8/2
4/2
00
3
9/2
4/2
00
3
100
1,000
10,000
1,00,000
Mean 0.5
Mean 5
FIGURE 7.6
Trend analysis. Plot of particles sampled in biomanufacturing room 121A, on specified dates
in the years 2002–2003. Each data point in this chart represents mean value of actual particle
counts (Chapter 6). A particle counter was used to measure the number of particles in a given
volume of air and the data from multiple mean values (particle counts on x axis) were entered
into a spreadsheet and then graphed with standard deviations (vertical lines). The upper line
presents counts for particles of 0.5 µm or less diameter, and the lower line represents particles
of 5 µm or less diameter.

312 Biotechnology Operations
Qualification, Validation, and Verification of
Analytical Methods
Validation was mentioned in Chapter 6 as an important aspect of bio-
manufacturing. Validation or the related process of qualification are also
performed on individual analytical tests. Typically, an assay is validated
during the late phase of the development cycle. Assays are often quali-
fied in mid phase, but some critical assays may be qualified even earlier
in development, and these tests may also be validated in early phases.
Critical assays are the ones that are important to the safety of the product
or to ensuring potency before use in clinical studies.
Regulatory agencies demand that analytical tests be validated before licen-
sure. The USP defines validation of an analytical procedure as “the process
by which it is established, by laboratory studies, that the performance char-
acteristics of the procedure meet the requirements for the intended ana-
lytical applications” (United States Pharmacopeia–National Formulary 2016).
The International Council on Harmonization of Technical Requirements for
Registration of Pharmaceuticals for Human Use adds that validation must
“ demonstrate that the (test) procedure is suitable for its intended purpose”
(United States Pharmacopeia–National Formulary 2016). Validation of an assay
also ensures the established specification is appropriate for a particular use
and product. Although tentative or working specifications may be estab-
lished long before assay validation begins, the validation process is a means
of confirming or adjusting those specifications. Hence, assay validation and
establishment of final specifications are highly integrated processes.
Verification applies to ensuring proper application and use of compendial
assays. Specifically, verification documents that a laboratory is, indeed, per-
forming an assay in the correct manner.
Assay validation is an experimental endeavor, and the process is always
performed under a written and approved protocol, one designed to achieve
specific purposes and perform exact experiments for each assay-product
combination. The protocol states a purpose and the scope and provides pass-
versus-fail rules for method validation outcomes. In general, the purpose
is to prove that the analytical method can perform adequately as a written
SOP for the intended purpose. The scope of use is also stated, along with the
specification that is currently under consideration. A series of experimental
procedures then challenge that assay to demonstrate suitability by a number
of criteria or traits that are established before testing. The most commonly
applied traits were briefly mentioned earlier, as they are important to con-
sider well before validation begins. These are described below as:
• System suitability: This is the ability for an analytical system to achieve
the objectives of the assay and is defined in several ways. First is the
need to ensure that equipment is suitable for the intended purpose

313Quality Control
and is installed and operational (see Installation Qualification and
Operational Qualification, Chapter 6). For some analytical tests using
highly sensitive instruments, the conditions and settings are carefully
defined. Reagents are shown to be suitable and reference standards
and controls adequate and well characterized. Interfering substances
in any reagent, including the sample matrix, are considered because
any substance could artificially enhance or inhibit an analytical sys-
tem. Sampling and sample preparation studies are completed to
ensure matrix compatibility with the test method and other reagents.
A system suitability report is prepared to demonstrate that the com-
plete analytical system, including instrumentation, is suitable for the
intended use and for further validation of the test method.
• Limit of detection and limit of quantitation: An early step in valida-
tion of quantitative assays is determination of the lowest amount
of analyte that can be detected, that is, the LOD. The amount that
can be accurately measured in a quantitative assay is the LOQ,
usually two values that bracket. For the r-protein CoA example of
total protein, for BCA assay (Table 7.2) in which the specification is
1.0 ± 0.1 mg/mL, an LOD might be 0.05 mg/mL, whereas the LOQ
might be 0.1–2.0  mg/mL of protein in phosphate-buffered saline.
These values of LOD and LOQ would be experimentally deter-
mined and would further ensure  that actual test measurements
are taken within the proper range of values.
• Linearity: For quantitative measurements, it is necessary to demon-
strate linearity, the ability, within a given range of analyte in sample,
to obtain test results that are directly proportional to the concentra-
tion of that analyte. In other words, a linear curve must be generated
within the dilution work area. Standards, shown to be of the high-
est quality by other methods, are prepared at concentrations in a
range and with a matrix that match that of the intended test sample.
For example, if one expects the r-protein to exist in a sample of BS
in phosphate-buffered saline at concentrations of 15  ±  5  mg/mL,
then one might establish test dilutions of standard BS in phosphate-
buffered saline from 5 to 30  mg/mL and then test them using the
standard assay. Duplicate assays might be performed on each of five
days and the results plotted as a linear regression. Acceptance cri-
teria for this example is based on statistical analysis and might be
a coefficient of determination, r2  >  0.98, and y-intercept of the lin-
ear regression. Linearity demonstrates that the assay results may be
extrapolated from the linear curve, within this range of values, and
that this can be achieved repeatedly.
• Precision: Analytical precision refers to the ability of an assay to
repeatedly produce the same or very similar measurements on
repeated testing when variables are held constant. Precision and

314 Biotechnology Operations
accuracy, described below, are visualized in Figure 7.7. Precision is
referred to as the degree of scatter. Critical variables, such as mea-
surement of sample volume or weight by a standard procedure, may
need to be evaluated first to ensure that pre-analytical procedures
are precise. Then the test is performed repeatedly in the same man-
ner. A test is precise if the results have little scatter. Acceptance cri-
teria for precision of an assay are usually given as the percentage
relative standard deviation for a given number of sequential tests
of the same sample. Along with robustness experiments, preci-
sion validation ensures repeatability, intermediate precision, and
reproducibility.
• Accuracy and range: Accuracy is a measure of how well an assay
agrees with a known true value, as visualized in Figure 7.7. Put
another way, it identifies the total error of the method, consider-
ing both the systemic or inherent technical errors and the random
errors for that test. Thus, range is the interval allowed between the
upper and lower concentrations of analyte in the sample. In a practi-
cal sense and for many assays, the acceptable range is demonstrated
as an outcome of linearity testing. To validate accuracy and range
by using the example of the r-protein, one might make several dilu-
tions, within, and just below and above, the limits used to produce
Precision and accuracy
Precise Imprecise
Accurate
Inaccurate
FIGURE 7.7
Precision and accuracy. This figure depicts, as holes in targets, the concepts of precision and
accuracy.

315Quality Control
the linearity curve. Each of these dilution points is repeatedly tested
several times to produce multiple results at each dilution point. The
linearity of the response is evaluated to determine whether, indeed,
the linearity testing results are confirmed and to learn the actual
range of acceptable accuracy values. The percentage relative stan-
dard deviation would be calculated for each dilution point and com-
pared to acceptance criteria, perhaps 95% to 105% of the theoretical
value. Typically, in such plots of multiple dilutions, the accuracy falls
outside these acceptance criteria, at dilutions above or below those
determined for the linear curve. This further determines a range of
values, calculated as baseline values, and here, as milligrams per
milliliter of total r-protein, which then are used to accept samples
for the assay. It also provides a percentage relative standard devia-
tion for each dilution within this range, a value that can be applied
to reference standards in the future.
• Specificity: Specificity is the degree to which the measurement is
due to the analyte of interest and not due to other substances that
could interfere with the assay or confound analytical results. Such
substances might include components of the matrix, such as mac-
romolecules or buffer salts, impurities or degradation products,
or similar but undesired molecules. The assay is shown to exactly
identify an analyte, to differentiate analyte from impurities, and,
when desired, to provide an accurate or exact result related to other
product attributes. Specificity validation requires input of these
substances by spiking a known pure sample. Purity of a known
sample, often the reference standard, is achieved by adding sub-
stances that are expected to interfere with the assay and might be
present in a sample. After multiple analyses of the various samples,
reference and reference plus substance, or other substance alone,
one can determine the degree to which the assay is specific for the
intended analyte. Acceptance criteria might be given as a percent-
age of the reference standard, such as ±10% of reference standard.
Any values obtained for a sample outside the reference standard
range or value would indicate undesirable interference.
• Robustness or ruggedness: This refers to the overall reproducibility
of the test results obtained when aliquots from a homogenous lot
of sample are analyzed under normal, expected operational condi-
tions, given that even the most consistent conditions introduce small
variations. Hence, variations in instruments, reagents, or test condi-
tions are introduced during the experiments used to validate the
assay. For example, a given reference standard might be tested under
the same procedure by different operators using one instrument on
the same day, might be tested using three different instruments but
by the same operator on the same day, or might be tested using the

316 Biotechnology Operations
same instrument and the same operator but on different days. An
experimental matrix is developed and parameters are carefully cho-
sen and then varied to ensure meaningful and affordable robustness
testing in multiple experiments.
Some assays require limited time or effort to validate, whereas other assay
validation protocols demand months of planning and experimentation and
consume significant resources. For example, an HPLC assay of a well-char-
acterized vaccine recombinant protein might be simple to validate, but an
immunopotency assay of the same protein performed in rabbits to deter-
mine the immunogenicity might require 12 months of effort and 10 times the
resources. Assay validation must be considered for each assay and be carefully
planned well in advance. Indeed, it takes much time and requires the input of
many experts to develop a good validation protocol even for one test method.
Validation applies to the assays used to test BDS and FDP, both for release and
stability purposes. A proper assay validation tests multiple batches or lots of
product, because consistency of results is important. In addition, fully quali-
fied controls and reference standards are always used. The product, BS or FP,
that is used in assay validation protocols is made by product manufacturing
processes that are or that exactly mimic commercial procedures. Clearly, QC
assay validation and manufacturing scale-up validation require a tremendous
effort, concise coordination, and a significant investment of time and money.
Owing to this, the QC plan must be carefully devised and the assays them-
selves must be scientifically sound before the sponsor begins assay validation
in late stages of the product development cycle.
Selected assays may be qualified before they are validated. Qualification is in
many respects a mini-validation, as it focuses only on important aspects of assay
validation and is performed under abbreviated protocols. In contrast to valida-
tion, qualification is completed earlier in development with only those assays
that are considered critical to demonstrating purity and potency or with those
in which confidence is lacking because of their newness, uniqueness, or com-
plexity. Qualification may also serve to establish product release specifications
for critical attributes, as multiple lots of product are tested using qualified assay
procedures. Another purpose for assay qualification is to give the sponsor
confidence that an assay is predictive of product quality for use in early clini-
cal trials. Results of qualification are predictive of validation. If an assay fails
qualification, then it is a bad candidate for full validation; the consequences of
assay validation failure can be great. Finally, qualification is sometimes recom-
mended by a regulatory agency to a product in early development, so as to
alleviate fears of using impure or subpotent product in clinical trials.
Assay verification refers to a process applied to commonly used assays,
notably those published as a standard method in a pharmacopeia or other
authoritative reference or regulation. Verification ensures that a method has
been established correctly when adapted into a new laboratory. A compen-
dial assay may be established in a biotechnology laboratory that has little or

317Quality Control
no experience with that test. In such cases, verification is a formal process,
similar to qualification, in which the sponsor ensures proper performance
and outcomes in the hands of less- experienced operators or at a new labora-
tory. Assays such as sterility test (USP <71>) and endotoxin test (USP <85>)
are candidates for verification, because they are critical to product safety yet
already well characterized and have highly detailed standard procedures.
Application of Statistics in Assay Performance and Validation
Utilizing good statistical practices throughout the assay development and
validation life cycle is important to ensuring correct performance while
minimizing bias. Perhaps the greatest threat to proper test performance and
interpretation of results is bias, a systemic distortion of results. Bias often
appears unbeknownst to the QC scientist; indeed, this is inherent in the defi-
nition. Factors generating or influencing bias must be identified; statistical
analysis is an important means of detecting bias. In addition, statistics is key
to correctly analyzing measurements, especially those considered a quanti-
tative measurement of an important attribute.
Statistical analysis is important to the QC scientist, because quantitative or
semiquantitative assays, and most potency assays and many purity tests fall
into these categories and require comparison of results to a standard curve.
This, in turn, requires constant calibration and ensuring linearity of these
tests. Demonstrating a linear response ensures that results are meaningful
and statistical analyses are applied to experimental results. For tests based
on linearity analysis, the statistical methods chosen have a great impact on
assay performance metrics such as accuracy or reportable range of values.
Statistical tests are also applied to assay qualification and validation, and
appropriate data analysis methods have been established for these endeavors.
Indeed, two metrics are considered for any assay, the measurements them-
selves and the variability of those measurements. The measurement must be
specific and accurate. Quantitative tests are either demonstrated to be linear
or, in some assays (e.g., dose response), they are nonlinear but require curve
fitting with a specific equation. Variability takes into consideration precision,
range, LOD, LOQ, and robustness. For these, statistical rules are applied to
interpretation of actual results. For example, assay precision is determined
by calculating the mean, the standard deviation, and the variance or coef-
ficient of variance. Certain statistical rules argue for focusing on the standard
deviation and its corresponding 95% confidence interval and considering
coefficient of variance of lesser importance. In the case of a potency assay for
a biopharmaceutical product, in which there may be considerable inherent
variance, such statistical rules must be considered in design, performance,
and validation of the assay. Acceptance criteria are established only after

318 Biotechnology Operations
careful statistical analysis of data generated by extensive use of the assay.
Good statistical practices are seriously considered from the outset of QC
planning and then throughout the QC cycle, because proper application of
statistical methods to analytical endeavors leads to reduced development
times, ensures that testing meets intended use, and prevents bias from enter-
ing into any analytical test.
Trend analysis is an important management tool, which is applied to most
test results in an effort to identify movement of values for controls, reference
standards, and test samples. A trend analysis for environmental monitoring
is shown in Figure 7.6. Data from a similar trend analysis of environmental
monitoring are analyzed with statistics, first to establish real-time data alert
and action limits, and second (Figure 7.8) to determine whether or not any
sample exceeds these limits.
Summary of Quality Control
Quality control is a technical or laboratory function to ensure, in part, the
purity, potency, and safety of biopharmaceuticals. Planning for and devel-
opment of QC for a given product is based on the attributes of that product,
because each test focuses on a particular attribute. Attributes are appear-
ance, safety, identity, strength, purity, and potency. Certain analytical tools
are available to sponsors at contract laboratories, but meaningful tests must
also be developed specifically for each product. This is especially true for
tests to measure purity and potency. A specification is established for each
test, beginning with early manufacture, and each specification serves as a
boundary to establish whether a product passes or fails testing. However,
as data are generated, a specification may change during the development
Alert limit: 95%
UCL = 10.860
Center line = 10.058
LCL = 9.256Q
ua
lit
y
ch
ar
ac
te
ri
st
ic
9.0
10.0
11.0
Sample
3 6 9 12 15
UCL-action limit: 99.7%
LCL-action limit: 99.7%
Alert limit: 95%
FIGURE 7.8
Application of statistics to trend analysis. Trend analysis data for an assay were statistically
analyzed and then graphed for upper (99.7%) and lower (95%) confidence levels (UCL and LCL),
on which action and alert levels, respectively, were established.

319Quality Control
cycle, as greater experience is gained on each test and with each batch or lot
of product. This information—attribute, test, and specification—is written
into a batch- or lot-specific document, referred to as a CoA, along with the
specific test results. This certificate compares specification to actual result
and is thus used to decide whether or not a batch or lot of product meets, by
test results, specifications and hence whether it may or may not be released
(pass or fail) for use.
A large number of analytical tools are available to QC scientists and many
more are being developed each year. In QC planning, it is incumbent on the
scientists to choose the correct tests to determine the quality of a product.
Other considerations early in QC development are use of reference standards
and test controls, samples and sampling, and the need to establish in-house,
special tests that are not available elsewhere. Quality control tests are used
not only to release product but also to measure stability of product after it
has been transported or stored under various conditions. Hence, stability
protocols are also developed for each product and for analytical methods
included in those protocols. The QC laboratory is also responsible for moni-
toring the environment of a manufacturing facility and operation and for
testing raw materials to ensure that whatever goes into a product is of appro-
priate quality. Assay qualification, verification, and validation are performed
during the development life cycle to ensure that analytical tools perform as
intended. Statistical analysis plays an important role in evaluating analytical
data, both the performance of tests and the test results.
Reference
British Pharmacopoeia. 2016. Medicines and Health Products Agency, London, UK.
Merck Index, 15th Edition. 2015. Royal Society for Chemistry. Cambridge, UK.
Merck Manual (of Diagnosis and Therapy), 19th Edition, 2011. Merck and Company,
Kenilworth, NJ.
Martindale: The Complete Drug Reference 38th Edition. 2015. Pharmaceutical Press,
Royal Pharmaceutical Society. London, UK.
Physician’s Desk Reference, 69th Edition, 2015. Medical Economics Company, Inc.
Montvale, NJ.
United States Pharmacopeia. 2015. United States Pharmacopeia. Rockville, MD.
United States Pharmacopeia–National Formulary. 2016. General Information/<1225>
Validation of Compendial Procedures. In: USP39-NF 34 Page 1640.
Pharmacopeial Forum: Volume No. 35(2) P. 444.

http://taylorandfrancis.com

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8
Nonclinical Studies
Nonclinical Studies and Risk Assessment
The assessments of risk and benefit for any candidate biotechnology prod-
uct are experimentally and progressively evaluated first in the laboratory,
then in animal models, and finely in people. Specifically, this involves under-
standing the nature of the biological construct or molecule, its purity, and
its potency after manufacture, as well as the safety and efficacy profile.
Nonclinical studies, performed in vitro and in animals, are primary means
of measuring the potential product risk, and much of this testing precedes
clinical trials. Results of nonclinical studies serve to better ensure that prod-
uct benefit will indeed outweigh risk once it reaches clinical studies and
the marketplace. Nonclinical study activities precede clinical research for
good reason. It is the user, often times the human subject enrolled in a clini-
cal trial, who bears the burden of risks associated with evaluating product
safety. Thus, the sponsor of a novel biopharmaceutical provides clear experi-
mental evidence that risks are tolerable and the product itself is unlikely to
result in disease or death to the human subjects or, on marketing approval,
to the public.
Information demonstrating safety and tolerability of a candidate biophar-
maceutical is presented in the Investigational New Drug Application (IND),
specifically in the Pharmacology and Toxicology section. Here, test results,
that is, in vitro laboratory and animal test data, demonstrate in various ways
both how the biopharmaceutical behaves in biological systems (pharmacol-
ogy) and whether or not it is toxic (toxicology). Some pharmacology and
toxicology test systems are simple and are applied to samples of the biophar-
maceutical in a laboratory setting, using tests focused on answering a single
question, for example, the mutagenic potential of a compound. Other tests
are performed in appropriate animal models and these are supplemented
with additional laboratory testing. An adequate and well-controlled panel
of nonclinical studies, an example of which is shown in a general scheme
in Figure 8.1, can demonstrate, beyond reasonable doubt, that the biophar-
maceutical possesses desired pharmacological attributes and the levels of
exposure at which it is safe, not toxic, and well tolerated in animals.

322 Biotechnology Operations
Humans are exposed to many biotechnology products that are not bio-
pharmaceuticals. Products used for environmental, industrial, or agricultural
(including food) purposes are studied in formal toxicology tests, both labora-
tory and animal. However, biopharmaceuticals are given the greatest safety
scrutiny and testing because they are directly given to large numbers of
humans, sometimes over long periods, and many are injected into the body.
Nonclinical testing of biopharmaceuticals has its foundation in the drug
industry, where a general understanding and appreciation for the value
of pharmacology and toxicology have led to successful development and
Pharmacology
Consider possible
animal models
Consider possible in vitro and
animal models
Select
animal models
(1 or 2)
Select in vitro
models
Select
animal models
(1 or 2)
ADME and pilot studies
1. Formulation, dose, and route delivery
2. Metabolism or catabolism
3. Biotransformation
4. Tissue distribution
Acute toxicology
Short-term dosing
In vitro toxicology
Assay development
Measure product in tissue and blood
Special toxicology
1. Tissue binding
2. DNA integration
Definitive pharmacokinetic and
pharmacodynamic studies
1. Cmax
2. Tmax
3. Concentration effect
Subchronic toxicology
Chronic toxicology
1. Long-term dosing
2. Reproductive
3. Immunotoxicology
4. Neonatal toxicology
TPP Toxicology
1. Medium-term dosing
2. Local tissue
3. Route delivery
FIGURE 8.1
Scheme of nonclinical activities in biopharmaceutical development. The targeted product pro-
file (TPP) is instrumental in developing a nonclinical plan. Both pharmacology and toxicology
studies are performed to identify a safe and effective dose. Assays are developed to measure
product and metabolites in blood and tissue, and appropriate animal models are used in vari-
ous types of studies.

323Nonclinical Studies
marketing of small molecule drugs. Studies of dose-response, pharmacoki-
netics, and pharmacodynamic relationships, and toxicology, as well as the
development and application of in vitro laboratory tests and animal models
are the scientific tools that are routinely used by scientists and described
in this chapter. However, because drugs and biopharmaceuticals differ in
many respects, the panel of tests required for biopharmaceuticals is often
unique, even if the basic principles are the same. Consider that biotechnol-
ogy products are typically large molecules, living cells, or microbial products
and have unique patterns of biodistribution and distinctive toxicities. Drugs
are usually small organic molecules. Biopharmaceuticals do not always lend
themselves to testing in traditional in vitro tests or animal models that have
been developed to assess the safety of drugs. Nonclinical biopharmaceutical
scientists apply knowledge and experience borrowed from the small mol-
ecule drug industry, but they have also developed unique methods to effec-
tively study the pharmacology and toxicology of biological molecules and
cells. Further, as compared to testing drugs, pharmacology and toxicology
testing of biopharmaceuticals often requires unique and expensive tests and
development of applicable animal models.
Biopharmaceutical Delivery, Pharmacokinetics,
and Pharmacodynamics
Product Delivery to the Body
Biopharmaceuticals pose unique formulation and delivery challenges
because of their large size, complex structure, and vulnerability to degra-
dation. Many injectable formulations are difficult to administer because of
high viscosities associated with concentrated formulations that are often
encountered when doses reach hundreds of milligrams required in the final
formulated product. Most biopharmaceuticals and drugs are transferred
from the final container, such as a vial or a syringe, to an initial target tis-
sue, and only then, it is distributed to the target organ or tissue, where
it has the intended therapeutic effect. There are many ways to achieve
this objective, some of which are listed in Box 8.1. Many drugs are given
orally because they are taken up in the digestive tract without first being
metabolized. Oral presentation is rarely the case with biopharmaceuticals
today and most are given parenterally. Products given intravenously are
designed and intended to be distributed throughout the body very rap-
idly. Other parental routes of delivery are intravenous, subcutaneous, and
intramuscular. Monoclonal antibodies and therapeutic proteins are often
given by one of these routes. Vaccines are usually given subcutaneously or
intramuscularly but some are given intranasally and others intradermally.

324 Biotechnology Operations
Certain  cellular therapies are delivered parenterally, often by direct injec-
tion into a target organ or tissue. Oral ingestion is by mouth, but in this case,
the biopharmaceutical must be specially formulated, so that gastric and
intestinal acids and enzymes do not degrade the product before it crosses
the intestinal or gastric mucosa. In addition, special consideration is given
to the size of a molecule taken orally, as large molecules such as an anti-
body would not be readily absorbed in appreciable amounts. Pulmonary
delivery for lung absorption is sometimes applied to smaller biomolecules
such as insulin. In addition, topical application of biopharmaceuticals is
another route of delivery and is exemplified by transcutaneous delivery
BOX 8.1 ROUTES OF ADMINISTRATION TO ANIMALS
OR MAN FOR A BIOPHARMACEUTICAL
1. Parenteral (injected)
a. Intravenous
b. Intra-arterial
c. Intramuscular
d. Subcutaneous
e. Intradermal
f. Intracardial
g. Intraocular
h. Intraperitoneal
i. Epidural
2. Oral
3. Inhalation
4. Body cavity
a. Intranasal
b. Sublingual
c. Rectal
d. Intravaginal
e. Intrauterine
f. Intraurethral
g. Intra-auricular
5. Topical
a. Percutaneous (transdermal)
b. Cutaneous
c. Ophthalmic

325Nonclinical Studies
of vaccines or therapeutic peptides. In development are a host of special
delivery methods for biopharmaceuticals such as patches, microneedles,
and special injection devices (e.g., pumps). Additional delivery methods
that require adjustments in formulation are being developed to enhance in
vivo accessibility; these include the use of controlled-release preparations
(e.g., microspheres and microparticles), protein modifications (e.g., albu-
min fusion), and genetic manipulations (e.g., site-directed mutagenesis).
Adsorption, Distribution, Elimination, and Metabolism (ADME)
Once a biopharmaceutical has crossed all barriers and is in the blood, it
must reach a target organ or tissue, an exact location to produce its thera-
peutic effect. For some biopharmaceuticals, this step can be challenging. It
must be absorbed, usually into the blood stream and remain stable if it is
to be distributed. A method of delivery may fail to achieve this objective.
For example, many therapeutic monoclonal antibodies are injected into the
subcutaneous tissue or muscle, even though the target organ, for example,
rheumatic joints, is some distance away. The biopharmaceutical must be
distributed and absorbed in adequate amounts, before it results in local
reactivity or is metabolized or otherwise eliminated. Biopharmaceutical
products are, therefore, designed to be absorbed, distributed, and then
metabolized and eliminated (but not too rapidly). These functions, known
as ADME, are studied and reported for each biopharmaceutical prod-
uct because these are of critical importance to success in clinical trials.
Figure 8.2 outlines the possible tissue relationships between each of these
functions.
Absorption
How is absorption defined for a biopharmaceutical? Oral-gastrointestinal
absorption is unlikely, because most biopharmaceuticals, composed of pro-
tein, cells, RNA, or DNA, are recognized as just another food substance, and
thus, the gastrointestinal juices and enzymes digest them. In addition, few
biopharmaceuticals might be absorbed across the skin (topical) or mucosal
surfaces (transmucosal), because they are simply too large to diffuse intact
across such barriers. Hence, most biopharmaceuticals are given by the paren-
teral route, which means that they are directly injected into either the blood
stream or a tissue. From here, biopharmaceuticals either have a local effect
or enter the blood or lymph systems, facilitating distribution to other tissues.
Distribution
After application of a product, it is very important for the biopharmaceuti-
cal to be distributed to the tissue or organ where it will have the greatest
therapeutic effect. It should not reach tissues, or build up, creating reservoirs,

326 Biotechnology Operations
where it may be toxic. It is also critical to sustain a certain level of biophar-
maceutical in the blood and tissue. Many biopharmaceuticals reach a state
of equilibrium on reaching the blood stream; whereas for other products,
there is a rapid drop in circulating levels after injection. The pharmacologi-
cal and toxicological implications of parenteral delivery may be considerable,
because a biopharmaceutical in the blood is rapidly distributed, allowing
exposure to many organs and tissues, and not only to the target tissue or
organ. Alternatively, a biopharmaceutical may be injected into a firm or
semisolid tissue, such as subcutaneous, where it resides in a depot and is
slowly released into and distributed by the blood stream. For some products,
Animal model
development
Biopharmaceutical
injection of product
(e.g., parenteral)
Heart Lungs Kidneys Brain
Available, free product
Metabolism
Tissue
bound Circulation Biotransformed
FIGURE 8.2
Outline of adsorption, distribution, metabolism, and excretion (ADME) studies. Using animal
models and sensitive and specific assays, product is measured in various tissues and blood.
Additional assays are used to measure metabolites.

327Nonclinical Studies
this can result in sustained levels of biopharmaceutical, with blood absorp-
tion and then distribution occurring over a longer period, as compared to an
intravenous injection approach.
However, for other biopharmaceuticals, it is not desirable to distribute the
product to certain tissues, as the molecule might be toxic to certain tissues or
organs. Some molecules tend to accumulate in one or another tissue or cellu-
lar compartment, a reservoir; this may be desirable or it may lead to toxicities.
For example, a product could rapidly accumulate in the liver, where it may
be hundreds of times more concentrated than in other tissues. If the product
is therapeutic in the liver, then it may be best to have that biopharmaceutical
largely concentrated right there and unequally distributed in the body. In
contrast, if the product is toxic to liver cells at high concentrations, then it is
not good for the biopharmaceutical to accumulate there. Sometimes, it is best
to avoid the blood stream, when possible, and deliver the biopharmaceutical
directly to the target organ. However, this can be challenging with certain
types of products that target hard to reach organs such as pancreas or brain.
Biodistribution studies to determine these pharmacological parameters are
critical to understanding pharmacology of a product. In addition, informa-
tion gleaned from distribution studies is used to support the development of
new formulations and delivery methods aimed at improving the therapeutic
value and reducing the toxicity of a biopharmaceutical.
Metabolism and Biotransformation
Biopharmaceuticals eventually change in the body to another form and
become metabolically inactive through normal processes such as enzymatic
degradation. A few biomolecules, such as the DNA in a genetic therapy or
a pluripotent-cell-derived product, may not follow this rule, because they
are developed for the purpose of longevity in the body. Yet, biotransforma-
tion, a term used to describe any biological process that converts the original
product to another molecular format, is the rule that applies to biopharma-
ceuticals. In some cases, biotransformation enhances the therapeutic activity,
whereas in others, it decreases, limits, or terminates the biological activity.
Physiological, genetic, and environmental factors may be, and often are,
involved in biotransformation. Although we can establish the average time
of biological activity in a given population, it has been nearly impossible
to reliably predict, for a single individual or animal, how long a particular
biopharmaceutical will remain active. Living organisms are quite diverse,
when it comes to processing biopharmaceuticals. Further complicating
the picture, the coadministration of two compounds can have unexpected
effects, because metabolic drug interactions are possible. Drug interactions
can impact absorption, distribution, pharmacokinetics, metabolism, or
excretion, and many patients take two or more drugs or biopharmaceuticals.
Metabolism and biotransformation studies can assist in understanding the
overall pharmacological profile of any product.

328 Biotechnology Operations
Excretion
Clearance is a process in which a biopharmaceutical is eliminated from fluid
phases, tissues, or organs. With most biopharmaceutical products, clearance
is expected to take place through the processes of metabolism and excretion,
but first, the molecule must remain in the target tissue or organ long enough
for it to have a therapeutic effect. Excretion cannot be too rapid. With many
small molecule drugs, the absolute rate of clearance is a linear function of
the concentration in blood. However, with biological molecules, this is not
always the case, and the rate of clearance is not simply the rate of elimination
divided by blood concentration. In addition, while small molecule drugs are
often cleared by liver and kidney, larger biological molecules are not often
metabolized in the liver and are retained, not excreted, as they pass through
the kidney. For many biopharmaceuticals, the sites of metabolism and excre-
tion are unknown, and it is assumed that components of degraded biophar-
maceuticals, such as polypeptides, amino acids, and nucleic acids, are simply
catabolized to a certain degree and then used by the body to produce energy
and to build other macromolecules.
Pharmacokinetics and Pharmacodynamics
The science of pharmacokinetics attempts, for a given biopharmaceutical, to
understand ADME and to explain the outcomes that follow dosage of that
product. Pharmacokinetics is the study of complex interactions that fate
between an active compound and the cells, tissues, and organs of the body.
Only certain aspects of pharmacokinetics are discussed here. Pharmacokinetic
studies for pharmaceuticals and drugs differ in many respects, because small
molecules and the large molecule biopharmaceuticals are often quite differ-
ent in biological properties and mechanisms of action.
However, some rules of pharmacokinetics do explain the behavior of many
biopharmaceuticals. For example, with specific and sensitive analytical tools,
we can measure the maximum concentration of a biopharmaceutical that is
reached after a certain dose is given by the route of injection. This value is
called the maximum concentration or Cmax and is shown in Figure 8.3. From
pharmacokinetic studies, we also determine the amount of time it takes
from injection of a biopharmaceutical, until Cmax has been reached. This is
referred to as Tmax, also shown in Figure 8.3. From the same experiment, it is
possible to measure the half-life of the biopharmaceutical in the blood, and
this is referred to as t1/2. Half-life is a derived value based on both clearance
and volume of distribution. The period from injection to Cmax is called the
absorption phase. Although this may be a very short time for biopharma-
ceuticals given intravenously, it is an important parameter for products that
are injected into subcutaneous or other tissues. The period beginning at Cmax
and lasting until all product has been eliminated from the blood is called the
elimination phase. If a product is given in multiple injections, then the blood
level rises, until it reaches a plateau, referred to as the steady state for that

329Nonclinical Studies
dose and dosing regimen. These rules do not apply to some biopharmaceuti-
cals. For example, vaccines seldom reach the blood in appreciable quantities,
and it is difficult or impossible to measure small amounts of recombinant
proteins or live cells in a solid tissue.
Clearance, discussed above, is an important aspect of any pharmacoki-
netic profile. The apparent volume of distribution is another parameter; it is
abbreviated as V (volume of distribution) and is equal to the amount of bio-
pharmaceutical administered, divided by C, the concentration of product in
drug or plasma. This value varies widely, depending on the amount of tissue
binding and the degree to which the product is hidden by or binds to other
materials. Using real-time models in animal or human studies and assuming
that assays are available to measure a biopharmaceutical in blood, it is pos-
sible to measure plasma concentration time curves for a product.
Bioavailability measures the amount of biopharmaceutical that is avail-
able for use by a tissue at any given time. For most products, bioavailability
is maintained through multiple doses; this keeps biopharmaceutical levels
at a reasonably constant, albeit fluctuating (within a range of values), level
in blood or tissue. Controlled bioavailability means that the product is con-
sistently available to the patient’s tissue and ensures therapeutic effect at
all times. Each of the factors—absorption, distribution, metabolism, and
excretion—has a great impact on bioavailability, as do calculated values such
as Tmax, Cmax, and t1/2. For successful therapy with many biopharmaceuticals,
2
5B
lo
od
c
on
ce
nt
ra
tio
n
of
p
ro
du
ct
(μ
g/
m
l)
50
100
4 6
Tmax
Cmax
t1/2
8
Time (hours)
10 12
FIGURE 8.3
Example of biopharmaceutical concentrations in blood over time. After injection of a biophar-
maceutical at Time 0, the concentration rises in blood, until it reaches a maximum concentra-
tion (Cmax) at the fourth hour (Tmax). The concentration then falls because of metabolism and
excretion, until one-half the maximum concentration is reached at the tenth hour, resulting in
value of t1/2 for the period.

330 Biotechnology Operations
pharmacokinetic experiments develop information that, in turn, allows one
to design and optimize delivery methods and dosing regimens, based on
the desired effect and the amount of product available to produce that effect.
This might sounds logical, even simple, but, in fact, deriving these val-
ues is a very complicated process, made more difficult by the lack of ade-
quate animal models in which to study most biopharmaceutical products.
Nonetheless, the product development program must take this information,
generated in animals, into consideration as the target level, maintenance
dose, loading dose, and individualized dose are calculated for man. Often
times, the human target dose calculated from pharmacokinetic studies in
animals differs significantly from the target dose estimates made, in the
absence of experimental data, in the targeted product profile (TPP). For large
differences, it is wise to ask why this happened and perhaps do further phar-
macokinetic experimentation. Each biopharmaceutical molecule is unique,
in molecular characteristics, how the product is dosed, and the indication.
For example, the loading dose of a gene therapy might be high and without
maintenance dose, whereas a monoclonal antibody to treat a chronic disease
might require a specific dose through years of treatment, without the need
for a higher loading dose at the onset of therapy. The possibilities are end-
less and must be experimentally determined for each product and intended
clinical indication.
In summary, the experimentally derived target level is simply the amount
of biopharmaceutical that, hypothetically, will produce the desired effect
when given in a particular formulation and route of delivery. The loading
dose is the amount of product that will be given at the initiation of therapy
to rapidly achieve the target level. The maintenance dose is the amount
that must be given at set intervals after the loading dose, to maintain the
target level.
In addition, speaking of the future, many traditional methods used to
measure pharmacokinetic parameters in small molecule drug studies have
not always been successfully applied to experimental pharmacokinetics of
biopharmaceuticals. Given that necessity is the mother of invention, there
are new methods that seem quite relevant for measuring various parameters
such as biodistribution and clearance of biological products. Optical imag-
ing using bioluminescence and dyes and variations of computer-assisted
tomography are promising in this respect. Future studies of biopharmaceu-
ticals will certainly be more informative; however, the measured endpoints
might seem quite different from those of small molecule drugs.
The science of pharmacodynamics studies biochemical and physiological
effects and the mechanism of action of biopharmaceuticals. The concept of
drug- receptor interactions underlies most pharmacodynamics for small mol-
ecules. Since most biological products exert their effects by interactions with
molecular or cellular components of an organism, pharmacodynamics is also
important for the development of biopharmaceuticals. To properly progress
a product to clinical development, a scientist should have some idea of how

331Nonclinical Studies
a therapeutic effect is generated within a complex organism. For some bio-
pharmaceuticals, such as a monoclonal antibody targeted to the receptor of a
particular cell type, therapeutic effect might be well known from discovery
research. Indeed, designer molecule biopharmaceuticals are developed for a
specific purpose, such as binding to a receptor having a known physiologi-
cal function. For other products, such as most recombinant protein vaccines,
the effectors’ mechanism remains unknown at the time it is first tested in
man. Some classes of molecules, often times given in one or a few doses, are
never completely understood, because their exact mechanism remains in a
mysterious and seemingly complex black box, even after market approval.
Therefore, many biopharmaceuticals are an exception to the recommen-
dation of clearly understanding the mechanism of action before using the
product in man. However, attempts are still made to understand pharmaco-
dynamics of a biopharmaceutical, and the information derived from these
experiments is applied to designing pharmacokinetic and nonclinical safety
studies and estimating clinical doses and dose regimens.
What types of pharmacodynamic information can be derived for a biophar-
maceutical during nonclinical development? It is important to understand
the relationship between the concentration of a product and the magnitude
of the response to that biopharmaceutical. However, the response may be
complex and even unpredictable in some individuals, animals, or man.
A concentration-effect curve can be constructed. As shown in Figure  8.4,
the experimentally derived information provides a wealth of information
regarding pharmacodynamic properties of a biopharmaceutical. Potency is
that part of the curve where an effect can be measured. Potency is clearly
Maximal effect
Potency range
Concentration of product
In
te
ns
ity
 o
f e
ffe
ct
 
No effect 
FIGURE 8.4
Concentration effect curve for a biopharmaceutical. The intensity of effect is proportional to the
blood (or tissue) concentration of the biopharmaceutical. By measuring physiological effect, the
potency range is determined, as are concentrations, with no or maximal effect.

332 Biotechnology Operations
based on the concentration of the biopharmaceutical but can be quite variable
within a given population. Maximal efficacy is that amount of biopharma-
ceutical that produces maximal effect in that individual or in a population.
There is also a slope to the concentration-effect relationship, and this reflects
the mechanism of action of the biopharmaceutical and is seen in data from a
population of animals or humans. Finally, there is biological variability, seen
as standard deviation from the line traced by the population value. Much
individual variability is due to genetic and other factors. At any given point
on the curve, this can be significant for some products and is referred to as
the individual effective concentration.
However, there are caveats to pharmacodynamic study results when bio-
pharmaceuticals are tested. The standard concentration-effect relationship
for a given biopharmaceutical considers a normal population, matched by
age, sex, disease status, and so on. As organisms (humans) age, our response
to a given biopharmaceutical changes and the concentration-effect relation-
ship of an aged population may look quite different from that of a young
adult population. The same could be said for populations composed of
infants, children, adolescents, and those with certain underlying diseases.
Pharmacodynamic variability demands that a biopharmaceutical developer
understand the kinetics and toxicity of each product and carefully consider
every population it might intend to treat. However, this is not an easy task.
Pharmacodynamic variability, the individual variation in response to a bio-
pharmaceutical, based on the mechanism of action, is an important issue in
product development. This variability is a factor even after extensive phar-
macokinetic and pharmacodynamic information is experimentally derived
from a population of animals or human volunteers.
Bioequivalence is a term that, in its purest definition, suggests that two dif-
ferent biopharmaceuticals have an equivalent effect. Stated in another way,
the concentration-effect relationship of two molecules is very much alike.
Consider two superimposed concentration effect lines, as pictured for the
single line in Figure 8.4. However, bioequivalence also has other, sometimes
more practical, meanings. It can mean that an active ingredient has the same
effect even when formulated in two different ways or that one biopharma-
ceutical has the same effect when given by two different routes of injection.
If a reliable model with minimal variability can be developed and applied,
bioequivalence testing can be an important aspect of nonclinical develop-
ment, as optimal formulations, routes of delivery, and other variables may be
tested, first in animals and then in man.
Bioavailability, first introduced under the pharmacokinetic discussion,
above, is also relevant to pharmacodynamics. Defined in pharmacology
as the fraction of the total amount of biopharmaceutical given (available to
systemic circulation), bioavailability has additional meaning for certain bio-
logical products that are given to produce a local effect and that rely little
on blood concentration. For example, a monoclonal antibody given intra-
venously has, on injection, 100% bioavailability to the circulatory system.

333Nonclinical Studies
However, if the same product must reach a tumor mass to produce an effect,
then it most likely has a much lower percentage of bioavailability where it
counts, that is, within the tumor. Experimental measurements of pharmaco-
dynamic properties of this monoclonal antibody are not meaningful, unless
pharmacokinetic information is available and considered. Thus, the effect
of bioavailability on bioequivalence is carefully considered with every new
biopharmaceutical and for each new indication for existing products.
For this and other reasons, biodynamic experimentation is challenging for
many biopharmaceuticals. Consider a few examples. A gene therapy should
replace a receptor, missing from birth, on a particular type of cell. This might
be achieved by inserting into host cells the gene for a molecular analogue or
by adding a (pluripotent-derived) cell that expresses the receptor. In either
case, the therapy replaces the missing activity. But how does one measure, in
a manner that is meaningful to the human situation, the pharmacodynamics
of either therapeutic approach? How does the biopharmaceutical scientist
determine which of the two approaches might be most successful at replac-
ing the receptor, and how is this tested in a whole body situation? Some
would take an experimental approach in an animal model, whereas others
would argue that animal studies are not relevant and the product should
forgo animal studies, and instead, pharmacodynamics should be consid-
ered first in human studies (and, of course, after safety studies have been
completed).
In a second example, the pharmacodynamics of a monoclonal antibody
are unknown. The antibody could directly bind and neutralize a molecule
excreted by a cell, or it could bind to a cell receptor and reduce the excretion
of the same molecule. How does one approach pharmacokinetic studies in
this case? Are the studies best done in animals, or should they be performed
in Phase 2 human clinical studies?
Application of Pharmacokinetics and Pharmacodynamics
in Biopharmaceutical Development
The pharmacokinetic and pharmacodynamic properties of each biopharma-
ceutical present important information, because this information is applied
to various decisions, including the selection of an efficacious dose and route
of delivery, to ensure that the new product is tested for safety at a correct
dosage. Indeed, nonclinical studies should demonstrate that new products
have little chance of causing unexpected and undesirable effect when given
to humans in subsequent clinical studies. If this is so, then it is necessary
to understand the pharmacokinetics and pharmacodynamics of the product
before embarking on an extensive program of safety testing in animals and
certainly before introducing the biopharmaceutical into man.
As suggested earlier, certain tools are required for pharmacokinetic and
pharmacodynamic development. One important evaluation is to directly
measure pharmacokinetics and pharmacodynamics in an applicable animal

334 Biotechnology Operations
model. A representative animal model may be available from studies of
other products for a similar clinical indication. However, no matter how well
proven the animal model is for another particular disease or class of product,
it is impossible to know if that model will be applicable to a particular bio-
pharmaceutical or its indication. For some biopharmaceuticals, it is difficult
to even begin the process, because of inherent biological complexity of the
product, animal physiology, potential immunogenicity issues associated with
a xenogenic environment, and the ability to mimic the human clinical disease
or underlying pathophysiology. For example, some of the most common bio-
pharmaceuticals, such as vaccines and genetic therapies, are very difficult to
classify, in part, because of the black box or great unknowns concerning the
exact mechanism of action. In addition, we often lack a full understanding of
pathogenesis of the disease that is to be prevented or treated. In the example
of a recombinant protein used as a vaccine to prevent an infectious disease,
we commonly lack knowledge on how the infectious organism is pathogenic
and how exactly the immune system of our body fights that disease.
Still, it is incumbent on the product developer to use available scientific
information and attempt to understand the pharmacokinetics and pharma-
codynamics of each new product. With proper methods to detect active bio-
pharmaceutical cells or cell receptors in blood and tissues, pharmacokinetic
and pharmacodynamic measurements can be made in animals. Using more
than one animal species, single-dose experiments can yield information on
ADME, preferred route of delivery, optimal dose, dose linearity (Figure 8.3),
concentration-effect (Figure 8.4) interspecies differences, metabolism, and
excretion. Multiple-dose pharmacokinetic and pharmacodynamic studies can
then be initiated or can be performed in conjunction with toxicology studies.
In addition to studies in animals, in vitro studies are often helpful. For
example, it is informative to determine whether a candidate therapeutic
monoclonal antibody binds specific cell types, and this might be achieved by
using human cells, derived from various tissues, in culture. In addition, tests
to study intracellular metabolism of compounds by using cell and organ
cultures are available. In vitro methods to screen for induction of immune
responses, again using cell or organ cultures, are also available.
To begin nonclinical experimentation, the biotechnology firm must have
a formulation (Chapter 6) for each candidate product, and there must be
enough material to allow extensive testing in the laboratory and in animals.
This is sometimes referred to as the optimized clinical formulation, which
means that it is the formulation of the product intended for Phase 1 clini-
cal studies. Clinical quality or comparable quality product is essential for
these studies to demonstrate the nonclinical safety that represents the same
product intended for use in human clinical studies. A decision on how the
product will eventually be administered to humans must also be made.
This brings to mind the need to have an animal model to evaluate alter-
native delivery methods, thus generating scientific data that will be used
later to support and justify the delivery method selected for human clinical

335Nonclinical Studies
studies. A pharmacokinetic and pharmacodynamic program should also be
designed to meet current regulatory requirements. Finally, there must be a
precise means of measuring the product, as it exists in a matrix such as ani-
mal blood or tissue. This means extensive analytical support (Chapter 7) to
detect and measure exactly the biopharmaceutical of interest.
Results of pharmacokinetic and pharmacodynamic studies are carefully
examined. Of particular importance are findings with safety implications,
such as undesirable localization, notably vital organs or tissues (e.g., the central
nervous system, the heart, and kidneys), of therapeutic molecules or cells. Such
information is then applied to the design of toxicology studies and to moni-
tor safety of subjects in human clinical trials. Nonclinical data also provide
a foundation on which to establish rational toxicological studies to support
clinical trials. It gives clues to development or application of the best animal
models or future pharmacokinetic, pharmacodynamic, or toxicology testing.
Specifically, study data may point toward an animal model that mimics the sit-
uation expected in man. For example, if a monoclonal antibody was expected
to have a therapeutic effect only if it remained in the human body for at least
2 weeks, it would be unwise to use, for toxicology testing, an animal in which
the same molecule was undetectable 1 day after it was injected. The same can
be said for the route of delivery. If a vaccine is to be given intramuscularly
to man as 200 µg in a volume of 1.0 mL, then one would not choose a mouse
model, because it is impossible to put this volume into a single mouse muscle.
Pharmacodynamic and pharmacokinetic studies often lead a developer to
change formulations according to the anticipated or most readily available
format. If a therapeutic DNA molecule did not remain, as required, at the
subcutaneous site of injection for 24 h in an animal model, then it might need
a new formulation, one that resulted in a depot effect to enhance longevity in
that tissue. Improvements in dosing, based on data from well-designed phar-
macokinetic studies, can be another positive outcome. Hence, well-designed
nonclinical studies of biopharmaceutical absorption, distribution, metabo-
lism, and excretion typically provide valuable information that allows for
improved and more efficient safety assessment studies and may also provide
a basis for the mechanism of action.
In conclusion, pharmacokinetic and pharmacodynamic studies are per-
formed, with careful planning, as a series of experiments and with close
coordination with other functional area experts, notably individuals from man-
ufacturing, regulatory affairs, quality assurance, and clinical studies. Primary
pharmacokinetic and pharmacodynamic effects are studied in animal models
by using a variety of in vitro (or laboratory) methods. Experimental design
focuses on the specificity of biopharmaceutical activity. If at all possible, levels
of biopharmaceutical should be tested in humans, based on the information
derived from these studies. For many types of cells and molecules, it is impor-
tant to determine where the molecule goes within the animal, to define tissue
or cellular interactions, and to identify how long it remains in any given loca-
tion and how and where it is metabolized or excreted.

336 Biotechnology Operations
Safety Assessment of Biopharmaceuticals
Toxicology
Toxicology is a science, specifically the study of adverse effects of agents—
physical, chemical, and biological—on living organisms. Since any molecule
can produce adverse effects, toxicology is important to all biotechnology prod-
ucts, not just to biopharmaceuticals. This science covers acute, chronic, and
long-term risks and uses a variety of established methods, many of which are
biological. Toxicology assesses risks, that is, the probability of adverse events,
caused by such effects. Toxicological studies go beyond measuring risks. These
studies provide data that determine the possible causal relationships and help
to establish limits of safety and design rationale and safe clinical studies. This
discussion covers general approaches to toxicology while using examples
derived from the safety testing of biopharmaceutical products.
The term toxicology immediately brings to mind chemical toxins, acids,
bases, or organic solvents created by man for the purpose of producing other
chemicals or lifestyle products. It also conjures images of physical agents
such as ionizing and nonionizing radiation and ultraviolet light. Further, we
consider the target of these agents to be a biological system, such as plants,
animals and, most notably, humans. Chemical and physical agents can
cause damage to living organisms. Drugs and medical devices are consid-
ered chemical agents and physical agents, respectively, and for decades, they
have been studied for toxic effects; many prove to be nontoxic but a surpris-
ing number are toxic, reflecting a range of toxicities from mild to severe.
Toxicology studies are routinely performed on drugs and medical devices by
using both laboratory (in vitro) and animal (in vivo) methods.
In the recent past, a third type of agent, that is biological, has been added
to the list of potential toxic agents. We have been aware of natural toxins,
for example, snake venoms and poisons from plants, but have not had, until
recently, the ability to manipulate the structure and function of biological
molecules or cells and then use them to prevent or treat disease. With the
advent of biotechnology, scientists began to develop biopharmaceuticals.
These compounds, produced by man (or at least designed by man) and some
being unique in nature, are intended for human exposure, sometimes repeat-
edly and over long dosing periods. Further, biopharmaceuticals are designed
to change the physiology or biological status quo of the user. However,
changing the physiological balance for the better in some ways can also have
undesirable effects in other ways. In addition, a recombinant molecule or
cell that produces a desirable effect in one tissue might cause an undesir-
able, or even toxic, effect in another tissue or organ of the same individual.
Hence, with the advent of biopharmaceuticals, it became clear that each mol-
ecule would be subjected to toxicology testing in the same manner as drugs
and medical devices. Clearly, biological substances could cause undesirable

337Nonclinical Studies
effects by interacting with cells or tissues, as much as chemical or physi-
cal agents. Biopharmaceuticals, if used properly or improperly, in excessive
dose, or at extreme exposure, can harm body structures or processes and
some might even pass these effects on to subsequent offspring. Thus, each
biopharmaceutical must be studied to evaluate toxicity, or the potential to be
toxic, when used in a particular manner for a given indication.
Design of a Safety Assessment Program
An effective safety assessment program must be carefully planned. Elements
of planning for biopharmaceutical safety studies are outlined in Chapter 1.
In this chapter, we delve into some factors that influence biopharmaceutical
toxicity, discuss the tools used in these studies, and consider common study
designs. The toxicologists have, or should have, four assets at their disposal.
These are as follows:
1. Scientific and design precedence established, over decades, for a host
of biological, chemical, and physical agents
2. In vitro methods to serve as rapid screening tests
3. Animal models and the ability to test agents in these complex
organisms
4. Established testing procedures to include acute, subchronic, chronic,
reproductive, carcinogenic, local tissue, immunological, and respira-
tory toxicological protocols
The key to completing a meaningful safety program for a new biopharma-
ceutical is to use these tools wisely under a product-specific and indication-
driven experimental strategy.
The nonclinical plan is thus based on elements of TPP, notably the intended
indication or disease, and intended product safety profile, as developed for
the candidate biopharmaceutical. Consider that some biopharmaceutical
products such as a therapeutic for a terminal illness, for example, metastatic
cancer, have a very different profile from a product such as a vaccine that is
intended to prevent a nonlife-threatening disease in infants. The plan is also
based on an estimate of the clinical dose and dosing schedule, as provided in
the draft clinical plan. From this information, and relying on experience with
similar products, on regulatory guidelines for the class of biopharmaceutical,
and on any available research data, the nonclinical professional can outline
the intended approach for safety testing. Using a recombinant therapeutic
protein as an example, Figure 8.5 presents a general scheme for nonclini-
cal testing of a biopharmaceutical by phase of development. It demonstrates
how the flow of events in a safety testing program structures a tiered test-
ing approach. With each tier or ascending phase of clinical development, the
product is used in both greater numbers and a more diverse population of

338 Biotechnology Operations
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339Nonclinical Studies
individuals. This, in turn, can demand more detailed and stringent safety
testing before each clinical phase.
The earliest nonclinical testing focuses on understanding the pharmacoki-
netic and pharmacodynamic properties of the biopharmaceutical, as noted
above. With this information, the intended human dose or doses can be esti-
mated for Phase 1 clinical study. Toxicity testing applies the intended clinical
doses and dosing regimen to the design of nonclinical studies that are com-
pleted and reported before filing an IND or initiating the first clinical study.
These early nonclinical studies include acute, subchronic, or other types of
studies that may be considered by a sponsor or might be required by regula-
tory agencies before initiating Phase 1 study with this class of product.
Subchronic and even some chronic testing may be required before enter-
ing Phase 2 human clinical studies. The route of delivery or the formulation
may be adjusted, based on Phase 1 study results, and therefore, additional
acute testing may be necessary or local tolerance testing may be advised.
In addition, because mid-stage clinical trials may expand into previously
untested human populations (e.g., women of childbearing potential or indi-
viduals with tumors or an underdeveloped immune response), it is wise to
consider specialized toxicity testing before initiating these clinical studies.
Since Phase 3 studies result in testing in a more diverse and much larger
population and involve doses given over longer periods of time, it may be
advisable to complete chronic toxicity studies at this stage of development.
Besides, at Phase 3, the dose and dosing regimen would have been estab-
lished, thus reducing the risk of having to repeat long, costly chronic toxicol-
ogy studies. On the basis of the intended population and use, it is also wise
to consider and plan for applicable specialty studies, such as those directed
toward an organ system (e.g., reproductive toxicology, neurological toxicol-
ogy, and immunotoxicology studies) and those focused on a product-related
issue (e.g., tissue-binding or DNA integration studies).
The planning process considers current regulatory guidelines, both
national (FDA) and international. It is important to consider that regulatory
agencies have responsibilities toward the safety and welfare of human sub-
jects who take investigational products and toward public health regarding
marketed products. Nonclinical safety testing plays a major role in meeting
these responsibilities. The International Conference on Harmonization  of
Technical Requirements for Registration of Pharmaceuticals for Human Use
(ICH) (Chapter 4) provides excellent guidance with regard to nonclinical
testing strategies, and this has the advantage of worldwide harmonization.
In addition, regulatory agencies ensure that adequate and well-controlled
nonclinical studies have been completed according to performance stan-
dards (e.g., current Good Laboratory Practices [cGLP]). Studies always meet
generally accepted scientific guidelines as well. This means, by way of exam-
ple, using experimental designs that test well-considered hypotheses, testing
adequate numbers of the correct animal species, dosing only with mate-
rial that matches the quality of product to be used in clinical studies, and

340 Biotechnology Operations
applying proper statistical tests when analyzing results. Conclusions drawn
in nonclinical study reports must be supported by data.
In Vitro Screens: Surrogate Measures of Toxicity
A relatively simple and inexpensive means of beginning a series of nonclini-
cal studies is to rely on screening tests. Unlike toxicology testing in whole
animals, in vitro screens are individual tests, each with a specific purpose
and activity criterion. In addition, in some cases, in vitro screening tests are
performed in a series to provide information on a single subject from a vari-
ety of tests. Many screens are available for chemical and drug compounds,
because of the large number of candidate products tested and a long history
of nonclinical development. Some in vitro tests are performed by contract
testing laboratories, whereas others are amenable to use in a sponsor’s labo-
ratory. Although plentiful and popular for drug and chemical development,
these screens may not be useful to measure activity criterion on biophar-
maceuticals because test criteria might not match assay requirements and
because biological molecules are often incompatible with the test matrix or
design. In addition, because of the limited experience, there may be ques-
tions regarding relevancy of test results when applied to biotechnology
products. Thus, specific concerns can focus on the sensitivity, specificity,
accuracy, and reproducibility of in vitro screening tests, when used with a
biopharmaceutical.
Nonetheless, when properly applied to a nonclinical safety testing pro-
gram, in vitro screening tests can provide valuable information that can be
used to design more complex in vivo studies. Examples of in vitro tests are
discussed here, and a longer list is provided in Box 8.2. Mutagenicity test-
ing, exemplified by the Ames tests, screens compounds for mutagenic poten-
tial. The Ames test relies on Salmonella bacteria as a substrate and measures
alteration in structure of a gene, after application of a test compound. Other
eukaryotic or prokaryotic cells may be used in the same manner, as long as
there is a reliable read-out for demonstrating mutagenicity. Carcinogenicity
testing takes mutagenicity one step further by asking whether the mutagenic
or genotoxic potential of a compound also results in the development of car-
cinogenic potential. Since not all mutagens are carcinogens and because not
all carcinogens are mutagens, the mutagenicity and carcinogenicity tests can
give distinct answers about product. Although screening tests can be helpful
to making early decisions, they are not definitive, and carcinogenicity test-
ing is considered in animals for compounds that might have carcinogenic
potential.
In its simplest format, lethality testing places test material in contact with
living, cultured cells and determines whether the material kills the cells.
Various cell types can be used in these studies. Variations of the in vitro
test measure biochemical or physiological parameters of cell health, which
would indicate that a cell might die, or at least not thrive, in the presence of

341Nonclinical Studies
BOX 8.2 EXAMPLES OF IN VITRO SAFETY TESTS USED
FOR DRUG OR BIOPHARMACEUTICAL SCREENING
In Vitro Safety Test Purpose of Test
Mutagenesis
Ames screening test Genetic toxicology for mutagenicity in bacteria
Mammalian cell mutation tests Genetic toxicology by mutagenicity of various
mammalian cell types
DNA Damage and Repair
Chromosome aberration test Chromosome aberrations and mitotic indices in
cells
Cytotoxicity tests Cytotoxic activity by using a variety of cell
types
Karyotype analysis Gross chromosomal analysis
Micronucleus tests Potential to induce genetic damage, measured
as induced micronuclei in a variety of cell types
DNA repair tests Potential to damage DNA
Sister chromatid exchange test Genetic damage, as manifested by sister
chromatid exchange in various cell types
Cell transformation tests Potential to cause genetic damage, as
manifested by induced morphological cell
transformation
Aneuploidy tests Chemically induced aneuploidy in cells
Other
Metabolism tests Evaluation of metabolic stability of various cells
Cellular anabolism Ability to affect protein anabolism in cells
Cellular respiration Ability to affect cellular respiration, measured
as ATP/ADP
In vitro drug metabolism Metabolism of products by cells
Human skin permeation test Prediction of dermal and ungula permeation
Mitochondrial toxicity test Damage to mitochondrial function
Hemolysis tests Damage to red blood cells
Drug interaction tests Ability of product to be metabolized in
presence of other drugs
Hepatotoxicity tests Measures drug-induced liver injury
Cytokine/chemokine secretion test Potential for triggering release of cytokines or
chemokines
Apoptosis assay Induction of cell death
Cellular proliferation Induction of cellular division and proliferation

342 Biotechnology Operations
the test product. Special types of cells—cardiomyocytes, neural, epidermal,
and gastrointestinal epithelial—can be used in such studies, giving rise to
test protocols with specific purposes, such as neurotoxicity testing. A num-
ber of screening assays, each providing a specific outcome, are currently
in development or validation with focused application. However, owing to
the unique nature of most biopharmaceuticals, their utility as a toxicology
screen is somewhat limited.
Developmental toxicology measures toxicity of a compound as it relates to
development of a fetus and, for biopharmaceuticals, is especially important
for molecules that might be used in women of childbearing age. In vitro
developmental toxicity screens would be considered insufficient by them-
selves for risk assessment in this area. However, some in vivo models that
use pregnant rodents can also be applied to screen compounds and are more
rapid but perhaps less sensitive, as compared with chronic developmental
toxicology studies.
In Vivo Safety Testing of Biopharmaceuticals
Although it is possible to perform some safety testing in various nonanimal
models, it is also necessary to test most biopharmaceuticals in live animals.
Toxicologists classify safety tests in three manners: (1) by the length of time
for which an animal is dosed, that is, acute, subchronic, and chronic studies;
(2) by an organ system of interest, such as neurotoxicology and immunotoxi-
cology; and (3) by a particular outcome, for example, carcinogenicity testing.
Since consumers demand thorough safety testing of each biopharmaceuti-
cal they use and because there are ethical questions regarding the use of
vertebrates in such tests, toxicology testing in animals is a serious scientific
endeavor, professionally performed and regulated by government agencies.
Animal Model Development
An animal model is a nonhuman, living vertebrate used in nonclinical
research to ensure that a product is reasonably safe and, in some cases, to
demonstrate efficacy or benefit before use in man. Consider that every model
is imperfect in some way, certainly as compared with the human situation;
this is why, it is referred to as a model. Nonetheless, an animal model allows
scientists to gain an understanding of a broad range of toxicological pro-
cesses and outcomes, and this means that the sponsor can make informed
decisions regarding the intended use of a biopharmaceutical in man. Thus,
animal study results help the sponsor to avoid the risk of causing harm to
humans, while at the same time, allowing the biopharmaceutical to provide
intended benefit at a particular dosing regimen. For example, if a biopharma-
ceutical dose of 1 mg/kg per week is toxic but a dose of 0.5 mg/kg per week
is not toxic in an animal model, then the sponsor can design a clinical trial
to test the lower dose and avoid the higher dose. Normally, animal models

343Nonclinical Studies
are chosen or developed for a specific study design, and not vice versa. As
discussed in more detail later, the sequence of planning events in safety
assessment is first a hypothesis or a question and then a well-considered
concept study design to drive the selection of the proper model. Selection of
the correct animal model or models is challenging, and in the end, compro-
mises are made and the best model is identified. Because a toxicology study
is designed to answer a specific question or a set of questions regarding the
safety of a particular biopharmaceutical, it is often necessary to use a differ-
ent animal species to answer each question posed.
For most drugs, safety testing requires the use of two or more animal spe-
cies over the development life cycle, whereas for other products, notably
many biopharmaceuticals, testing in one animal might meet all regulatory
requirements. Normal or healthy animals are usually tested first to dem-
onstrate safety. In addition, it may also be necessary to use a second model,
an animal with a similar or identical disease, because the disease process
may greatly modify the toxicology profile of the product. General guidelines
for the selection of animal model species are listed in Box 8.3. Overall, it
can be very challenging to identify two excellent animal models for acute
and chronic safety testing of a biopharmaceutical. Typically, there are many
trade-offs in the selection process and the perfect model may never be found.
Identifying an animal model with the disease is especially challenging,
because the animal species selected must be appropriate and also the dis-
ease must be relevant to the human condition and must have a similar etiol-
ogy, pathogenesis, and clinical outcome. For example, to examine the safety
of a therapeutic vaccine to treat Alzheimer’s disease, one must identify or
develop a model of the disease that arises and progresses in the same man-
ner as the human disease and the animal must exhibit the same immune
response as would be expected in a human (having this condition) treated
with that vaccine. Advances in development, notably the use of transgenic or
knock-out animals, offer appropriate models, but these animals can be very
expensive. Having chosen an animal model, it is then important to carefully
design the nonclinical study to take advantage of any attributes the animal
may possess, while at the same time, using the correct number of animals
for each question posed.
Animal models should be validated, or at least qualified, for a particu-
lar application. Unless an animal model has been widely used for a par-
ticular class of biopharmaceuticals or disease, it is advisable to perform
pilot studies and determine whether or not the chosen species and strain
is, in fact, suitable for the intended purpose. Experience with an animal
model will also help to better understand and more accurately plan for
animal numbers needed in a pivotal preclinical study, especially if the ani-
mal attrition rates associated with the creation of the animal model are
unknown. Studies performed in the research laboratory environment help
to ensure that when later used in an expensive and lengthy toxicology
study, there will be no technical issues and the results will be meaningful.

344 Biotechnology Operations
Use of animal models proves very useful in identifying any delivery issues
or specific challenge with measuring a proposed clinical end point before
evaluation in the clinical setting. Indeed, small pilot studies of a compound
in a given animal model often lead to improvement in both the application
of the model and the ultimate toxicology study design.
Test Product Formulations, Routes of Delivery, and Dosing Designs
Development and selection of animal models are not the only things that
need to be considered before performing a nonclinical toxicology study.
There is also the need to have a final product (Chapters 5 and 6) that matches
the formulation and quality of the biopharmaceutical intended for use in
humans. Far too often, nonclinical studies are considered invalid because
they apply to a product that differs in strength or quality from the intended
BOX 8.3 CONSIDERATIONS FOR SELECTION OF
ANIMAL MODELS FOR TOXICOLOGY STUDIES
• Observe taxonomic, anatomical, and physiological similarities
to humans
• Consider overall anatomy and physiology of animal
• Anatomy and physiology of target organ, tissue, and cells
• Demonstrates pharmacology, pharmacokinetics, and pharma-
codynamics, similar to the intended human population
• Metabolizes drug in similar manner
• Has same receptors or mechanism of action
• Expresses same target organ, tissue, or cell responses
• When possible, provides a model of the human disease
• Dosing parameters match human condition
• Ability to give full human dose and dose regimen to animal
• Consider route of delivery
• Economics
• Use enough animals of this species to fully answer the
question
• Consider cost of maintaining the animals over the period
of study design
• Ethics
• Is it necessary to use an animal to answer the question(s) or
an in vitro system would suffice?
• Is it possible to use a species lower on the taxonomic chart?

345Nonclinical Studies
clinical material. Formulation, strength, and quality can greatly impact the
biological activity, including parameters related to safety, of a molecule or
cell. Hence, it makes sense to use the same or a comparable formulation in
nonclinical studies as will be used in clinical studies. This can have a signifi-
cant impact on scheduling a nonclinical study, since formulation, manufac-
turing, and quality control timelines impact the nonclinical plan. In addition,
there may be trade-offs. For example, if it is absolutely necessary to use a
mouse to test a human dose of a therapeutic protein, it may not be possible
to give a full human dose because of volume constraints in the animal. The
result could be to split doses or to concentrate the formulation. These issues
arise constantly in design of safety studies and must be addressed scientifi-
cally in consultation with other development scientists.
Route of delivery presents another hurdle in design of nonclinical studies.
For the example given above, it might be necessary to give the product to a
mouse by the intraperitonal route, because it is impossible to give a full dose
to this animal by the intended human route, that is. intravenously. Indeed,
sometimes an animal species is chosen based on matching the intended
route of delivery for man. Because pig skin is very similar in microanatomy
and function to human skin, the pig is used in many nonclinical studies of
biopharmaceuticals that are to be delivered to human epidermis.
Nonclinical animal studies reflect a vocabulary that is unique to this
endeavor. These terms are explained here because they are commonly used
in study protocols. First are the trade terms. Test article is the product or final
product, as described in Chapters 6 and 7. It is the final formulation of the
material that is being tested. Neat means an article is used in full strength or
undiluted. Placebo represents inactive ingredient and might be referred to
as control or control article. A diluent is a defined solution, buffer, or formu-
lation, such as physiological saline, used to titrate the test article or control.
An excipient is a nonactive ingredient included in a formulation. Common
excipients for biopharmaceuticals include detergents that prevent protein
precipitation and sugars that preserve the integrity of cells in a solution. A
particular type of excipient, the vehicle, is a chemical that serves to enhance
transfer, absorption, or distribution of the biopharmaceutical. Tween 80, a
detergent-like molecule is used to prevent aggregation and as such consid-
ered a vehicle in formulations of certain protein biopharmaceuticals.
A second consideration is the route by which a biopharmaceutical is given
to an animal or human; the most commonly used routes are listed in Box 8.1.
Attention is also given to physiological variables that can have an impact on
dosing of a product to an animal or to man. Local effects are the physiologi-
cal responses of the recipient to the test article when it first reaches the recipi-
ent tissue or organ. Absorption and distribution are the processes by which
the test material moves away from the site of delivery and establishes itself
in various tissues and organs. Formulation can have a major impact on how
efficiently absorption and distribution occur and where a biopharmaceuti-
cal goes in the body. Next, there is the issue of metabolism, the process by

346 Biotechnology Operations
which the biopharmaceutical is chemically changed, broken down, or other-
wise used by the body. Absorption and distribution, and hence also the local
effects, influence metabolism of many products and were discussed earlier.
Calculating dose is an underappreciated skill but can have a major impact
on the outcome of a nonclinical study. At the outset, the design of a nonclinical
study has, from the TPP, a target dose or a range of doses that clinical experts
suggest might be used in human volunteers. For proposed nonclinical studies,
the study design brackets the target human dose, based on an understanding
of the product and the chosen animal model, as well as the dose equivalents
with respect to understanding the relative body weight or surface area of an
animal versus a human. If body weight is chosen as a comparator, then dosing
calculations are made as milligram of biopharmaceutical per kilogram of body
mass. Alternatively, dose estimates based on body surface area are becoming
more common, perhaps because of obesity in society, disease being treated, or
factors related to pharmacokinetics or pharmacodynamics of biopharmaceuti-
cal products. The chosen dose can impact the calculated relative dose for some
biopharmaceuticals and where small animals, for example, mice, are employed.
Selecting a close approximation of the intended dose, dosing schedule, route of
administration, and rate of administration to the future human clinical study
design is important to consider in the design of animal studies. Calculation
and rationale of dose and approximation of human dose equivalents also need
to be carefully planned. Finally, it is important to calculate from a study design
the total test article, control article, and vehicle requirements, as well as speci-
fications and tests for quality, After this, the outcomes and decisions on study
design should be discussed with colleagues responsible for biomanufacture
and quality control, focusing on the total quantity and number of individual
lots of product, diluents, and placebo.
Protocols and Performance of Biopharmaceutical
Safety Studies in Animals
A concept nonclinical study design, once found acceptable to a product
development team, is then written further in a nonclinical study design doc-
ument, that is, the protocol. The purpose of the nonclinical study protocol is
to guide the investigative team in performance of a study. Elements of a non-
clinical study protocol are given in Box 8.4. Responsibility for preparing the
protocol is given to an individual, the study director, who is responsible for
ensuring the performance of the study according to the study protocol. This
individual is also responsible for completing a study report at the end of the
investigation. Nonclinical protocols are also reviewed by other scientists
who serve on the nonclinical study team and are approved by the quality
assurance unit. Further, any use of animals in research requires review by an
Institutional Animal Care and Use Committee (Chapter 4), which is respon-
sible for the ethical and proper use of laboratory animals. A nonclinical
study always results in a report, a prospectively written scientific document

347Nonclinical Studies
that identifies the design and justification of the study, as mandated under
the protocol. The study report contains the experimental results, statisti-
cal analysis, list of deviations, and conclusions made by the study director
and provides, in appendices, all tabulated raw data derived from the study.
A supplemental report may be incorporated in the final study report; the
supplemental report includes expert opinions about the clinical relevance of
results by a physician or clinical pathologist. Nonclinical study reports are
typically large documents, hundreds of pages, even for a small and simple
studies it is common for these reports to be submitted to the FDA as part
of an IND application. Finally, it is worth noting that a nonclinical study in
animals costs a significant amount of money and takes considerable time
(6–12  months for acute and subacute studies and much longer for chronic
studies) from concept protocol design to the final study report; this is the
reason that much care is taken for study design and execution.
Elements of a Nonclinical Study Design
Adequate and well-controlled nonclinical study designs have common ele-
ments, no matter how the study is classified (e.g., acute, subchronic, and
chronic), and each element is considered in the study protocol. Content of
the protocol is, to some extent, mandated by cGLP regulations, and this
alone harmonizes the format (Box 8.4). To begin, there is a facility and it is
staffed with qualified individuals capable of performing functions called
BOX 8.4 ELEMENTS OF A NONCLINICAL STUDY PROTOCOL
FOR SAFETY TESTING OF BIOPHARMACEUTICALS
• Title, purpose, sponsor, and testing facility
• Detailed identification of test and control articles and animals
• Methods of identification
• Description of materials used in the study, including the
animal diet
• Dosing levels of test material
• Experimental design and methods used to control bias
• Type and frequency of tests and measurements
• Records to be maintained
• Statistical methods
• Dated approval of protocol by sponsor, study director, and
quality assurance
• Any changes made
• Approvals

348 Biotechnology Operations
for under the protocol. The facility is properly designed and equipped
for the types of studies performed. As noted above, each study is defined
under a protocol and has a responsible study director. A quality assurance
unit is also an integral component of nonclinical programs by conducting
audits, reviews, and approvals among other functions (Chapter 5). If the
study calls for laboratory analysis, which most studies do, then there are
adequate laboratory facilities identified to perform the work either at the
study site or, for specialized tasks, at qualified contractor sites. Animals are
involved in most studies and all their support is met; this involves hous-
ing, feeding, treating, and inspecting or analyzing each animal, as well as
ensuring quality care and well-being. In addition to a protocol, routine
procedures—everything from handling animals to archiving records—are
fully described in instructional documents such as standard operating
procedures (SOPs). In addition, every bit of information and all data are
captured on source documents and may be transferred to study data cap-
ture forms, most designed uniquely for that study. All of this information
is reviewed by qualified professionals, condensed into summaries, written
into a final study report, and properly reviewed and approved. Quality
assurance involvement is essential to this process.
However, each protocol is scientifically unique in purpose, scope, and
design and is written by the study director, with much scientific and tech-
nical input and based on a hypothesis. One reason is that each nonclini-
cal study is related to a unique biopharmaceutical. To test the hypothesis
and answer every question posed in the study objectives, a study design
includes a unique mixture of animal and laboratory treatments, tests, and
procedures. The design is given at three or more levels of variables. One
level defines the dose and respective controls, the dosing scheme and sched-
ule, and the length of time for which animals are on study. This leads to
the designation of animal groups with each group receiving a different
treatment.
The second level of design provides further instruction for carrying out
the essential elements of the study. An example is animal care and treat-
ment, baseline pathogen screening, observations, shaving or clipping,
anesthesia, food, weight, and physical environment are written into the
protocol in an effort to support the scientific design. Another example is
laboratory testing. Clinical laboratory testing—hematology, clinical chem-
istry, urinalysis, and immunology—measures the health of the animals.
Other laboratory testing may measure the level of product or metabolites
from that product.
The third level of design enters into the example of laboratory testing,
because the analytical laboratory must develop or have on hand an assay
or set of assays that are robust and both selective and sensitive for the
compound being studied, when it exists in animal blood or tissues. Assay
qualification (Chapter 7) is a common practice to support nonclinical stud-
ies. Thus, laboratories must be capable of performing a variety of analytical

349Nonclinical Studies
methods. Very special laboratory studies may be performed at another
site, at either a contract laboratory or a sponsor’s laboratory. For example, it
might be necessary to send animal blood or tissue to a contract laboratory
to measure levels of the excipient Tween 80 if there is a concern that the
excipient might be concentrated in the body of an animal. A special assay
to measure the effect of a product on animals might be performed by the
sponsor of a study if this test requires special expertise or laboratory equip-
ment. For example, a cellular assay to measure proliferation of lymphocytes
in response to a recombinant protein might have to be performed by the
sponsor’s laboratory on specimens taken from animals immunized with a
new vaccine.
Returning to the second level of design, the planner must consider other
procedures in the in-life phase of the study. The in-life phase of an ani-
mal study is defined as the time beginning with the initial assignment of
animals to the study, including dosing of the animal, until the time when
it is euthanized or released from the protocol. Most toxicology protocols
demand frequent examination of animals during the in-life phase of the
study, and this involves close observation and measurements of general
animal health, such as weight and amount of food consumed per day. It
may be helpful to use or develop qualitative assessment scales to better
define and capture subjective data. For example, animal appearance is a
very good indicator of overall animal health, but it is hard to quantify and
usually not standardized; therefore, provision of a general appearance
scale or of predetermined defined criteria are meaningful and provide
a valid way to capture this assessment. Some commonly used measures
of animal health are given, as in-life and postlife measures, in Box 8.5.
After the in-life phase, defined by the protocol, most animals, perhaps
with the exception of some large species and nonhuman primates, are
euthanized. This begins the postlife portion of the study. For most safety
studies, a formal necropsy is performed, and rigorous gross inspection
is followed, after tissue preparation, by histopathological examination of
every organ. Parameters to measure animal health or toxicity are listed in
a protocol, and any abnormality in each of these parameters is recorded
and the results are analyzed by a certified veterinary pathologist. Plans
are also included for data handling, analysis by a statistician, and report-
ing. Records are exact and complete, and all procedures fall under cGLP
guidelines. Data and reports follow stringent criteria for analysis and
review by the laboratory and sponsor and for approval and reporting to
regulatory authorities.
Hence, the design and procedures provided in nonclinical and clinical
studies are carefully considered, are rigorous, and are provided in detail.
Nonclinical designs and protocols are instructive and directive, because both
types of protocols are important to human subjects and users of the biophar-
maceutical. These studies are time consuming and very costly, so there is little
room and no justification for error.

350 Biotechnology Operations
BOX 8.5 COMMON MEASURES OF IN-LIFE ANIMAL HEALTH
AND POSTMORTEM CHANGES IN NONCLINICAL
TOXICITY TESTING
• In-life measures of animal health and well-being
• Clinical signs
– Weight loss and weight gain
– Appearance or behavior (ruffled fur and hunching)
– Agonal events
– Neuromuscular, ophthalmic, and other special tests
• Clinical laboratory pathology
– Hematological abnormalities
– Clinical chemistry abnormalities
– Abnormalities in urine, secretions, or other samples
• Postmortem measures of animal health and well-being
• Death (mortality)
– LD50
– Time to death
– Cause of death
• Gross observations at necropsy
– Edema and fluid in tissues and body cavities
– Color, size, and appearance of organs
– Whole body weight
– Whole organ weights
– Obvious signs of systemic disease
– Tumors
– Developmental abnormalities
• Histopathological examination of tissues
– Tissue or cellular changes
– Signs of infection or inflammation
– Malignancies or nonmalignant tumors
– Developmental abnormalities
– Signs of systemic disease or stress to a system or the
body as a whole
(Continued)

351Nonclinical Studies
Nonclinical Safety Testing
The extent and level of nonclinical safety testing are largely based on the
type of product being developed, in addition to what is known about the
predicted safety profile of that product. There are a number of traditional
nonclinical safety tests that have been utilized for decades that the USFDA
embraces as being validated and dependable and that yield predictive
results that are likely to correlate with human clinical safety. Examples of
these common nonclinical tests are provided in Box 8.6. Although this list
of common tests is provided, they are not all required for every biotechnol-
ogy product, but rather, a subset of the most relevant tests is selected to be
performed. The selection of testing and extent of testing are based on prod-
uct profile and other characteristic attributes of the product and include the
product’s biological function. Brief descriptions and examples of the most
common nonclinical safety studies are given in the following sections.
Acute Toxicity Testing
Acute toxicity testing has been defined as the short-term evaluation of toxic-
ity in animals after a single dose of a biopharmaceutical. Today, the defini-
tion would, by most accounts, include study designs with multiple doses of
the product but over a brief period and again with short-term evaluation.
If the devil is in the study details, then the devils here are the definitions
of the terms brief period and short-term evaluation. For a therapeutic protein,
an acute study might involve three doses over three days, with completion
of in-life studies on the sixth day. In contrast, an acute study of a vaccine
might be three doses over ten days, with completion of in-life studies on
the eighteenth day. Hence, the definition may be adjusted, depending on
the type of compound, expected dose and dosing schedule, possible toxic
effects, indication, and animal model. However, there are established com-
mon elements and guidelines for acute toxicity tests. These guidelines are
as follows: (1) Findings are suggestive and never definitive as to the overall
toxicity of the biopharmaceutical; (2) It is a screening toxicity and may be
BOX 8.5 (Continued) COMMON MEASURES
OF IN-LIFE ANIMAL HEALTH AND POSTMORTEM
CHANGES IN NONCLINICAL TOXICITY TESTING
• In-life or postlife observations and tests
• Infection demonstrated by microbiological examination or
culture
• Immune response to various stimuli
• Pharmacokinetic or pharmacodynamic measurements

352 Biotechnology Operations
BOX 8.6 COMMON NONCLINICAL SAFETY TESTS USED
FOR DRUG OR BIOPHARMACEUTICAL EVALUATION
Safety Test Purpose of Test
General toxicology
Maximum tolerated dose
(MTD)
Dose-limiting toxicity
(DLT)
No observed adverse
effect level (NOAEL)
No observable effect level
(NOEL)
• Establish administration schedule and approximate
clinical exposure.
• Identify reversible or irreversible side effects.
• Identify extent and severity of pathologic lesions.
• Evaluate regenerative capacity of organs and organ
systems.
• Typically one rodent and one nonrodent species.
• Minimum of three animals/sex/group.
• Early stage of clinical development.
Acute toxicity testing • Establish short-term exposure toxicity, based on
intended human clinical exposure.
• Confirm administration and delivery schedule.
• Rodent and nonrodent species.
• Single-dose toxicology study or repeat-dose-range
finding study (MTD).
• Early stages of clinical development.
Subchronic toxicity
testing
• Establish dose and dose regimen.
• Increased exposure relative to acute toxicity.
• Repeat dose with study duration of 1–3 months.
• Definitive toxicity profile to be obtained.
• Early stage of clinical development.
Chronic toxicity testing • Establish long-term exposure toxicity and correlate
with intended human exposure.
• Use larger number of animals to allow statistical
evaluation and accommodate for animal attrition.
• Early to mid stage of clinical development.
Reproductive toxicology
testing
• Evaluate male spermatogenesis, female follicular
development, fertilization, implantation, and early fetal
development.
• One pharmacologically relevant species is usually
sufficient.
• Biopharmaceutical product intended for adults of
childbearing potential or children with developing
reproductive systems.
• Animal evaluation to include both sexes.
• Focus on reproductive system and/or organs.
• Fertility of both sexes, early embryonic development to
implantation.
• Early to mid-stage of clinical development.
(Continued)

353Nonclinical Studies
BOX 8.6 (Continued) COMMON NONCLINICAL SAFETY TESTS
USED FOR DRUG OR BIOPHARMACEUTICAL EVALUATION
Safety Test Purpose of Test
Developmental
toxicology testing
• Establish mortality, structural abnormalities, functional
impairment, and growth alterations in fetus or neonate.
• Typically use rats or rabbits.
• Identify effect on embryo-fetal development.
• Pre- and postnatal developmental effects.
• Examine female parturition and lactation.
• Used for selected biopharmaceutical products.
• Mid- to late stage of clinical development.
Carcinogenicity testing • Establish carcinogenic potential.
• Prolonged exposure or extraordinarily high
physiological doses.
• Determine if the biopharmaceutical product resembles
similar chemical class or structure of known
carcinogen.
• Use two different rodent species.
• Route, dose, and dose regimen used.
• Biopharmaceutical product specific.
• Later stages of clinical development.
Immunotoxicity testing • Establish potential to cause immunopathology.
• For immunomodulatory pharmaceuticals, consider
immunophenotyping via flow cytometry.
• Used for selected biopharmaceutical products.
• Evaluate anaphylactic response, delayed
hypersensitivity, acute immune response to product,
potential to elicit autoimmunity, and potential of
immune suppression.
• Choice of animal species and immune status is critical
for validity of these studies.
• Early stages of clinical development.
Genetic toxicity testing • Evaluate potential to negatively affect genetic material.
• Examine for specific mutations or more general
chromosomal breaks or rearrangements.
• Utilize both in vitro and in vivo assay formats.
• Early stages of clinical development.
Tissue binding or local
tissue tolerance
• Evaluate local reactions to multiple doses.
• Long- or short-term and based on clinical intent.
• Used for selected biopharmaceutical products.
• May be combined with acute, subchronic, or chronic
toxicity study.
• Surrogate in vitro studies may be useful.
• Animal studies are likely most appropriate.
• Early stages of clinical development.

354 Biotechnology Operations
useful to rank toxicity, as variables are adjusted (e.g., dose, number of doses,
timing of each dose, and route of delivery); and (3) It represents only an
assessment of potential toxicity. To further confuse the definition, acute tox-
icity is defined in one manner for drugs, in another manner for compounds
intended for environmental release, and in yet another manner for certain
types of biopharmaceuticals.
An acute toxicity study design does, however, contain elements that
deserve mention. First, some are range-finding studies, meaning they are
designed to define a dose level that is suitable for the proposed clinical dose
and on which to base more definitive range-finding studies. Second, end
points and measurements are as important in acute toxicity testing as they
are in any other study. One end point may be finding the dose level that
results in significant or measurable harm or disease (requires careful testing
and definition) or the dose level that might be lethal (easy to measure and
define). Here, the term LD50, the dose of a product that causes the death of
50% of animals receiving the product, may be used. Although this end point
is seldom measured for biopharmaceuticals, a similar concept, here referred
to as XD50 can be applied, where X is a particular measurement of declining
health, such as 50 g of weight loss in a rat. Toxicologist have identified and
used numerous acute toxicology range-finding study designs, end points,
and measurements, and now, some of these are applied to nonclinical stud-
ies for biopharmaceuticals, notably to therapeutic products. An example of
a typical acute toxicity study design for a biopharmaceutical product is pro-
vided in Box 8.7.
How are the results of acute toxicity testing of a biopharmaceutical consid-
ered and acted upon with regard to further product development of the bio-
pharmaceutical? Some outcomes are easily interpreted, and at other times,
they confound, more than clarifying, the interpretation of results. A lethal
dose (e.g., 100 mg/kg) of any biopharmaceutical in a relevant animal species
would not be considered for a Phase 1 clinical study. In addition, if that lethal
dose were near or in the range that had been chosen for clinical therapy, then
this product would probably not be progressed to clinical trials unless it
were first reformulated or otherwise changed to significantly reduce the tox-
icity, while retaining the therapeutic effect. Yet, there are caveats even with
this example. Should the product be indicated for a life-threatening disease
for which there is no other possible therapeutic intervention, then it might
be progressed to more definitive toxicology studies. Every result must be
considered in context.
Although not definitive, the results of acute toxicity testing certainly
aid the selection of dosages and perhaps dose regimens, or, at least, they
should, if the study was designed properly, the animal model was care-
fully chosen, and the dosages bracketed the proposed human dose. Acute
studies that give multiple doses are also instructive on additive effects of
the biopharmaceutical in an animal, an information that can be applied
to later studies. Acute studies may also provide information on proper

355Nonclinical Studies
timing of doses and, if properly designed, may yield meaningful phar-
macokinetic and pharmacodynamic data. Again, these objectives must
be considered in the acute study design and completed in the in-life and
laboratory phases of the study. It is worth noting that acute toxicity stud-
ies of biopharmaceuticals are occasionally performed in research labora-
tories and without regard for GLP. They are considered pilot studies on
which to base dose selection for an adequate and well-controlled (cGLP)
subchronic and definitive toxicology study, and most often, they are not
be acceptable to regulatory authorities as definitive acute toxicity studies.
In such cases, results that demonstrate lack of toxicity are not compelling,
but when toxicity is noted, it must be reported to regulatory authorities
and considered in the overall picture of product safety. Significant benefit
is derived in savings of time and money, but there is some regulatory
and scientific risk involved in performing such pilot studies. Often times,
non-GLP studies that demonstrate good documentation practices through
the generation of a prospectively defined study protocol, quality control,
accurate data capture, and generation of a final study report associated
with these pilot studies may go a long way in gaining the acceptance of
these study results by FDA.
BOX 8.7 ACUTE TOXICITY STUDY DESIGN FOR
A BIOPHARMACEUTICAL PRODUCT—EXAMPLE
Acute Toxicity
Rationale • Determine maximum tolerated dose or no observable effect
level
• Identify potential organ toxicity, determine reversible or
irreversible damage, and determine clinical safety end points
• Determine dose level (basis for repeat dose toxicity study)
In-life duration • 1–2 days up to 15 days
Test system • One rodent animal species
• One nonrodent animal species
Administration • Closely mimic that intended for human clinical
administration
End points • Weight change
• Physical appearance
• Gross necropsy
• Clinical laboratories
• Clinical pathology
• Other indicators of toxicity
• Mortality
Cost estimate Low to medium*
*Depending on study elements such as animal species, in-life duration, controls, dose
range, and end point analysis.

356 Biotechnology Operations
Subchronic and Chronic Toxicity Testing
There are several different definitions for chronic, subchronic, and sub-
acute toxicity studies that cover any number of nonclinical safety testing
when applied to biopharmaceuticals. The terms subacute and subchronic
are subjective, but progression from subacute through chronic studies
reflects an ever-increasing exposure of animals to the biopharmaceutical,
beyond those applied in acute studies. The subacute study, a term used
more often with drugs than with biopharmaceuticals, refers to the studies
that are done as repeat dose and at dose levels between those of acute and
subchronic studies, with durations of 1–3 months. Subchronic studies are,
perhaps, 3–6  months in duration and typically involve multiple doses, if
indicated. They look for cumulative biological or health changes in ani-
mals. They can, however, be broad explorations, examining animals for a
wide range of symptoms or diseases. They should, in the end, define toxic-
ity as well as add to the pharmacologic body of information. Subchronic
study results are both qualitative and quantitative and the studies attempt
to, and should in fact, meet statistical end points to clearly demonstrate
toxicity if it exists. The anticipated result of a subchronic study, the one
supported by unequivocal data, is safety of the product at a dose and dos-
age regimen that are desirable and feasible in humans. This is referred to
as a clean dose level of the biopharmaceutical. Finally, a well-designed sub-
chronic study forms the foundation for designing required for follow-on
chronic and specialty toxicity studies.
The study design of subacute and subchronic studies are driven by many
variables, not to mention the indication and nature of the product, and no
single design should be considered authoritative. The selection of an ani-
mal model is very important to the success of a subchronic study, and the
data from acute studies and the research laboratory are most supportive.
Regulatory guidelines for drugs and often pharmaceuticals specify test-
ing in two animal species, including a rodent (rat) and a larger nonrodent
animal (dog or nonhuman primate). Today, therapeutic biopharmaceuticals
reviewed by the Center for Drug Evaluation and Research (Chapter 3) often
follow this guideline. However, other biopharmaceuticals, such as vaccines
and genetic therapies, have followed precedent established at the Center
for Biologics Evaluation and Research and used a single species, shown to
be suitable for the product, notably for the intended indication, dose, and
dosing regimen. For subchronic studies with these biopharmaceuticals, it is
important to perform pilot studies to test variables and the animal model
before the definitive subchronic study is performed.
Design may be single dose or, more commonly, repeat dose. It is important
to ensure that the chosen design is statistically valid, so that enough animals of
each sex are assigned to each dosing group. Single-sex studies are acceptable
but only if a good rationale and justification for why sex-specific differences
are not anticipated. Different doses are tested for most biopharmaceuticals to

357Nonclinical Studies
ensure that the dose and dose regimen taken to human studies are safe and
well tolerated and that the next higher dose is not unsafe. An example of a
typical subchronic and chronic toxicity study design for a biopharmaceutical
product is provided in Box 8.8.
Statistical planning is an important consideration in determining adequate
animal numbers and dose escalation in prospective study design; this is
especially important to be able to rely on statistical power in the design and
data analysis for any unexpected results. In other words, upfront involve-
ment of a biostatistician in the design of a lengthy, expensive, and labor-
intensive study may allow valid statistical interpretation of unclear results
via statistical analysis. Likewise, building a margin of safety into a toxicol-
ogy study not only represents good science but also provides the opportu-
nity to demonstrate failure (e.g., toxicological findings at high dose levels).
A wide safety margin also serves as a control that demonstrates that the test
system is, in fact, capable of identifying toxicities at dose levels higher than
those anticipated to be used in human clinical studies. Demonstration of a
broad safety range or plateau provides an added level of confidence in safety,
which supports the scientific integrity of the study, rather than potentially
unknowingly approaching a sharp cutoff, which represents a narrow win-
dow of safety. A graphical representation of this is presented in Figure 8.6,
where toxicities or incidence of safety are plotted relative to dose escalation.
Additional plateaus may be identified, especially if small dose increments
BOX 8.8 SUBCHRONIC AND CHRONIC TOXICITY STUDY
DESIGN FOR A BIOPHARMACEUTICAL PRODUCT—EXAMPLE
Rationale • To determine no observable effect level (NOEL)
• To evaluate dose response of multiple subsequent dosing
regimens
• To identify and characterize specific organ toxicities
• To predict optimal human clinical dose
In-life duration 2 weeks (subchronic), up to 6 months (chronic)
Test system • One rodent animal species, the rat
• One nonrodent species, the dog
Administration Three dose levels, to include levels likely to produce no toxicity
and high toxicity (but <10% mortality) End points and measurements • Weight change • Physical appearance • Gross necropsy • Clinical laboratories • Clinical pathology • Other indicators of toxicity such as appetence or lethargy • Mortality Cost estimate Medium to high 358 Biotechnology Operations are used. Note that other areas along the dose escalation scheme, for exam- ple, between points 1 and 2 or points 3 and 4 of the x-axis carry the risk of more variable incidence of adverse effects and do not represent good areas to select a starting dose. Controls are always included in subchronic studies, and for many bio- pharmaceuticals, this means at least two additional groups of animals, one dosed with the formulation minus the active ingredient and another, the null control, dosed with normal saline or nothing at all. Details regarding per- formance of a subchronic study, such as dosing animals in all groups at the same time, can be important. Defining study termination and animal eutha- nasia is also critical to study design. It is important to leave enough time after dosing to allow toxicity to occur, but then again, this is not a chronic toxic- ity design. For some biopharmaceuticals, guidelines recommend sacrificing one group of animals after the last dose is given and euthanizing another group, treated and controlled, in a similar manner, weeks after the last dose is given. These nuances in study design demand that the sponsor is very familiar with precedent within a class of biopharmaceuticals, has read all the regulatory guidelines, and intends to meet with FDA (Chapter 3) soon after the concept design is drafted and well before the subchronic study begins. One definition of a chronic study is long study, which takes months to even years and examines animals repeatedly and closely for death, changes in health, or signs of chronic disease. Collectively, long-term or prolonged peri- ods are defined in guidance documents as continuous dosing for 6 months, with intermittent dosing. For many biopharmaceuticals, chronic studies are not worthwhile because the product is not given over a long period of 1 0 200 400 800 1000 600 2 3 4 T ox ic iti es (s af et y) Dose (efficacy) Dose selection Plateau A C B FIGURE 8.6 Using safety and efficacy in selecting a dose. Determining the most appropriate starting dose that is likely to demonstrate an acceptable range of safety and efficacy can be achieved by identifying a plateau. In an animal toxicity study, as the dose increases, so do the associated toxicities (A); in many cases, the incidence of adverse effects may level off between dose levels, which is referred to as a plateau (B). After reaching a plateau, subsequently higher doses are usually associated with increased toxicities (C). 359Nonclinical Studies time to patients. A recombinant therapeutic protein, intended to be dosed just three times, on Days 0, 30, and 60, is a fine example of a product that might not require a long, chronic study. In contrast, a therapeutic monoclo- nal antibody intended to treat patients with chronic inflammation by dos- ing biweekly and for many years deserves chronic toxicity testing, as does a genetic therapy that is designed to incorporate foreign DNA into the human genome and produce a lasting effect, even though it is given one time as a single dose. Another important criterion to consider while determining the requirements of performing a chronic toxicity study may be whether long- term tissue retention of the biopharmaceutical product is anticipated when administered in humans. To know when, in the product life cycle, the chronic studies are designed and performed is not as challenging as it is to know how they are designed and performed. In chronic toxicity testing, the biopharmaceutical is administered over much of the animal’s lifetime. Animals are kept on protocol, and are housed, fed, and observed daily, for a substantial portion of the animals expected life. It is no surprise then that small animals, especially mice, which has a lifespan of less than 2 years, are selected for chronic studies. Chronic toxicol- ogy studies are often confounded by findings that are a normal part of aging. For example, spontaneous events such as sudden and unexplained deaths of individual animals are a reality in all species and cancers—common find- ings of aging—inbred animals. Well-controlled studies are the key to dis- tinguishing product-related adverse events and disease from those simply associated with aging or a common environment. Therefore, statistically valid designs require large numbers of animals in each group to distinguish an incidence of disease or events that occur by chance from the incidents related to product toxicity. This concern drives the design of large stud- ies with many animals to account for attrition and significant numbers of samples and tests, applicable to both in-life and postlife evaluations. Hence, chronic studies tend to be large and expensive, because of which it becomes crucial to ensure proper design, to focus on the toxicity that really matters, and to ensure study protocol reviews by regulatory agencies. Reproductive, Developmental, and Teratogenicity Toxicity Testing Biopharmaceuticals intended for use in individuals of childbearing age or in children with developing reproductive systems are further tested to ensure that the product will have no undesirable effect on reproductive tissues or on a developing fetus. Consumers are extremely sensitive about develop- mental and reproductive toxicology for good reason. Guidelines (e.g., ICH and FDA) suggest that a sponsor consider at least three types of studies if the product can reach the gonads or fetus. The first type of study, Segment I, is toxicity to male and female fertility and of early embryonic develop- ment to implantation. End points measured in such animal studies are mat- uration of sperm or eggs, gonadal integrity, and, in females, normalcy of 360 Biotechnology Operations gestation until the time of implantation. This calls for using an animal model of both sexes and recently mated female animals of an appropriate model species. Measurements focus on the reproductive cells and tissue. The  sec- ond type of toxicology test, Segment II, is for embryo-fetal development. In these studies and after treatment with the biopharmaceutical, organogenesis is studied in pregnant animals from the time of implantation till the second gestational period. Here, the study is designed to measure abnormalities that might develop in the fetus and the associated organs, such as placenta. The third type of study, Segment III, focuses on pre- and postnatal development. Here, dosing of animals begins in the earliest phase of gestation and contin- ues through birth of the animal. Examinations are performed at various times during the development of the fetus and include examination of neonates. Other study designs may be used, especially if the class of biopharmaceutical is suspected to cause reproductive or development abnormalities. The need for product to actually reach the reproductive system or the developing fetus is a consideration when selecting an animal model. Since there is no typical biopharmaceutical, each product must be consid- ered on a case-by-case basis. It is worthwhile to examine precedence and regulatory guidelines before designing a study and to consider a study laboratory and animal facility with experience in reproductive and devel- opmental toxicology. Two examples on how and when to perform a study are instructive. For the first example, the biopharmaceutical is a therapeu- tic protein, intended for long-term, monthly, intravenous dosing at 100 mg/ dose. Since the molecule binds receptors of white blood cells, is able to cross the placenta, and is indicated for use by women during childbearing years, reproductive and developmental toxicology testing is considered advisable, and probably even necessary, before Phase 1 clinical studies and certainly before Phase 2. The second example is a recombinant protein of a virus, a vaccine intended for the general population, including women of childbear- ing age and children older than 2 years. It is given intramuscularly at 5 µg per dose and in three total doses. The sponsor intends to add a label warning stating that the vaccine should not be taken if a woman might conceive in the near future or is already pregnant. This vaccine may be tested for devel- opmental toxicology in young animals before clinical studies in children; however, for the adult population, and given the warning, it may never need reproductive or developmental toxicology. For either product, ADME studies would be helpful in making a decision, because they would demonstrate the distribution of the product after injection. These examples point out the need to consider all aspects of a product, notably pharmacology and the intended treatment population, before designing a toxicology protocol. Carcinogenicity Testing Biopharmaceuticals are not commonly thought of as carcinogens; however, in theory and sometimes in practice, they might be found to be associated with 361Nonclinical Studies cancer. Carcinogenicity testing is a long and expensive process, much like chronic toxicity testing. Guidelines concerning when and how to perform carcinogenicity testing on a particular biopharmaceutical product are avail- able, and there is precedence for most classes of product. Mice and rats are used almost exclusively, and strains of each species must be selected on the basis of many factors: longevity, spontaneous tumors, capacity to develop tumors in response to known carcinogens, and tolerability of the biopharmaceutical to be tested. Design issues such as route of administra- tion, doses, dosing regimens, and termination are complex, and the sponsor considering carcinogenicity testing is well advised to seek expert guidance and regulatory guidance before embarking on study design. The field is rife with pitfalls, complications, uncertainties, controversies, and changes in recommended practices. Interpretation of results presents another oppor- tunity to seek expert opinion, especially if controls were limited in scope or number and if criteria for carcinogenicity were not well considered in the design and protocol. Although complex and difficult, carcinogenicity studies are simply necessary for some products, sometimes before mid- or late-phase clinical studies. Immunotoxicology This relatively new field evolved out of observations and studies on the toxic effects of chemicals on the immune system. With a large number of biopharmaceuticals targeted, directly or indirectly, to the immune system and with other biologicals likely to interface with immune cells and tis- sues at some point during their distribution throughout the body, immu- notoxicological studies deserve consideration for many classes of product. Adding to the situation is the complexity of the immune system, notably the fact that scientists do not yet understanding the intricacies and control mechanisms of this system. Major immunotoxicological concerns are as fol- lows: (1) Adverse allergic responses to the biopharmaceutical because the product or an excipient is perceived as foreign: This is manifested as imme- diate or delayed hypersensitivity reactions, some of which can be immedi- ately life threatening. (2) Immune responses to the biopharmaceutical that neutralize the molecule and make it ineffective: This is not commonly seen with products that are taken over a long period of time. Further, when bio- pharmaceuticals in solution change format, such as going from soluble to microparticulate while in storage, the propensity to elicit both allergic and neutralizing immune responses may increase significantly. (3) Upregulation of the immune response or an inappropriate immune response resulting in immunity to self and thus leading to autoimmune disease: Immediate upregulation caused by biopharmaceuticals that act to release, immedi- ately and in large amounts, cytokines or other mediators of inflammation is of special concern. Use of recombinant cytokines is especially suspected in this regard. (4) Downregulation or suppression of the recipient’s immune 362 Biotechnology Operations response: This is common in patients with pre-existing conditions such as cancer or immunodeficiency. Immunotoxicology testing is highly recommended for certain biophar- maceuticals or for any product derived from and possibly containing mol- ecules from certain sources. A few examples are cytokines or cytokine-like molecules, vaccines and vaccine adjuvants, monoclonal antibodies or immu- noglobulin-like molecules, allergens, products mimicking or derived from microbes that themselves stimulate untoward responses, products derived from certain plants or those mimicking plant allergens (latex, peanut, etc.), and molecules that bind to cells or receptors on cells comprising the reticu- loendothelial system. Given the complete list, it becomes clear that immuno- toxicology is increasingly considered in design of a safety testing program. In addition, there are no simple templates for routine testing of a molecule as there are for acute toxicology studies of certain products. An example of a typical immune toxicity study design for a biopharmaceutical product is provided in Box 8.9. However, note that studies are best designed based on an understanding of the immunological properties, or potential, of the molecule. From this knowledge, it is possible to consider how relevant tests may be performed, in vitro or in vivo. An efficient approach is to piggy-back immunotoxicology studies with acute and subchronic toxicology studies, whenever possible, by adding immunological measurements to the protocol. BOX 8.9 IMMUNOLOGICAL TOXICITY STUDY DESIGN FOR A BIOPHARMACEUTICAL PRODUCT—EXAMPLE Immune Toxicity Rationale To demonstrate that product is neither toxic to the immune response nor elicits an untoward immune response or reaction In-life duration 2 weeks (acute/innate response) or 3 months (subchronic/ adaptive response) Test system One rodent species, mouse or rat Administration Repeat dose, as determined in acute or subchronic toxicity study End points • Clinical laboratories (e.g., hematology) • Measure immune cells, numbers, and functional parameters • Cytokine profiles • Mixed lymphocyte reaction • Macrophage functional assays • Histology of lymphoid organs • Immunohistological examination of cell types and molecules, in situ Cost estimate High 363Nonclinical Studies However, these measurements always require the application of immuno- logical or cytochemical assays, some of which are expensive, time consum- ing, and technically challenging. In addition, given the species specificity of cells and molecules involved in the immune response, it may be difficult to draw valid conclusions, no matter how well designed the study or how compelling the data. For example, a humanized monoclonal antibody tested in an otherwise appropriate rat model might be expected to be highly immu- nogenic in that species. In addition, a recombinant vaccine antigen that is immunogenic and could lead to hypersensitivity reactions in man might not be immunogenic or allergenic in rabbit, rat, or mouse. The possibilities are endless and suggest that immunotoxicology testing must be carefully considered in concept and experimental design, that pilot studies are desir- able once a model has been selected, and that results be expertly interpreted before conclusions are made. In addition, even when all appropriate pre- cautions are taken, there is always a chance that the animal model cannot accurately predict immune reactions in humans. One notable example is the tragic results of a first-in-human clinical study performed by TeGenero, a pharmaceutical company in Würzburg, Germany. Severe unanticipated side effects occurred in March 2006, which resulted in what has been termed a cytokine storm, which threatened the lives of six health volunteers and ulti- mately resulted in the bankruptcy of TeGenero. The investigational product was a humanized monoclonal antibody developed to activate T regulatory cells of the immune system. In summary, preclinical safety assessments included in vitro studies, as well as a number of animal studies. Efficacy was demonstrated in rodent models and further supported by nonhuman primate studies. Immune safety was of primary importance, as the investi- gations continued toward conducting human clinical studies. A randomized first-in-human safety study was initiated with an intravenous infusion of TGN1412 at a conservatively low dose, which represented a very generous safety margin. The issues, challenges, and lessons learned from this horrific incident are summarized in Box 8.10. A reference for additional reading on this case study is provided in the additional reading section of this book. Genetic Toxicology Another relatively new subspecialty, genetic toxicology, studies the effects of chemical, biological, or physical agents on nucleic acids, genes, and chromo- somes. Biopharmaceuticals can profoundly affect genetic material, but the mode of action is usually quite different from that of small molecule drugs or ionizing radiation, insults that result in chemical changes to nucleic acids, producing mutations, chromosomal breakage, or abnormalities in controls. Cancer chemotherapeutic agents are examples. In contrast, certain biologi- cal products, notably genetic therapies and products containing DNA as the active ingredient, are designed to alter the genome through biological pro- cesses. They may deliver therapeutic DNA to the nucleus and even insert 364 Biotechnology Operations BOX 8.10 ISSUES, CHALLENGES, AND LESSONS LEARNED FROM THE TEGENERO INCIDENT • Preclinical testing performed • In vitro safety profile: Flow cytometry, binding affinity, and antigen-specificity studies • Safety and efficacy in rodents—normal healthy, rheuma- toid arthritis, and autoimmune models • Safety and efficacy in nonhuman primates—demonstrate that TGN1412 binds to primate CD28, NOAEL • Preclinical study conclusions • No issues noted in a repeat-dose toxicity study at 4-week intervals • No issues noted in a dose escalation study at weekly intervals • No issues noted as a result of conducting numerous phar- macology studies in rats • No observable adverse effect level (NOAEL) determined in cynomolgus monkey studies • No sign of a first-dose cytokine-release syndrome (e.g., no immune system activation) • No increase in cytokines (e.g., TNF) • Clinical study design • Blinded, controlled, single center safety, and tolerance of single ascending dose • Assess pharmacokinetics of the single dose at each of the four dose cohorts • Determine effects on immune system (e.g., lymphocytes, cytokine profile, and immune response) • Eight healthy, normal volunteers—two  placebo and six investigational product (TGN1412) • Intravenous infusion of TGN1412; eight subjects in the first cohort dosed 10 min apart • Clinical adverse events • Adverse events began within 90  min post dose and esca- lated in severity • Stage 1—Immune cell activation and cytokine release (Continued) 365Nonclinical Studies BOX 8.10 (Continued) ISSUES, CHALLENGES, AND LESSONS LEARNED FROM THE TEGENERO INCIDENT • Stage 2—Shock, perfusion failure, hypoxia, and cytokine- mediated injury • Stage 3—Multiple organ failure • Symptomatic subjects transferred to hospital critical care unit 12–16 h post dose • Drug-related adverse events included mechanical ventila- tion, recurrent fever, increased peripheral vascular perme- ability, and peripheral ischemia with necrotic fingers and toes • Clinical laboratory findings • Significant and rapid increase in all cytokines on days 1 and 2 • Up to 1000-fold increase in TNF and IFN-γ at 2 h and on day 2 • TNF spiked within 1 h of TGN1412 infusion • Cytokines decreased by day 3 • Significant decrease in CD3+, CD4+, and CD8+ • Lessons learned • Classification and sequence of adverse events: Cytokine storm (1–24  h), reactive or transient (5–8  days), recovery (3–20 days), and long-term (15–30 days) • Perform preclinical studies in accordance with Good Laboratory Practices (GLPs) with accurate data collection, reporting, and interpretation • Highlight the future need for testing of biologics to be per- formed in a pharmacologically relevant species • Reliance of dose calculation from preclinical safety studies using NOEL or NOAEL may not be sufficient and one needs to consider a broader approach, using all relevant informa- tion (e.g., product characteristics, biological potency, mech- anism of action, species specificity, and dose-response data) • Clinical studies should be initiated with caution and be conducted in accordance with good clinical practice (Continued) 366 Biotechnology Operations that DNA into the genome, making them suspect of causing genetic toxic- ity but by unique mechanisms. One example of a biopharmaceutical with the potential to cause genetic toxicity is a DNA molecule intended to repair or replace a gene within a cell of the target tissue, such as a white blood cell in the bone marrow. The molecular delivery system enhances the chance that foreign DNA in the product will enter a host cell nucleus and integrate into the host’s genome, thus enhancing its therapeutic potential. However, with this product, it is possible that the DNA would be delivered to the wrong tissue or cell, perhaps a gonad, enter the nucleus, and insert into cellular DNA of gonadal cells, perhaps even sperm or eggs. It might then be expressed in an uncontrolled or inappropriate manner or might even be passed to the next generation. Much of the genetic toxicity testing of biopharmaceuticals focuses on such possibilities, mainly on errors in well- intended gene delivery. Genetic toxicity of a biopharmaceutical is typically studied both in vitro and in vivo, if a suitable animal model is available. An in vitro protocol might use mammalian cells in culture to determine the frequency at which inappropriate insertion or expression occurs. More definitive animal stud- ies are designed to inject biopharmaceutical into an animal and, using BOX 8.10 (Continued) ISSUES, CHALLENGES, AND LESSONS LEARNED FROM THE TEGENERO INCIDENT • Hospitalized subjects continued to improve. Five sub- jects were released approximately 30  days post dose; one remaining patient was hospitalized for almost two addi- tional months • Clinical design for first-in-human study should be science based and carefully justified based on the compound tested • Care must be taken in determining the route and rate of investigational product administration • Timing of subject dosing is especially important to high-risk studies; in this study, a staggered dose administration by a couple of hours would have minimized the risk to perhaps only a couple of subjects • The clinic must be prepared to provide appropriate and adequate emergency support under current practices, and this must be documented. In this study, the clinical research unit was located on the premises, and perhaps, this provided a false sense of security, which contributed to the lack of urgency to get the subjects to the hospital critical care unit 367Nonclinical Studies sensitive nucleic acid probes, measure in various types of tissues or cells the nucleic acid that has been introduced. Additional studies of cells or tissues can determine whether the therapeutic nucleic acid is actually inserted into the genome of living cells. Returning to the example, a therapeutic plasmid DNA, intended to deliver a missing gene to myeloid cells in the bone marrow, could be injected into mouse bone marrow and then located and identified with nucleic acid probes. Locating the gene in bone marrow or blood cells 3 days after injection would be an expected finding and considered a desir- able event. However, discovery of this DNA in testicular or ovarian tissue of the same mouse would be a cause for concern, because the injected product might have entered the nucleus and even inserted into the genomic DNA of the germ cells. Although most biopharmaceuticals have little potential for influencing the genome or altering DNA or RNA, a few products have significant potential to do this. Here again, it is advisable for the sponsor to review regulatory guidance, identify and study precedence for their class of product, and seek expert advice, because genetic toxicology studies are long, arduous, and expensive, and failure to conduct them when required can lead to significant delays in development. Tissue Binding or Local Tissue Tolerance Biopharmaceuticals are sometimes given in large amounts to a single site on the body; most of them are injected. For example, a monoclonal antibody may be periodically injected subcutaneously in doses, each over 100 mg. A number of untoward reactions can result, and nonclinical study designs consider how local reactions are detected at the site of injection or deposition in an animal model. The mechanism of action can be quite different for each biopharmaceu- tical and tissue. Local immune and inflammatory reactions can result, espe- cially after multiple doses, and these may be chronic or acute. Cells or vaccine antigens can, by product design, remain at the site of injection and cause prob- lems that are not anticipated and are not immunologic, such as proliferation of fibrous or adipose tissue. A common method for studying tissue binding or local tissue tolerance is to add measurements of local reactivity to already des- ignated acute, subchronic, and chronic toxicology protocols. In one example, tissue samples are taken periodically by biopsy and again at the time of sacri- fice and studied for signs of local toxicity. Inappropriate cell or tissue binding by a product could result in damage to tissue or even lead to untoward reactions or disease. What can be done to ensure that a biopharmaceutical, designed to bind a particular receptor or cellular molecule, will bind only to the intended target and not to the inno- cent bystander cells or tissues? Evaluation of nonspecific binding needs to be closely evaluated since many therapeutic biopharmaceuticals are developed for the purpose of binding to a certain cell surface molecule, and monoclo- nal antibodies directed against a number of proteins which places emphasis on this concern. Toxicology studies to determine tissue-binding patterns are 368 Biotechnology Operations called for whenever a product could inappropriately bind to normal cells. These studies are performed using immunohistochemistry and other meth- ods that clearly demonstrate tissue or cell binding or the lack thereof. Various human tissues (cadaver material) are used as substrates in these studies. Other approaches may be applied for certain types of molecules. As these studies are somewhat artificial, that is, they are performed in vitro and not in a living organism, the significance of cross-reactive binding study results may be unclear. In this case, it may become necessary to perform additional experimentation in live animals. As noted earlier, methods are now available to track biopharmaceuticals in a living animal. Quality of Nonclinical Studies: Current Good Laboratory Practices Consumers and the government recognized in the 1970s a need for quality in preclinical testing of drugs. The response was institution, by FDA, of a qual- ity system known as cGLP. This set of regulations, outlined in Chapter 4, is applied to all safety testing of biopharmaceuticals. For non-FDA-regulated products, such testing may be required by other government agencies respon- sible for licensing (Chapter 4) products released into the environment and simply contacting humans. It is important to consider the scope of the GLP regulation, stated in 21 CFR 58 “for conducting nonclinical laboratory stud- ies that support or are intended to support applications for research (clini- cal investigation) or marketing permits for products regulated by FDA” (CFR 1978). The scope further defines nonclinical laboratory studies as “in vitro or in vivo experiments in which test articles are studied prospectively in test systems under laboratory conditions to determine their safety” (CFR 1978). The definition goes on to exclude clinical studies and laboratory studies that are designed as exploratory or to determine potential utility or product char- acteristics. The scope of GLPs is important in three important respects. First, it does not apply to research, early development, or clinical trials. Second, it excludes quality control of product (which falls under cGMP). Third, it does apply to all laboratory studies, including animal studies, in which a claim is made for product safety. Thus, GLPs apply to most of the work we discussed in this chapter, with the possible exception (if safety claims are not made on the results) of pharmacology studies. At the heart of GLP regulations are requirements for (1) a quality assur- ance unit (defined in Chapter 5) with broad authority to review and approve or disapprove of just anything and everything; (2) a study protocol; and (3) a study director. In  addition, cGLPs are comprehensive, with every other aspect of laboratory operations and procedures, from facility standards to 369Nonclinical Studies elements of animal feed, found in these regulations. Elements of cGLP are listed in Chapter 4. Finally, many of the terms used throughout this chapter to guide the scientific, management, and administrative aspects of a nonclin- ical study, including this term itself, were either introduced or institutional- ized by cGLPs on their introduction in 1976. Summary of Nonclinical Studies This chapter reviews methods used to assess the risk and benefit of a candi- date biotechnology product, with emphasis on biopharmaceuticals, as per- formed in nonclinical laboratory and animal studies. These studies begin once the nature of the biological construct or molecule, its purity, and its potency after early production have been characterized and defined. Initial safety studies are performed to determine product risk in animals before being evaluated in human clinical studies. Nonclinical study results are used to assess benefit versus risk and provide valuable information for designing the first-in-human clinical study. For pharmaceuticals, nonclini- cal testing provides the foundation for understanding the pharmacological and toxicological properties of the product in animals and in vitro assays. Of importance is to understand the different testing requirements associated with product-specific attributes. For example, in vitro laboratory tests are used to characterize the investigational product, both during the early stages of product development and throughout commercialization. The laboratory testing is then typically used to evaluate each lot of product that is intended to be used, to support additional preclinical safety testing platforms such as animal studies. Animal testing is conducted to support preclinical safety; these studies typically include absorption, distribution, elimination, and metabolism (ADME) studies used to determine the pharmacokinetics of a product and pharmacodynamic studies used to demonstrate how the prod- uct interacts with cells and tissues. In addition, a number of safety tests are performed to measure the toxicity of the product at predetermined doses and dosing regimens intended to match or exceed those to be used in the pro- posed future human clinical study. Toxicity studies are designed to measure acute, subchronic, or chronic effects of the product, as well as to screen for specific types of toxicities. Nonclinical studies are designed and performed to predict human risk. This very important safety testing is conducted under a quality system, cGLP. In a nutshell, studies conducted in compliance with cGLP are prospectively designed, written, approved, and well documented. A final study report is typically generated to include results, summaries, conclusions, tabulated data, figures, and tables. The data in the final study report are verified by an independent reviewer, usually representing quality 370 Biotechnology Operations assurance, and the report is finalized with a required signature approval from both the study investigator, the study director, and a quality assurance representative. Reference CFR. 1978. Good Laboratory Practice for nonclinical laboratory studies. Title 21, CFR Part 58. US Government Printing Office, Washington, DC, Source: 43 FR 60013. 371 9 Clinical Trials Introduction to Clinical Trials A clinical trial, also referred to as clinical study or clinical research, is the overall process of evaluating the safety and efficacy of a medical product or an intervention in humans. Importantly, it is investigational and the purpose of a clinical trial is to learn about a product and how it impact humans. The intention is not to treat patients but rather to provide data regarding safety and efficacy in humans. Successful completion of several clinical trials is required for market approval of drugs, biologics, and some medical devices by FDA, and hence, clinical studies are used in the biotechnology industry to support market approval of biopharmaceuticals. However, the concepts developed for clinical studies incorporate scientific and design elements shared with field trials of other biotechnology products for which there is no testing in humans, such as field studies of environmental or agricultural products. In the scheme of biopharmaceutical development, clinical research fol- lows animal studies and other preclinical testing and evaluation, because a product is always sufficiently tested for safety and performance in labora- tory studies before it can be evaluated in human clinical studies. Human clinical research, the first research that uses healthy volunteers, is divided into several phases of clinical development (Figure 9.1). Each subsequent phase becomes increasingly large or complex and shows a continual shift in focus from measuring product safety in small studies—often in healthy individuals—to measuring both efficacy and safety in a larger patient popu- lation. Collectively, clinical development is a long, complex, and expensive process based on scientific methods, data integrity, and a high degree of reg- ulatory oversight. Clinical trials are an important aspect of biotechnology development, because a large number of firms develop medical products—drugs, bio- logicals, or medical devices—and because before commercialization, each medical product must be extensively tested in humans. Some biotechnol- ogy firms plan to take their product through all phases of clinical develop- ment to market approval. Others have a different business strategy and plan 372 Biotechnology Operations Phase 1 regulatory review and approval (initial IND) IRB review and approval Protocol and supporting documents Phase 1: Concept protocol and design Phase 2 protocol regulatory submission and review Phase 2 supportive clinical studies Pharmacokinetics Food effects QT/QTc Special populations Mass balance Clinical Study Report (CSR) Phase 1/2 supportive clinical studies Pharmacokinetic Pharmacodynamic Dosing and dose schedule IRB review and approval Phase 2: Concept protocol and design Phase 1 clinical trial Phase 3: Concept protocol and design Phase 2 clinical trial Phase 3 protocol regulatory submission and review Phase 3 clinical trial License application and approval Postmarket approval/ Phase 4 clinical trials Regulatory requirements IRB review and approval Elective studies Follow-on safety New indications New populations: children, elderly, and so on New formulations, delivery systems, and so on CSR CSR Protocol and supporting documents Protocol and supporting documents FIGURE 9.1 A typical scheme of clinical trials in biopharmaceutical development. In an electrocardiogram trace the QT interval is a measure of time between the start of Q wave and the end of the T wave in the heart’s electrical cycle (QT). The QTc represents a heart rate “corrected” QT measure. 373Clinical Trials to evaluate the product only in early phases of clinical development, until there is added value through proof of product safety in man. Most often, a biotechnology firm with candidate biopharmaceutical product will strive to add further value to that product by initiating human clinical research studies. Despite this strong desire to increase product value by performing a clinical study, small biotechnology firms often have little or no experience in this endeavor. In addition to lack of experience, many biotechnology firms have a culture of intellectual liveliness, in contrast to rather somber tone of clinical development, where there is an emphasis on scientific proof beyond reasonable doubt and thorough and sometimes detailed or rigid quality pro- cedures based on government regulations and guidelines. The result is that to be successful in clinical research, many biotechnology firms find that they must change, to some degree, their culture and means of doing business. Another option for a biotechnology firm would be to form a strategic alliance with a large industry partner who has experience and resources to conduct the necessary clinical studies. This strategy would surely provide the most expeditious route to biopharmaceutical product development and associated human clinical studies. However, this mechanism comes with a price, usu- ally in the form of the partner obtaining an equity share in the biotechnology firm and gaining some level of control over decision making and the devel- opment path forward. This chapter attempts to explain the basic elements of clinical research, as they apply to the development of medical products. It begins with a brief his- tory of the field, provides an overview to introduce concepts and terms used in clinical research, and progresses to a section on clinical planning. Further information is provided on the design and conduct of clinical studies, with extensive discussions concerning the people and institutions involved in a typical trial. A section is dedicated to clinical trial operations, which dis- cusses each phase of clinical development. Another section covers quality systems for clinical trials, that is, current Good Clinical Practices (cGCPs). This chapter ends with a discussion of ethical behavior and the importance of ensuring the well-being of human subjects enrolled in clinical research. Background of Clinical Research Introduction Historically, clinical research has been considered as either observa- tional or experimental. In observational studies, the investigator has no control over the study conditions, and investigational drugs and placebo are not given to the subjects. These studies may be referred to as epide- miological, because they measure current conditions absent novel medi- cal interventions. Although observational studies may form a foundation 374 Biotechnology Operations for conducting an experimental clinical study or for following safety of a biopharmaceutical post licensure, they are not used to study the safety and efficacy of a particular biopharmaceutical during the development process. For experimental clinical studies, also referred to as controlled studies, the investigator designs the conditions for the study and follows that design in an exact manner. Clinical studies of biotechnology products performed for the purpose of market application, that is, premarketing studies, are always controlled clinical studies. Most controlled clinical trials compare two or more treatment modalities. The specific treatment schedule with the test product is predetermined, and all other treatments to or medical con- ditions of the subjects are managed as similarly as possible. The design of a clinical study for a biopharmaceutical may thus compare the test product with a marketed product used to treat the same indication (a comparator) or, if no appropriate comparator is available, to a placebo (a sugar pill) used to minimize or control study bias. Historical Information on Clinical Trials Comparative drug studies were first reported in the eighteenth century but used infrequently until the twentieth century when, as noted in Chapter 3, laws and regulations were developed to ensure that drugs and medical devices were adequately tested for safety and effectiveness. Indeed, these regulations have, in part, influenced clinical trial designs and are based on good scientific research practices that had been used in laboratories, nota- bly the need to test a hypothesis in a formal manner. Another process called randomization or the blinding process, that is, assigning a patient to receive one or another drug, was first used in laboratory and agricultural field research and then was adopted as good scientific practice for clinical trials. Monitoring and auditing, now standard practices for clinical trials, were earlier used in nonclinical studies and were found to be an effective means of ensuring the quality of data. Indeed, as drug development became more complex and clinical studies grew larger, many scientific and quality prac- tices were applied to clinical trials, which have now become the norm for biopharmaceutical clinical research. In the 1970s, the quality and ethical aspects of clinical trials came to be known as cGCPs, and by the 1990s, cGCPs had gained international accep- tance, as the quality system for clinical trials. During this period, the num- ber of clinical trials grew dramatically because more drugs, medical devices, and biotechnology products entered clinical development, more countries required studies to be performed on their own soil, and regulatory agencies demanded greater numbers of ever-larger studies for each product. Further, clinical investigators and statisticians devised better clinical study designs, more effective means of conducting studies, and improved means of col- lecting and analyzing data. Today, many thousands of clinical studies are underway each day in dozens of nations and throughout the world. 375Clinical Trials Clinical research plays an increasingly important role in the development of biopharmaceutical products and also in their postmarket approval evalu- ation. Each clinical trial is also becoming increasingly complicated, expen- sive, and publically announced. Indeed, results of clinical trials are relayed almost daily in newspapers and on television. The increased complexity and demands of clinical research have led to more regulatory requirements and an increased need for documentation. Indeed, some would argue that expan- sion of clinical research could someday outgrow the ability of the clinical research community to provide infrastructure for all ongoing trials. Others disagree and feel that careful planning and exact execution of clinical devel- opment makes for a successful clinical study, allowing a sponsor to deter- mine whether or not a product is safe and efficacious in man and, therefore, fit to be marketed for the intended purpose. Generalizations in either case are not correct. Well-designed and executed clinical studies are and always will be required to demonstrate the safety and effectiveness profile of any biopharmaceutical. Clinical research requires careful planning to ensure that a valid study is completed and to fully meet the rights and interests of each volunteer enrolled in any study. To provide an overview of clinical trials, this chapter examines the structure of clinical research principles and activities, as they are applied to the devel- opment of medical products in the biotechnology industry. We focus on key issues that one must consider for successful clinical development of any prod- uct: organization, planning, personnel, operations and processes, documenta- tion, quality, ethics, and resources. Organization of Clinical Research Phases of Clinical Trials Clinical development of biopharmaceutical products is divided into four distinct and sequential phases, identified in Figure 9.1. These phases pro- ceed from the most controlled conditions and environment (Phases 1 and 2) to a scenario that is closer to the real world (Phases 3 and 4) in which the product is used to treat patients. Other terms, including the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use nomenclature, are provided. • Phase 1. Early phase. Clinical safety and toxicology. Clinical pharmacol- ogy. Human pharmacology: The first administration of a new prod- uct to man at a fixed route and schedule is considered a Phase 1 trial. Typically, the four goals of a Phase 1 study are: (1) to estimate the maximum tolerated or safe dose; (2) to determine whether any organ systems are affected by the product; (3) to identify any toxicity 376 Biotechnology Operations related to the product, and if toxicity is identified, to measure the extent, duration, and reversibility; and (4) to observe any unantici- pated (desirable or undesirable) activity of the product. • Phase 2. Expanded dosing. Pharmacokinetics. Therapeutic exploratory: The concept and definition applied to Phase 2 study has been somewhat misused and broadened by the biotechnology community. However, a textbook definition suggests that this phase of clinical develop- ment explores different dosages of the product, compares the effects of doses of the product to those of a placebo and, ultimately, deter- mines which dose has the best safety and efficacy profile. Results of Phase 2 set the stage for design of the Phase 3 clinical trial. • Phase 3. Pivotal clinical trial. Therapeutic confirmatory: In this phase of clinical development, the selected dose is given to a much larger number of individuals, each representing the target patient popula- tion that was established in the product labeling. A Phase 3 trial is referred to by regulatory agencies as a pivotal trial, because it is the clinical basis for marketing approval or disapproval decisions. • Phase 4. Therapeutic use: Postmarketing studies aim to further study safety and efficacy or to extend the indication or use of the product. The Science of Clinical Research A clinical program is designed just as any scientific research project, and the design must be scientifically sound, that is, it should test a hypothesis or a series of hypotheses, have clear objectives, and identify measurable out- comes. Quality in performance of clinical research is also important, and proper collection of data is essential. Use of other scientific tools, such as blinding of procedures and application of placebo or comparator, statistical analysis of data, and objective interpretation of results, is an essential ele- ment of clinical research. Unfortunately, some in biotechnology may forget that good clinical research is based on the scientific method and ethical guidelines and is not simply a business or entrepreneurial endeavor; hence, clinical research should be highly regulated and structured. Clinical research is the definitive step in evaluating new biotechnology products for safety and efficacy, that is, the prevention, diagnosis, or treat- ment of disease. Clinical trials, and the systems that support them, are com- plex endeavors and require collaboration among investigators, industry, academic institutions, and government agencies. Adding to their complex- ity, clinical trials have undergone a number of changes in the past decade. Progress in biomedical sciences and biotechnology has accelerated and increased the need for clinical studies and created new opportunities to improve clinical trial processes. Advances in informatics, laboratory and clinical diagnosis, and data management have led to new ways of evaluat- ing human subjects and reporting information and data. However, a new 377Clinical Trials biopharmaceutical product can only be brought to market by a firm if the product is first shown to be safe and effective. Stated another way, clinical development is outcome driven and the outcomes are issue focused and, ultimately, based on meaningful scientific data, which, in turn, are based on a relevant hypothesis and clinical study design. Quality in Clinical Research and Current Good Clinical Practices Current Good Clinical Practice is an ethical and scientific quality standard and a quality system applied to designing, conducting, recording, and reporting clinical trials. Although cGCP has taken on a definition of regula- tory compliance, it is actually more than that and represents an established means of conducting a clinical trial. There are national and international standards for cGCP but the ICH standard is the most commonly accepted version and the one followed today by most biotechnology firms and in most nations. Quality and cGCP in clinical trials are discussed later in this chap- ter, and the elements are included throughout the text. Clinical Development Planning The key to successful clinical research programs in this ever-changing and fast-paced environment is effective clinical development planning. Planning must focus on the clinical claim or claims and, as noted in Chapter 1, this is best stated in the targeted product profile (TPP). The primary purpose of the label claim in the TPP is to inform prescribers and patients about the documented benefits of a product. Clinical outcomes, derived from clini- cal trials, provide the basis for label claims. Further requirements for clini- cal planning are based on FDA regulations and guidelines. For example, a major requirement of an Investigational New Drug Application (IND) is the investigational plan, a section of the document that outlines the sponsor’s intended clinical research program (Chapter 3). The TPP for a biopharma- ceutical states medical objectives of the product, including the indication and therapeutic and safety profiles. Using a well-conceived and well-written TPP with a list of desirable out- comes, a clinical development plan can be written as a predecessor to the product development plan (PDP), both described in Chapter 1. Note that the clinical development plan is not a stand-alone document; it is instead an important but integral part of and is woven into the overall PDP. In addi- tion, the clinical development plan is a living document and, like the overall PDP, can be changed at any time, as long as the changes are coordinated with other aspects of the PDP and with members of the product develop- ment team. The clinical development plan is a written document that describes how a new biotechnology product can be progressed, in an orderly and timely manner, from first administration in man to postlicensure studies. 378 Biotechnology Operations It explains to colleagues, management, and regulatory agencies, the pro- posed product’s clinical development scheme, based on current knowledge, and also provides critical information to the development team, such as the rationale; time frame of Go/No Go decision points; costs, both internal and external; and an outline of the proposed clinical studies. A well-constructed clinical development plan also addresses, in a concept protocol, the important scientific, medical, and operational issues or factors, as listed in Chapter 1. Thus, the clinical development plan brings together all elements— scientific, management, and operational—into a cohesive document. As a living docu- ment, the clinical development plan provides the opportunity for adjust- ment, as new information, including preclinical, manufacturing, and clinical information, is obtained from the ongoing research. Furthermore, a complete and current clinical development plan allows the firm, investors and/or partners, and FDA the opportunity to provide valuable feedback on the future clinical milestones and regulatory path (e.g., recognize an alter- native development strategy such as qualify for orphan drug status or fast- track approval process). Infrastructure for a Clinical Trial: Individuals, Documents, and Investigational Product Earlier, we noted that experimental clinical studies test a hypothesis, have a written design or protocol, use scientific research methods, and are carefully controlled endeavors, employing human volunteers and professional staff. The planning aspect is referred to as clinical study design. The elements of a study design, the individuals involved in a clinical study, and the documents that support and control a study are discussed in this section. The qualifications of clinical study staff and their responsibilities are given in Box 9.1. A list of clini- cal study documents and the primary purpose of each are given in Box 9.2. Design of Clinical Trials and the Clinical Protocol Once a clinical plan has been completed, the clinical professionals at a bio- technology firm now focus on each clinical study identified in that plan. The basic elements of a study are outlined in a document, usually not exceeding 10 pages, referred to as the concept protocol. This is really the scientific basis for a study design and represents the initial proposal of experienced profes- sionals, such as a medical director, a clinical project manager, and various investigators. Elements of a concept clinical protocol are listed in Box 9.3. The hypothesis being tested is paramount, but other matters are also important. Objectives are keys to success. Study size, patient population, and indication are also critical to experimental design. As noted below in greater detail, 379Clinical Trials Phase 1 studies are smaller and focus only on safety, whereas Phase 3 tri- als are typically large, multicenter endeavors that evaluate both the efficacy and the safety of a product. Once the design has been drafted, the firm must consider the management and operational elements that will support the study. This evaluation sometimes reveals the design to be overly complex and demands that the clinical protocol be revised to make it more feasible, from an operational point of view. Alternatively, it might suggest that the number of subjects in each group is too low if a meaningful conclusion is to be drawn or that the enrollment targets are too aggressive, with historic patient populations not likely to be enrolled in the designated timeline. It is especially important to ensure that the nonclinical safety study plans cover the recommended dose and dosing schedule. Noting the nonclinical product needs, it is important to ensure availability and sufficient supply of investiga- tional product that is needed to support the proposed human study design, as it relates to dose and manufacturing scale. Normally, the biotechnology firm asks experts, such as statisticians, toxicology scientists, experienced clinical investigators, and, often, FDA, to review and comment on the design BOX 9.1 CLINICAL TRIAL: INDIVIDUALS AND RESPONSIBILITIES • Volunteers, patients, and human subjects • Sponsor and staff • Medical director • Safety monitor • Auditor • Medical writer • Clinical project manager • Regulatory staff • Manufacturing and clinical trial materials • Principal investigator and staff • Subinvestigator(s) • Nursing staff • Recruiter • Statistician • Institutional review board • Board chair and board members • Administrative staff • Quality assurance unit 380 Biotechnology Operations BOX 9.2 CLINICAL TRIAL: DOCUMENTS • Concept protocol: A brief design of a clinical study used as the basis for discussions between sponsor, investigator, and regulatory authorities. It is the foundation for preparing the full protocol. • Clinical protocol or protocol: An instructive document that identi- fies exactly why, how, and with whom a clinical study will be performed and provides schedules of events. • Informed consent (IC): Detailed procedures used and the time frame required to obtain informed consent. • Informed consent form (ICF or CF): This document explains to a volunteer the potential risks and benefits of a clinical study. To enroll in a study, a volunteer must understand and sign the CF. • Investigator’s brochure (IB): An informative document that identi- fies for each member of the investigative staff the information on the clinical study, the product being tested in the study, and the possible risks and benefits to the volunteers enrolled in the study. • Form FDA 1572: Statement of investigator captures information on the investigative teams and is an agreement by the investi- gator to follow the protocol and regulations regarding clinical studies. • Curricula vitae: Resumes of the principal investigator and key investigational staff. • Clinical trials agreements: This agreement between a sponsor and an investigator or a clinical contract research organization out- lines responsibilities of each party to perform a clinical study. • Form FDA 3674: This represents certification by a sponsor to disclose clinical trials information to clinicaltrials.gov, accord- ing to the U.S. law. • Forms FDA 3454 and 5455: Financial Disclosure. In these doc- uments, the sponsor discloses to FDA the financial arrange- ments that exist between clinical investigators and the sponsor. • Case report forms (CRF): These are paper or electronic forms on which the investigator enters medical information gath- ered during a clinical trial. • Patient diary: These are the forms completed by subjects during the outpatient phase of a clinical trial to capture data on pos- sible AEs and general medical condition of the individual. (Continued) 381Clinical Trials contained in the concept protocol. Thus, the overall clinical objective and the objectives of each phase of development, both provided in the clinical plan, drive the full clinical study design, as outlined in the concept protocol. The document that ultimately describes in detail the clinical study design is referred to as the clinical protocol, and this is written by clinical study staff once a concept protocol is acceptable. The most important step in any clinical study is to prepare a complete, well-organized, and scientifically sound pro- tocol. Protocols can be changed, or amended, but amendments take time and incur cost. Therefore, to avoid delays, the protocol and other clinical docu- ments consider, and provide for, every eventuality likely to occur once the study begins. Responsibility for writing a protocol may rest with the sponsor or the principal investigator (PI). The sponsor is a representative of the biotech- nology firm, whereas the (physician) investigator is the person responsible for conducting the study in accordance with the protocol. Thus, an investigator is retained by the sponsor. Today, national and international guidelines provide a standard organization, a template, for the clinical protocol. The elements of a protocol are listed in Box 9.4. A heading sheet gives a fully descriptive title, names the investigator and their institute or employer (affiliation), and identifies the sponsor. Most institutions give a unique num- ber to each protocol. The second sheet, a signature page, provides the names and contact information of everyone responsible for the protocol and, under a statement of compliance, prompts for the signatures of both the PI and the sponsor. A summary of the protocol is typically provided in the next section, and this begins with a statement of the objectives and, in most protocols, the formal hypothesis to be tested and is followed by a brief summary of infor- mation on the product under investigation. BOX 9.2 (Continued) CLINICAL TRIAL: DOCUMENTS • Operations or administrative manual: This collection of adminis- trative and management information and instructions guides performance of the clinical trial and supplements the protocol. • Form FDA 3500 MedWatch: This is a standard form used to report safety information and adverse events to FDA. • Investigational product accountability log: It is used to document the receipt and distribution of all investigational products. • Transfer of obligation log: It serves to document any or all obliga- tions transferred by the clinical investigator to other clinical staff members. • Screening log: It is used to document all subject-screening events and usually includes documentation of subjects that meet study participation criteria. 382 Biotechnology Operations The study design is then described in some detail, because it is the sci- entific heart of clinical research. Since most studies compare the treatment under investigation to another treatment (comparator) or to no active treat- ment at all (placebo), the design describes how the comparison will be made in the study design. It includes a description of the dose or doses of product and placebo or comparator, duration and intervals for giving doses (dos- ing), and a description of dosage forms. Typically, patients are divided into groups, or cohorts, and members of each group are given one or another treatment or dose. Further, design criteria may include use of randomization, whereby patients are randomly assigned to one or another group, or blinding, the process of keeping the exact treatment for each patient hidden from the subjects, PI, study staff, and sponsor. These elements of an experimental design prevent bias from entering into a study and are absolute require- ments for late-stage studies performed in the U.S. Bias, a predisposition to BOX 9.3 ELEMENTS OF A CONCEPT CLINICAL PROTOCOL • Description of the biopharmaceutical investigational product • Previous use in man or animals • Stated indication • Protocol title • Potential clinical investigator(s) • Anticipated study duration • Study phase • Intervention regimens • Study objectives • Study hypothesis • Subject population and general characteristics • Major inclusion and exclusion criteria • Study design and schedule or duration, number of subjects and groups, and • study site(s) • Study schema • Study end points: Safety and direct or surrogate efficacy • Study procedures and methods (primary, in general) • Assessments • Stopping rules • Unique scientific, ethical, or medical aspects 383Clinical Trials a particular outcome or a prejudice, and any element of design in a protocol that might lead to bias are carefully avoided in the study. Other methods may be applied to a clinical study design to avoid bias, improve study per- formance and validity, or ensure safety and well-being of human volunteers. Stopping rules are descriptions of how a study will be halted, temporarily BOX 9.4 ELEMENTS OF A CLINICAL PROTOCOL • General information: Title, numbers, names of investigator, and sponsor, version-controlled. • Background information: Description of the product and how and when to administer. • Trial objectives, with purpose and stated hypothesis. • Trial design: Scientific design and factors that ensure or enhance the design. • Selection and withdrawal of subjects: Inclusion and exclusion crite- ria; withdrawal of subjects. • Treatment of subjects: Administering medications and monitor- ing subjects. • Assessment of efficacy: Efficacy measurements and end points. • Assessment of safety: Safety measurements and end points. • Statistics: Data sets and statistical analyses. • Access to source documents or data. • Quality control or quality assurance: All aspects of compliance and quality; monitoring procedures. • Ethics: Ethical standards for the study. • Stopping rules: Clearly defined stopping rules resulting from safety signals or adverse events. • Safety reporting: Specific adverse event-reporting requirements to clinical investigator, IRB, and FDA. • Data handling and record keeping: Management of data during and after the trial. • Finance and insurance: Responsibilities for payments and liabil- ity insurance. • Publication policy: Anticipated publication and authorship policies. • Appendices: For example, treatment and test charts and sched- ules, consent form, standard medical guidelines, publication policy, and references. 384 Biotechnology Operations or permanently, should a certain type or series of unanticipated events or adverse events (AEs) be noted during the study. These rules provide a means of enhancing study safety and well-being of human participants. Stopping rules are explicit and fully described in the protocol, and once approved by FDA, they serve as an important agreement between the PI, the sponsor, and their regulatory agency. Other important aspects of a clinical study design are rules for selection and enrollment of patients, that is, inclusion or exclusion criteria, and for with- drawal of the subjects. It is important to carefully select subjects, enrolling only those who meet stringent criteria. In many Phase 1 studies, the investi- gator may wish to enroll only healthy or normal subjects, who are best suited for studying a product on its first introduction to man. In many other studies, the subjects represent the actual patient population, as described in the TPP, that the biopharmaceutical is intended to treat. In either situation, it is impor- tant to include in the study a specific type of individual and to exclude those who have other medical conditions or a disease that could put them at risk of undesirable reactions to the product or that could potentially confound the study results. Hence, inclusion and exclusion criteria are written into a protocol. Inclusion criteria identify attributes that the patients must have to enter, or enroll in, the study, whereas exclusion criteria identify issues that make a potential subject ineligible to enroll or participate in the study. For example, if one were to study a biotechnology product that was intended to lower blood pressure in otherwise healthy individuals, hypertension (high blood pressure) would be an inclusion criterion, whereas severe or advanced cardiac disease might be an exclusion criterion. A list of inclusion and exclu- sion criteria that might be applied to a Phase 1 clinical study in which nor- mal, healthy individuals were enrolled is given in Box 9.5. A number of other rules, for example, how to replace, with a new sub- ject, those who withdraw from a study, may be given in the design section of a protocol. Sometimes, clinical studies must be terminated or particular subjects must withdraw, either voluntarily or on request of PI. The protocol describes in the design section how these types of enrollment decisions are to be made and carried out. The treatment of subjects with the test product is normally described in great detail in the protocol. To prevent bias, product is administered to each subject in exactly the same manner, at a prescribed amount, and on an established schedule. Assessment of safety and efficacy is essential to the success of a clinical study and a protocol explicitly describes how each is measured. Outcomes, broad results, or visible effects that form the basis for the study hypothesis are described in medical terms. An example of an outcome in the case of a biophar- maceutical product intended for treatment of lung cancer might be to remain free of tumor for 1  year. Second, one or more end points are clearly stated. End point is the term used to identify a measurable parameter, again exactly medically defined. End points must reflect the objectives and the disease that 385Clinical Trials BOX 9.5 EXAMPLES OF INCLUSION AND EXCLUSION CRITERIA • Inclusion criteria: • Age: 18–50 years • Sex: Male or nonpregnant female • Good general health, as demonstrated by medical history, baseline laboratory tests (urinalysis, clinical chemistry, and hematology), and physical examination • Laboratory values within 1.25 times institutional stated normal values • Negative test results for HIV-1 and Hepatitis-A, -B, and -C • Low risk of coronary heart disease based on National Health and Nutrition Examination Survey-1 cardiovascular risk assessment and screening electrocardiogram • Negative tests for autoimmune diseases, rheumatoid arthri- tis, and antinuclear antibody • Reliable access to the clinical test facility and availability to participate for the duration of the study • Assessment of the understanding of questionnaire com- pleted before enrollment and demonstration of understand- ing of risks and benefits associated with study participation • Ability and willingness to provide informed consent • If the participant is female and of reproductive potential, she should: – Have a negative serum or urine beta human chorionic gonadotropin pregnancy test performed within 3  days before study initiation – Agree to consistently use effective contraception from 21  days before study initiation and for the duration of the study • Exclusion criteria: • Prior receipt of similar biopharmaceutical • History of confirmed diagnosis of (disease or condition) within the last 2 year • Use of (specific drugs) within 5  months of enrollment or use of (specific drugs) within 2 months of enrollment (Continued) 386 Biotechnology Operations BOX 9.5 (Continued) EXAMPLES OF INCLUSION AND EXCLUSION CRITERIA • Recent (within 2  weeks) use of (specific drugs) with (spe- cific effects or drug indications) • Anticipated use of medications known to interact with (investigational class of biopharmaceutical) • Use of any investigational or nonregistered drug or vac- cine or whole blood or blood product within 90  days of enrollment. • Use of systemic immunosuppressive medications or cancer chemotherapeutic compounds within past 90 days • Current or past diagnosis of Type I or Type II diabetes • History of severe allergic reactions • Screening laboratory abnormalities beyond the limits defined in the inclusion criteria • Clinically significant medical condition, physical examina- tion findings, other clinically significant abnormal labora- tory results, or past medic history that may have clinically significant implications for current health status in the opinion of the investigator • Any contraindication to phlebotomy • Body mass index <19% or >30%
• Acute illness at the time of enrollment
• Pregnant or lactating female or female who intends to
become pregnant during the study period
• Serologic positivity for Hepatitis B or C or HIV-1
• Psychiatric condition that precludes compliance with the
protocol, including ongoing risk for suicide or psychosis
• Suspected or know current alcohol abuse or recreational
intravenous drug use within the last 12 months
• Acute illness at the time of enrollment
• Any other condition that, in the judgment of the investi-
gator, would interfere with or serve as a contraindication
to protocol adherence, assessment of safety or reactogenic-
ity, or a participant’s inability to give informed consent, or
increase the risk of having an adverse experience to the
study drug.

387Clinical Trials
is being treated, prevented, or diagnosed. In the lung cancer example, an end
point might be tumor mass found in lung. Third, to adequately evaluate end
points, that is, the measurements, the act of determining an amount or quan-
tifiable dimension of that end point is necessary. In the lung cancer example,
the measurement of tumor mass, number, location, and size of each, once each
month, by a radiological method, might constitute a valid measurement. Safety
end points are also measured. For example, to determine whether the patients
became allergic to a biopharmaceutical product, the protocol might direct the
investigator to carefully search for rashes after each treatment. The success of
a clinical study rests on establishing in the protocol meaningful and exact out-
come, end points, and measurements. Hence, medical experts are often asked to
advise this phase of protocol development, and in later phases of clinical devel-
opment, these issues lead to important discussions with regulatory authorities.
Safety, as well as efficacy, end points are described in the protocol. Should
they occur in a subject, these are recorded as AEs if they are mild or limited in
scope and severity and as serious AEs (SAEs) if they are severe, such as ana-
phylactic shock. When faced with an AE or SAE, the investigator or another
physician must determine whether the reaction is, or might be, related to the
investigational product. Indeed, each protocol, no matter the phase, states a
large number of safety measurements, such as clinical laboratory tests and
physical examination, that must be taken for each subject to determine the
safety and tolerability of the test product. Other measurements focus on the
efficacy of the product being tested.
The statistical section of the protocol describes, in detail, the analyses that
will be used to make comparisons of end points in the overall subject popula-
tion. It also applies statistical principles to support the design of the study. For
example, the statistical discussion provides rationale for the number of subjects
enrolled in each group, treatment or placebo, of a Phase 3 clinical trial. Another
section of the protocol deals with administrative issues, such as control of the
test product review, approval of study documents, methods for collecting and
recording raw data, and details such as insurance, or publication policy.
Most clinical trials enroll the total subjects numbering in dozens (Phase 1),
hundreds (Phase 2 or Phase 3), or low thousands (Phase 3). Yet, other clinical
studies of biotechnology products, notably pivotal Phase 3 trials, are quite
large and are conducted simultaneously by many investigators at several
sites. Today, Phase 3 international trials may enroll in excess of 60,000 sub-
jects at more than 100 sites in more than 20 countries. Such big trials demand
much administrative support and instruction. For these purposes, a clinical
operations manual is used in addition to the protocol. The Ops Manual is an
extension of the protocol and describes in greater detail all administrative
aspects of the study and provides detailed medial and managerial instruc-
tion to the staff involved in the clinical study. Using operations manuals is
a good business practice, because these manuals further ensure success of
a scientifically well-designed study by providing consistent procedures at
each trial site.

388 Biotechnology Operations
Human Subjects, Patients, and Volunteers
A clinical trial includes humans willing to participate and receive either
the product or, perhaps, the placebo. Later in this chapter, we describe the
rights of those individuals who volunteer to receive investigational prod-
ucts on behalf of the sponsor and the PI and, hopefully, for the betterment
of the health and well-being of all mankind. For this, we, in biotechnology,
greatly appreciate their participation. Since every volunteer enrolls in a clini-
cal study by his or her own free will, we refer to these individuals with the
general term volunteer. For products that could provide some benefit and
are used in individuals with a disease or a medical condition for which the
product is indicated, the volunteers are referred to as patients. In some stud-
ies, such as with preventive biopharmaceuticals (e.g., a vaccine) or where the
studies enroll healthy individuals (e.g., Phase 1), the volunteers are referred
to as subjects. We use the terms volunteer, subject, and patient without fur-
ther definitions in this chapter.
The Sponsor
In the case of industry studies, the sponsor typically is not only the finan-
cial and resource backer of a clinical study but is also the IND holder and, as
such, takes ultimate responsibility for the study. Responsibilities of sponsors
before, during, and after a clinical study are clearly defined in regulatory
guidelines. First and foremost is the responsibility of ensuring the rights and
well-being of every human subject or patient and of maintaining quality,
through quality assurance and quality control, of the trial. To meet this obli-
gation, the sponsor of a clinical study has policies and procedures that dem-
onstrate exact intentions. A sponsor may delegate responsibilities to another
party, but this must be specific and in writing. Such is the case when an
individual known as PI is retained to perform the study or when a contract
research organization (CRO) assumes various functions and responsibili-
ties of the clinical trial. Smaller biotechnology firms often delegate most or
all clinical trial functions to others, but they can never transfer the ultimate
responsibilities of ensuring that a study is conducted, recorded, and reported
properly or that patients are always treated according to medical and ethical
standards. A biotechnology firm engaged in clinical studies always has, on
staff or retainer, clinical trial experts. Indeed, most sponsoring biotechnol-
ogy firms retain internally the monitoring and auditing function of clinical
trial and data management, thus ensuring the integrity of data and attend-
ing to financial and general administrative duties.
An important and yet often overlooked responsibility of the sponsor is the
selection of a qualified PI and, along with the investigator, the institution or
the CRO at which the study is to be performed. This is often a difficult task
for the biotechnology firm because, with both an exciting technology and ade-
quate investment, the firm’s management may be faced with several quali-
fied investigators, each of whom wishes to perform the study. Some may be

389Clinical Trials
inexperienced or otherwise unqualified to head an important clinical study
but appear knowledgeable about the product. Others may have years of expe-
rience as investigators but be inexperienced with this type of product. The
sponsor must find a person who is both qualified scientifically and has the
proper experience with the product type; finding or selecting the right PI can
be a challenge for the Sponsor and is further complicated with multicenter
trials, where several qualified investigators must be identified.
Sponsors ensure that all the paper work is completed during the trial. In
addition, as one might expect, a great amount of paper work is, in fact, gen-
erated before, during, and after each clinical study, no matter how small the
trial. The IND sponsor, not the PI, communicates directly with regulatory
agencies. The sponsor confirms that review was completed and approval
was given by the  institutional review board (IRB). Through the Investigator’s
Brochure (IB), the sponsor informs the investigator and his or her staff about
the product to be tested. Both the IB and IRB are described later.
The sponsor also plays important roles in relating safety information in a
timely manner. First, there must be a system in place to receive review from
investigators and, if necessary, report any safety information that is generated
during the study. The AEs and SAEs are collected by the sponsor in a timely
manner, and SAEs are immediately investigated and promptly reported to
those whose job it is to influence, make, or review medical decisions, that is,
the investigator, the medial monitor, the IRB, and the regulatory authorities.
The sponsor has in place a system of expert review for AEs and SAEs; this is
typically the job of the medical (or safety) monitor, a physician who examines
each event and reports his or her opinion regarding the significance of and
the relationship between SAE and the product to the sponsor. If, in the eyes
of the investigator or the medical monitor, AEs or SAEs are related to the test
product and certainly if the safety of the patients is at risk, the sponsor is
responsible for reporting the events to all investigative staff and regulatory
authorities and, in some cases, for stopping the study. Termination of a study,
meaning that the product can no longer be given, is driven by detailed rules
or study stop criteria, which are also provided in the protocol.
To ensure that the clinical study is being conducted properly, all aspects of
the study must be audited or monitored by an experienced and knowledge-
able individual, on behalf of the sponsor. Clinical trial monitoring is not the
same as the role ascribed to the medical (safety) monitor, described above.
In contrast to the medical (safety) monitor, who reviews AEs or SAEs pro-
vided to them, trial or study monitoring is a process that involves visits by a
professional auditor to the clinical study site at regular intervals to inspect
the clinical study documents and medical records. This auditing or moni-
toring process further ensures, among other things, that the study is being
conducted according to the protocol and within the guidelines established
to protect the rights and safety of the subjects or patients. The trial monitor
reviews study records for completeness and accuracy, and auditors inter-
view the study staff to verify that everyone is qualified to perform his or her

390 Biotechnology Operations
assigned role in the clinical study. Deviations, variance, and deficiencies are
noted by a monitor and reported to the sponsor, who, in turn, is responsible
for immediately correcting the issues or, alternatively, for stopping the study,
until corrections are made.
Another major responsibility of a sponsor is to prepare, update, and dis-
seminate a summary of clinical, nonclinical, and other pertinent product
information to the PI and his or her staff. This is done with a document
called the IB, written by the sponsor before the first clinical trial and updated
as new information becomes available. The elements of an IB are provided
Box 9.6.
An important issue that can arise with a clinical study is conflict of inter-
est, real or perceived and usually financial in nature, on the part of either
PI or sponsor or both. To perform an unbiased study, it is important that
the PI not be beholden to the sponsor and that any investigator assigned
to a study has no significant financial interest in the sponsoring biotech-
nology firm. Financial interest could result in bias on the part of inves-
tigational staff, and even a perception of conflict of interest or potential
bias in the sponsor–investigator relationship, particularly where it involves
substantial sums of money, can call into question the merit of a clinical
trial. Of course, investigators are remunerated for their time, expenses, and
BOX 9.6 ELEMENTS OF AN INVESTIGATOR’S BROCHURE
• Table of contents
• Summary
• Introduction
• Physical, chemical, and pharmaceutical properties and formu-
lation of the product
• Results of nonclinical (i.e., safety, pharmacology, and toxicol-
ogy) studies
• Effects (including safety, pharmacology, and pharmacokinet-
ics) in humans known from previous clinical studies
• Marketing experience, if any
• Summary of data and guidance for the investigator
• Anticipated risks and adverse reactions
• Summary of clinical data
• Assessment and treatment
• Toxicity management
• Additional risks associated with this or similar products
• References

391Clinical Trials
professional expertise. However, compensation must be fair and open and
a clinical investigator should not have a substantial interest in corporate
stock options.
The Principal Investigator and His or Her Study Staff
The principal investigator, often referred to as the PI or simply the investi-
gator, is the individual responsible for conduct of a clinical study at each
clinical trial site. She or he is retained by the sponsor, who delegates specific
clinical responsibilities to that PI. In return, the PI receives reimbursement for
expenses, including salary for the time spent in executing the study, under the
agreed protocol. There may be other benefits to PIs, such as publication of sci-
entific articles and ability to work at the cutting edge of their profession. A PI
may be employed at an academic institution or in private practice or he or she
may be associated with a CRO. Whichever the case, the agreement between
sponsor and PI typically includes funding for additional study staff, such as
nurses, administrators, clerical assistants, and individuals, to recruit subjects.
In some cases, the PI will be asked to prepare the protocol and other clinical
documents, but larger sponsoring firms frequently provide these documents
and ask the PI to follow these instructional documents. A physician may, in
the U.S., be both the sponsor and the investigator, or, as happens with some
biopharmaceutical firms, the sponsor may directly employ an investigator.
In effect, a PI is responsible for everything that happens at the clinical
trial site, including activities by his or her staff. In the United States, a PI
formally accepts this responsibility in one of the two ways, under a contract
with the sponsor or, in an abbreviated manner, by signing an agreement,
Form 1572, with FDA. Among varied investigator responsibilities, the most
important is to exercise clinical oversight and medical judgment at the site.
The PI ensures that everyone on his or her investigational team conforms
to the protocol and any other instructions (e.g., Operations Manual) con-
cerning the study and that subjects’ rights are fully met. Principal investi-
gators are responsible for submitting the protocol to the IRB for approval
and then beginning the study only after receiving this approval. The inves-
tigator is also responsible for enrolling and then treating patients in the
proper manner; for patient compliance in taking the investigational prod-
uct throughout the study; for ensuring that clinical documents, such as case
report forms (CRFs), are correctly completed; and for accountability of the
investigational product. Certain administrative functions are also required
of the investigator; these include maintaining professional credentials,
managing the research staff, communicating with the IRB, participating in
study meetings or conference calls, and maintaining good relations with
the sponsor and other parties involved in the study. Last but not least, the
PI is the principal scientist in a clinical study. In the end, each of the PI’s
responsibilities focuses on maintaining the scientific integrity of the study,
safety of the patients, and integrity of the data.

392 Biotechnology Operations
Clearly, a busy physician investigator cannot complete a clinical trial
without help, and so, investigational staff is employed at each study site.
Principal investigators often enlist other physicians to work on a study; these
are referred to as subinvestigators. Subinvestigators are qualified to serve in
this capacity by education and training, and in many cases, they are profes-
sionals working closely with the PI, such as medical residents or junior staff.
Although the PI may delegate certain medical responsibilities to subinves-
tigators, they still accept full responsibilities with this regard. Study nurses
are typically registered or licensed nurses and are employed because of their
medical training and experience and because a clinical trial involves medical
procedures and measurements. Study nurses originate many study records;
these documents are then reviewed and co-signed by the PI. They educate the
volunteers, ensure that informed consent (IC) is always properly adminis-
tered, take patient histories, and consider AEs and SAEs. Patients or subjects
do not just appear magically at the study site, but they must be recruited by
someone adept at identifying potential volunteers and at coordinating their
initial visit. This team member is the volunteer recruiter. Administrative
staff manage and organize records and files and assist recruiters and nurses.
Investigational product is usually maintained and distributed by a clinical
study pharmacist, and for some investigational products, this individual
prepares medication according to the sponsor’s instructions. Larger studies
also employ data specialists, individuals who are dedicated to transferring
data from paper to electronic databases and who ensure data integrity and
accuracy.
Institutional Review Boards, the Process of IC, and IC Form
We, as a society, have, appropriately, given significant rights to individuals
who volunteer for and participate in clinical trials. These rights derive from a
very important document, the 1964 Declaration of Helsinki. The Declaration
itself is based on the Nuremberg Code of 1947. The Code was drafted in
response to horrific situations that occurred during the Second World War,
specifically when Nazi investigators conducted biomedical experiments on
prisoners without the consent of those individuals. The heart of the Code is
the requirement for full understanding of the risks by written consent from
any human volunteer. This means that the person who is receiving any inves-
tigational treatment, no  matter how minor, must have the legal capacity to
give consent (or, in the case of children, have a parent or legal guardian give
consent), be so situated to exercise free power of choice without coercion,
and have a clear understanding of the investigation, including possible risks
and benefits.
Under the 1964 Declaration of Helsinki, a guide to physicians and others
involved in biomedical research involving human subjects, regulations for
clinical research now state that legally effective IC (Box 9.7) must be obtained
by an investigator before involving that subject in any clinical research.

393Clinical Trials
Conditions for IC have been established in most countries for usual condi-
tions as well as for unusual situations, such as children, those with demen-
tia, and those in emergency situations, that is, when the individual receiving
the investigational product may not be capable of being fully informed. In
the United States, protection of human subjects is mandated by the Code of
Federal Regulations, 21 CFR Part 50, a regulation with broad application.
In practice, IC is requested by the PI from each subject immediately before
enrolling that person into a clinical trial. Human subjects are asked to review
a description of the clinical study, including the design, potential benefits,
and possible risks. In some cases, such as novel investigational products, sub-
jects are queried or quizzed by written examination, to demonstrate that they
clearly understand the study and any risks to which they may be exposed
during the course of the clinical trial. Subjects are always given the opportu-
nity to ask questions from the PI, even if his or her staff is administering IC.
Once satisfied and willing to enroll, the subject signs an IC form (ICF or CF)
in the presence of a witness. However, the consent is always reversible and,
should the subject change their mind, it may be negated at any time in the
study. In effect, this means that a subject may leave a clinical research study
at any time and for any reason or for no stated reason.
The ICF is written after the protocol has been drafted and reviewed by the
PI and the sponsor and once nonclinical toxicology information or data from
previous clinical studies are available. Consent forms may be written by the
PI or the sponsor. Since the CF must be approved by an IRB, this board’s pre-
ferred institutional format should be considered for each clinical study site.
The IRBs often request changes to a CF, and so, it is not unusual to have sev-
eral slightly different versions, one for each site, in a multisite clinical trial.
BOX 9.7 ELEMENTS OF IC
• Statements that the study involves an investigation and pur-
poses for the research
• Description of risks or discomforts
• Description of possible benefits
• Disclosure of possible alternative treatments available to the
subject
• Description of processes used to maintain confidentiality
• Explanation of potential compensation or medical treatments
• Individual to contact for answers to pertinent questions about
the research or risks and benefits
• Statement that participation is voluntary and that refusal or
withdrawal will result in no penalty

394 Biotechnology Operations
For a variety of reasons, it is sometimes necessary to obtain the approval of
two or even three IRBs for some investigational sites. It is a necessary, but
sometimes a challenge, for both sponsor and investigator to ensure that each
form has correct content and is acceptable under current regulations.
The IRB (known as independent ethics committee in some countries) is a
committee, comprising usually five to ten medical professionals, clerics or
lay persons, responsible for ensuring and protecting the rights and welfare of
human subjects who participate in biomedical research. The IRB reviews pro-
tocols, the IC, the IB, and related materials, such as recruiting advertisements
and compensation. In doing so, the committee helps to ensure the rights of
subjects. The IRB must judge whether or not possible risks to the subject
outweigh potential benefits or the knowledge gained through the study. The
responsibilities of the IRB do not end with approval of the study and study
documents, as the IRB continues to review the program as clinical research
progresses and always considers reports or changes, such as SAEs and study
termination. Once the study begins, the IRB must review SAEs and other
significant issues that arise. Annual reviews of each study are mandatory,
whether or not there are issues related to the product, the subjects, or the
study itself. Of course, no member of the committee should have a conflict of
interest with any study he or she reviews.
Most institutions that conduct clinical research—universities, hospitals,
research centers, and CROs—have established IRBs. Independent IRBs are also
available and are used by sponsors when the investigational site has no insti-
tutional affiliation. Although no accreditation is required for IRBs, their records
are reviewed by national regulatory agencies, and in recent years, IRBs at some
notable institutions have been suspended for failure to follow regulations. In
the United States, the Department of Health and Human Services is ultimately
responsible for ensuring compliance with human use regulations, but this
department designates agencies under its supervision, such as FDA and the
National Institutes of Health, to be involved. In a practical sense, each IRB is
composed of individuals from different walks of life—ethicists, clerics, scien-
tists, and lay persons—so that the review is balanced in nature and considers
various professional and social aspects of the proposal. The committee meets
periodically; this gives each member an opportunity to review the clinical doc-
uments noted earlier. After review, these documents are discussed in an IRB
meeting, and it is not unusual for the committee to ask for additional informa-
tion on a particular concern or recommend changes to a document. By working
together, IRBs, sponsors, and PIs support each other, ensure the integrity of a
clinical study, and protect the right of human subjects enrolled in that study.
Investigational Product
Clinical trial supplies or materials include the biopharmaceutical, the investi-
gational product, placebo or comparator, or diluents, and any device used to
apply or deliver the product or otherwise ensure correct use and safety of the

395Clinical Trials
product as it is given to the volunteer. The investigational product must meet
specifications in terms of identity, purity, strength, and quality, as discussed
in Chapters 6 and 7. It is very important that the investigational product be
of consistent quality for all clinical trial sites and at all times throughout the
duration of the study. Investigational product used for the clinical study is
provided by the sponsor, that is, the biotechnology firm manufacturing the
biopharmaceutical product, in a timely manner, is properly labeled, and is
kept in a secure storage location, maintaining proper environmental condi-
tions (e.g., temperature) throughout the study. In a blinded study, steps are
taken to identify the product and placebo or comparator correctly and yet
maintain the blind. For example, a label on a vial of investigational prod-
uct in a blinded study may be changed by the pharmacy to a code, so that
the PI or the study nurses are not aware of the treatment, active product or
placebo, given to each subject. All these clinical supply operations are care-
fully managed, tracked, and documented, so the disposition of all product is
accounted for. As noted earlier, a pharmacist with experience in clinical trials
often manages these tasks and ensures that all clinical supplies are of the
highest quality, correctly labeled for the study, fully accounted for in records,
and are properly stored and distributed to study staff. He or she also ensures
that any unused clinical supplies are returned to the sponsor.
Collection of Clinical Data: Case Report Forms and the Patient Diary
Accurate and timely collection of all clinical data is an absolute requirement
for any clinical study. The process can be divided into four major stages:
• Preparation of document formats, forms, and media to collect the
data
• Collection of data during the clinical trial
• Review or audit of data to ensure completeness, accuracy, and
integrity
• Analysis of data
It takes considerable planning and effort to fully and properly collect clini-
cal trial data. The initial or raw data are referred to as the source informa-
tion or a source document, the original document on which an observation is
recorded. This includes records such as laboratory reports, clinical or patient
charts, memoranda, patient’s diaries, and pharmacy dispensing records. The
raw data from a source document may be initially recorded on a CRF or it
may be transferred from a source document to a CRF by study personnel.
The CRF is a printed, optical, or electronic document designed to record all
the information, no matter what the source, required by instructions pro-
vided in the protocol. Case report forms, and there are many for each clinical
study, are drafted after the protocol has been completed and after it is known

396 Biotechnology Operations
what data will be collected, in what format, by whom, and how frequently.
Once finalized, CRFs are printed in final format and distributed to each clin-
ical trial site. A CRF is issued to each subject participating in a study.
If patients or subjects are hospitalized throughout the course of treatment,
then data are easily collected in an environment conducive to keeping com-
plete and accurate medical records. More often than not, the investigational
product is given during brief clinic visits, and if the patient is feeling well,
he or she is sent home after a few hours (or days) at the treatment site. For
other products, the patient takes the product at home and only visits the clinic
initially and then periodically for follow-up physical examinations or tests.
When the patient is away from the clinic, the subject diary may be used to col-
lect data. In a diary, each subject records any symptoms he or she has noted
during the study. Although this is not a highly reliable means of collecting
data, it does sometimes reveal drug-associated AEs that occur between clinic
visits.
Patient diaries are reviewed and CRFs are completed by study staff, par-
ticularly by study nurses and physician investigators. These documents are
then subjected to final review and approval by the PI. During the course of
a study, and after CRFs have been completed by the investigational staff,
they are audited by an outside representative, the clinical auditor or moni-
tor, a representative of the sponsor. Whether paper or electronic, CRFs are
carefully reviewed against source documents to ensure accuracy of the data.
Today, data on paper records are usually entered into an electronic database
to facilitate statistical analysis. This is often performed using double data
entry methods, in which the same source data is entered twice by two inde-
pendent people and then electronically compared for consistency of data
entry. Since the transfer of data from source documents to CRFs or to elec-
tronic databases is prone to human error, electronic data collection, that is,
directly recording information, for example, blood pressure, from a source
document into an electronic database is becoming routine practice. Although
this reduces errors of transcription, it requires a validated electronic system,
including both hardware and software, and well-trained clinical staff.
No matter what the format, data are analyzed according to an analyti-
cal or statistical plan that is prepared by a statistician before beginning
the trial. Several computer programs are commonly used to analyze data
and to prepare tabular and graphic presentations of the information.
Statisticians, experienced in clinical trial data management and analysis,
are employed by the sponsor for these tasks.
Clinical Testing Laboratories
Clinical laboratory data are important to all clinical trials. Body tissues and
fluids, notably blood, are collected and sent to a laboratory, where they are
tested for various parameters. For most of these tests, the laboratory is in a
hospital or other medical center and is therefore certified by an accreditation

397Clinical Trials
agency such as the College of American Pathologists and regulations such as
the Clinical Laboratory Improvement Amendments. However, many clinical
investigations also necessitate the performance of unique laboratory tests.
These may be performed in a specialty laboratory or, in some cases, in an
academic laboratory or the sponsor’s laboratory. In such cases, tests must be
initially qualified for accuracy and specificity, and in later stages of clinical
development, they must be fully validated. Indeed, most of the quality crite-
ria applied to product quality control tests (Chapter 7) are applicable to the
tests used to measure clinical end points.
The sponsor is responsible for ensuring that each clinical testing laboratory
meets all requirements and that laboratory testing is completely and accu-
rately documented. The PI and staff ensure that samples are taken exactly as
mandated by the protocol and then properly stored and shipped to the clini-
cal laboratory. The PI also reviews the results, takes proper medical action,
and ensures that test results reach the patient’s records as a source document.
Reporting Results of Clinical Trials: Clinical Summary Reports
Once clinical trial data have been audited, analyzed, and tabulated, these
are included in a clinical summary report (CSR). This document, normally
prepared by a medical writer with the help of biostatisticians, describes the
clinical trial and reports all important aspects of the study. Since each report
is reviewed by the sponsor and by regulatory agencies, it must be clear, com-
plete, and well written. The data are tabulated and presented in an unbi-
ased, yet clear and concise manner. A report relates essential elements of
the protocol, clearly describing the design, treatments with investigational
product, and the population of human subjects. It discusses results of the
study, presenting data in tables and figures, and drawing conclusions made
by the PI and statistician with concurrence of the sponsor. Safety issues are
discussed in detail and statistically significant differences between treat-
ment groups, with regard to safety and efficacy data, are analyzed, usually
by several statistical tests. Conclusions and discussion of the data are written
in a CSR, and in many cases, a manuscript describing the study, its results,
and the conclusions is submitted to a scientific journal for peer review and
publication.
Clinical Trial Operations
Study resources and people involved in clinical research of a biopharmaceu-
tical product were reviewed in earlier sections. We now integrate this infor-
mation by describing the planning and performance of clinical studies, first
giving an overview of a typical clinical trial operation and then focusing,

398 Biotechnology Operations
more specifically, on each phase of development (Figure 9.1). The discus-
sion on clinical trial operations focuses on tasks that are often performed
in-house, by the biotechnology firm, and that are performed for the sponsor
by CROs.
Activities Leading to a Clinical Trial
Early in the development life cycle of a biopharmaceutical, the clinical plan is
written and it then becomes part of the overall PDP (Chapter 1). The decision
of when to enter and exactly how to design the first, or Phase 1, clinical trial
may not be established by the sponsor until a later date, perhaps after sev-
eral preclinical and very early development milestones have been achieved.
For example, before the design of the Phase 1 trial is completed, the dates
and schedules for the nonclinical studies, the manufacture and control of the
clinical trial product, and filing of the IND are established. Once a tentative
schedule has been set and there is a high degree of confidence that investi-
gational product will be available, it is possible to prepare a detailed Phase 1
clinical trial plan and the Phase 1 protocol.
Even the simplest clinical study requires quite a lot of coordination. Even
if the biotechnology firms do not have a formal medical affairs department,
they often have someone on staff who has experience in managing clini-
cal trials, and this individual has the responsibility for clinical planning.
Alternatively, a highly qualified and recommended consultant may be
retained to provide early clinical guidance. As soon as Phase 1 planning
process begins, the biotechnology firm decides if all elements of the study
will be performed by CROs or some aspects will be kept in-house. Seldom
does a biotechnology firm have the resources to hire enough professionals
to directly do all aspects of clinical work themselves. Thus, early decisions
in clinical planning are usually to identify what, if anything, will be done
in-house, and if clinical support is to be performed by CROs, how this will
be established. If all clinical work is to be contracted, the efforts should be
divided and functional areas, such as trial performance and quality efforts
(i.e., auditing), should go to a second contractor. This distribution of oversight
ensures implementation of the checks and balances that are very important
for a successful clinical study.
Now, an experienced clinician must design the study and write a concept
protocol. This may be done by a consultant or in-house staff or it may wait
until the investigator and investigative site have been selected. For clinical
research of many biotechnology products, the sponsor can choose from doz-
ens of academic sites, usually medical schools, and CROs. For other prod-
ucts, for example, in the case of a cancer treatment, the sponsor might only
consider sites that specialize in treating those patients. It is really important
to identify a clinical site that has access to right patients, a sufficient pool
of patients, experienced staff, and the infrastructure to completely perform
Phase 1 clinical trial. It is not uncommon to find an excellent investigator

399Clinical Trials
who works at an unqualified site or vice versa, that is, the ideal site but with
mediocre investigators. Once potential sites are selected, site visits are con-
ducted by the sponsor. A site is chosen, the PI is designated and agrees to do
the study, and, after negotiation over the scope of work, budget, and sched-
ules, a contract is signed. This is usually followed by selection of a CRO to
perform monitoring and perhaps a third group and a fourth group to pro-
vide other services (e.g., central laboratory). Now, the clinical trial team has
been established for that study and site.
Laboratory support is a hallmark of any clinical trial and it comes in
two types: (1) standard or routine clinical laboratory support; and (2) spe-
cialty laboratory analytics. Routine clinical laboratory support is offered
by almost any hospital laboratory and includes analysis such as hematol-
ogy, clinical chemistry, and basic immunodiagnostics. Small or early phase
trials, in fact, often use hospital laboratories. However, in large clinical tri-
als, a central laboratory, represented by a single contract laboratory, is used
to process, in the same technical manner, samples provided by multiple
clinical study sites. Most studies also require specialty diagnostics or ana-
lytical techniques. For example, it is often necessary to measure the bio-
pharmaceutical in samples of blood during a pharmacokinetic (PK) study.
In addition, for vaccine studies, the immune response to the product must
be measured with a variety of immunological assays, mostly unique and
some even difficult to perform. Specialty laboratories may offer these
unique testing services, but more often, these assays are adapted to or
developed for clinical studies of specific products. Biotechnology firms
may either do specialized assays in-house, at the firm’s internal laboratory,
or they may identify a contract laboratory capable of developing the tests.
There is no standard solution, and the sponsor must carefully plan exactly
how it is best achieved.
Once the clinical site has been identified, the sponsor’s representative,
working on behalf of the product development team, drafts the full clinical
protocol. The investigator also identifies staff, for example, sub-investigators,
recruiters, study nurses, and statistician, to assist in the study. Once the pro-
tocol has been written, the CF and the CRFs are drafted and, along with
the protocol, submitted by the investigator to the IRB. Institutional review
boards typically meet once or twice each month, and it is normal for an IRB
to request that changes be made in one or more documents before they are
approved. Hence, the process of protocol approval can take weeks or even
months to complete.
While the PI is leading these study and protocol development and review
activities, the sponsor is actively recruiting a medical (safety) monitor and a
clinical monitor or auditor. These individuals review the clinical trial docu-
ments before submission to the IRB and before ensuring quality and compli-
ance at each study site through a prestudy site visit. At the same time, the
sponsor is completing manufacture, labeling, and control of the investiga-
tional product and making arrangements to have it delivered to the clinical

400 Biotechnology Operations
site. In addition, the sponsor is actively preparing and then submitting the
regulatory documents, such as the IND, to FDA.
Once the regulatory agency accepts the IND and gives permission to begin
the clinical study, the sponsor delivers the product to the clinical site and
the investigator begins the sequential processes of screening, accepting,
consenting, and enrolling subjects. The dosing phase of the clinical trial may
now begin. Volunteers are given the number of doses specified in the proto-
col and efficacy end points are measured per the protocol. The volunteers are
closely followed throughout the study for any sign of reaction to the product.
If an SAE or a series of suspicious AEs are noted by the investigative staff,
then dosing and further enrollment may be halted. In such cases, the medi-
cal (safety) monitor, the sponsor, and, subsequently, the regulatory authori-
ties are notified. This leads to investigation and discussions; if the safety
of subjects can be ensured, the study may begin once again. However, if it
appears that subjects may be at undue risk or that the product is unsafe, then
the study may be terminated. Fortunately, studies of most biotechnology
products are not halted in early clinical studies because of safety concerns
and most studies progress to completion, as specified in the protocol. Yet,
patient follow-up is often a long process, and subjects may be asked to return
for physical examinations or laboratory tests for months or even years after
the last dose of investigational product has been given. Extensive examina-
tion of subjects further ensures the safe and tolerable nature of a new prod-
uct. Throughout the study, the clinical monitor visits the site to ensure that
the study is being performed according to the protocol.
Once all data have been entered into CRFs, each form is screened for accu-
racy and completeness and the information is transferred to an electronic
database. Statisticians are normally responsible for these steps and the statis-
tical data analyses that follows. In the case of biotechnology firms, the spon-
sor often retains a consultant statistician to perform analyses and to prepare
tables and figures that reflect the data. A CSR is then written by the investi-
gator or a medical writer. This CSR is first provided to the sponsor for review
and then to regulatory authorities as definitive results of the clinical study.
The above description lists only the most important tasks, and their inte-
gration, involved in a typical clinical study. A host of other issues—some
financial, others medical, and many administrative—must be considered in
the design and execution of every clinical trial.
Phase 1 Clinical Trial: First-In-Human Study
A Phase 1 study represents the first time a biopharmaceutical is used in
human. Phase 1 studies focus largely on safety and tolerability of the prod-
uct but may also include measurement of efficacy end points. The num-
bers of subjects enrolled in a Phase 1 study is small, usually less than 50
and often less than 25. A sponsor may elect to do several Phase 1 studies
(i.e., Phase 1a, Phase 1b, etc.) in sequence, each focusing on a particular

401Clinical Trials
scientific question. This is often the case with complex and novel biotech-
nology products. Phase 1 trials may be conducted on an outpatient basis
or an impatient basis, or both. In the case of a Phase 1 clinical research
unit, healthy volunteers may be required to remain in the onsite clinic for a
few days, weeks, or, sometimes, a couple of months during an early phase
dosing study, which requires a strict time-sensitive administration and
sample collection schedule. In these cases, volunteers are compensated for
their time and typically reasonable living accommodations are provided.
An initial Phase 1 study is usually conducted with healthy individuals.
Exceptions are the products that have an excellent safety profile and are
intended to treat life-threatening diseases, such as a study of a gene ther-
apy to treat rapid progression of a cancer or the study of an antiviral agent
to treat a chronic infection such as human immunodeficiency virus infec-
tion. In such cases, actual patients, having exhausted all traditional thera-
pies, are enrolled into a Phase 1 study.
The design of a Phase 1 study may be open-label, meaning that both patient
and investigative staff members know the nature of the treatment (i.e., inves-
tigational product or placebo) when it is given or it may be blinded or double
blinded, in which case a placebo (sugar pill) is given to one group of subjects,
without this knowledge being disclosed. Product dose may be escalated in
Phase 1 studies, but the scheme is quite conservative and only a few indi-
viduals are enrolled in each dosing group. Indeed, standard dosing schemes,
such as single rising dose or multiple rising doses, are selected for each new
biotechnology product. These study designs are shown in Figures 9.2 and 9.3,
respectively. In a single rising dose study, subjects are randomly assigned to
groups, perhaps five to ten subjects per group. The lowest dose is given to
subjects in the first group and the next (higher) dose is given to individuals
in the second group. The process continues, until the highest dose is reached,
which is determined from toxicology study results as the maximum toler-
ated dose. In a multiple rising dose design, the dose is constant for any given
individual but the individual returns to the clinical trial site to receive an
additional dose or doses. In the interest of safety and to monitor the possibil-
ity that a particular dose might result in acute reactions, only two or three
subjects in a group may be dosed with the product. This is done hours or even
days before the remainder of individuals assigned to this group are dosed in
the same manner.
Phase 1 trial measurements focus on safety end points, but measures of
efficacy are typically performed whenever possible. Subjects may be kept
in a clinic for days or even weeks after the treatment, so that they can be
carefully evaluated at specified intervals. For example, frequent physical
examination of subjects, use of electrocardiograms to identify changes in the
heartbeat, and regular clinical laboratory testing are the hallmarks of Phase
1 studies of novel biotechnology products. Criteria for ending the treatment
or dosing of human subjects whenever an SAE or multiple AEs are identified,
that is, stopping rules, are very important elements of Phase 1 studies.

402 Biotechnology Operations
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403Clinical Trials
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404 Biotechnology Operations
Clinical Pharmacology Studies of Biopharmaceuticals in Human
Additional studies are often required to fully understand a biotechnology
product before it can enter Phase 3 trials. These studies are often given cre-
ative and complex numbers and letters, such as Phase 1c or Phase 2a by their
sponsors. Although numbered, each study is designed to specifically sup-
port the overall clinical development plan and is best referred to by its pur-
pose (e.g., Pharmacokinetic Study in Normal Adults).
Pharmacokinetic studies are almost always performed if a new biotechnol-
ogy product is to be given repeatedly or in significant amounts. In PK studies
with human subjects, the product is given in a carefully controlled manner
and then blood or another body fluid is taken at regular intervals after dos-
ing, and these samples are tested to determine the half-life of the product
in circulation. Although PK studies may be a part of the Phase 1 or Phase
2 investigations, they are also performed as standalone studies, designed
strictly for that purpose.
Pharmacokinetic studies have been best developed for drugs, but they are
used extensively for studies with monoclonal antibodies and other biophar-
maceuticals intended for distribution throughout the body. Pharmacokinetic
and pharmacodynamic studies are further described in Chapter 8; they are
typically performed in animals during nonclinical studies before they are
conducted in human.
Mass balance studies are designed to determine where in the human body
a new biotechnology product goes and how long it remains in each location.
To perform these studies, product may be labeled with radioisotopes (hav-
ing extremely short half-lives) and then given. Product metabolism, excre-
tion and even localization in organs can then be followed with radiometric
devices. For biotechnology products that target a particular tissue, mass
balance studies may be performed in conjunction with imaging methods
that allow the molecule to be identified in a particular organ. For example,
it would be important to understand if a molecule aimed at cancerous cells
in lung bound largely to the tumor mass and not to critical and unaffected
organs, such as heart or kidney.
Food effect studies determine whether a particular type or amount of food
has an effect on the uptake and effectiveness of a new biopharmaceutical. It
is from food effect studies that we learn whether a patient should ingest a
product on a full or an empty stomach. Although quite important for orally
ingested products, such as many drugs, food effect studies may also help to
explain PK observations of biopharmaceutical products, such as unexpected
patterns of excretion or binding to components of serum.
Additional Phase 1 studies may focus on subpopulations, such as a racial or
geographic population, the elderly, adolescents, or children. Controlled stud-
ies may also be performed to determine whether a biotechnology product
will have greater or lesser effect when taken with another drug. These con-
comitant medication studies measure the effects of drug-drug interactions.

405Clinical Trials
Some classes of products are known to cause very unique types of reactions
and these may be studied in more detail with additional pharmacology stud-
ies in human subjects.
Phase 2 Clinical Trial: Proof-of-Concept Study
The second phase of clinical development includes one or more therapeutic
exploratory or proof-of-concept studies, referred to as such because they are
designed to provide sufficient data to suggest that a biopharmaceutical prod-
uct may well have the intended effect. Phase 2 studies may be dose ranging
and demonstrate the dose that is optimal to take forward into later studies.
With certain other Phase 2 trial designs, the intention is to determine the
minimal effective dose, or threshold effect of the biopharmaceutical. Another
intention of Phase 2 study may be to determine the maximum effective toler-
ated dose, at least within the dosing criteria identified in Phase 1. In addition,
Phase 2 study designs may examine various end points and measurements
for those end points, searching for ones that will provide the best estimate of
drug safety and efficacy in subsequent studies.
Phase 2 studies are often performed at five or more clinical study centers
( multicenter study) because there is a need of more patients—50 to 500 is a
typical number—with a single disease and to determine whether results vary
by study site. A single center cannot often recruit these many qualified indi-
viduals. Thus, Phase 2 studies are typically multiarm studies, designed with
several arms or groups (cohorts) of patients, each receiving a set dose level of
the product. Whenever possible, Phase 2 studies are placebo controlled and
double blinded, meaning that neither the patient nor the investigator and staff
knows which treatment or placebo a patient has received. Note that in cases
where studies require a patient population (diseased individuals), the control
group is not kept from treatment; in place of placebo, the current standard of
care is provided, which offers a benchmark for the treatment arm (investiga-
tional product). If the treatment involves multiple doses of a product, as is
often the case, the treatment period will be much longer in Phase 2 then it was
in Phase 1, so as to determine a more realistic effect of the biopharmaceutical
on both safety and efficacy. Hence, Phase 2 provides several benefits beyond
evaluation of product safety and efficacy. It is a means of determining whether
enough patients with the condition exist, so that a larger definitive study could
be conducted. Another ancillary benefit of Phase 2 studies, those performed at
more than one site by several teams of investigators, is a real-world evaluation
of each site. In addition, the range of subjects’ medical conditions enrolled in a
Phase 2 study may be very informative for developing a Phase 3 study design.
However, results from Phase 2 studies are seldom definitive because they
do not enroll enough patients to absolutely demonstrate safety, tolerability,
and efficacy. Some argue that a Phase 2 clinical study is actually a mini-
Phase 3, that is, a rehearsal for the pivotal study, and therefore, the results
matter greatly for business development and the decision to move forward.

406 Biotechnology Operations
Others suggest that Phase 2 is often not predictive of the outcomes in Phase
3 but is a means of determining the best dosing regimens and a valuable
lesson. Whatever the case may be for a given biopharmaceutical, Phase 2 is
an important step in the clinical development of any product, and therefore,
each study must be carefully designed and executed, with thorough analysis
and discussion of the results.
Phase 3 Clinical Trial: Therapeutic Confirmatory
After successful completion of Phase 2, the sponsor will almost certainly
hold a meeting with regulatory authorities to discuss findings and to pro-
pose the design of a pivotal or Phase 3 human clinical study. Discussions
between sponsor and agency typically follow this meeting, and within a few
weeks, both parties should agree on the design of the all-important Phase
3 or pivotal clinical trial for the biopharmaceutical. Phase 3 studies are also
referred to as adequate and well controlled, as they must be just that. They
are, in fact, the study based on which the product will be registered and
labeling claims will be supported.
Phase 3 trials are carefully considered, with significant input from medical
experts, statisticians, and those who manage and operate the study. Phase 3
studies are always large and multicenter, and today, most of these studies
are multinational. Some drugs are tested in two Phase 3 trials, both using
the same product and indication. The study is statistically powered, that is, it
includes enough patients, so that definitive answers as to safety, tolerability,
and efficacy of product can be obtained from a single study. Placebo or com-
parator is typically used and double blinding and other means of preventing
bias are always included in design, where possible.
The adaptive design may also be considered, with regulatory agency con-
currence, for mid- and late-stage clinical trials of certain products. Adaptive
design means that changes may be made in the design of a clinical study if
such change is guided by examination of data, accumulated at a particular
interim milestone. An adaptive design can reduce the duration of a study
or decrease the total number of patients required, and because it is based
on recent information and experience, it can enhance the value of data that
are generated by study completion. Although the greatest interest in adap-
tive design has been with adequate and well-controlled late-stage (Phase 3)
studies, this approach has also worked well with ascending-dose or other
mid-stage studies. However, there are caveats. Adaptive clinical study
designs are prospective and must be carefully considered with regulatory
authorities before initiation. As noted above, the interim analysis on which
change is based is itself carefully selected and protocol revisions are previ-
ously planned, and certain changes may not be acceptable under any cir-
cumstances. Nonetheless, given a wide range of acceptable design changes,
the adaptive design offers numerous opportunities when properly planned
and applied.

407Clinical Trials
As one might imagine, no matter what the size or design, a Phase 3 study
can take years to complete and generates millions of data points and huge
volumes of source documents as well as IC, CRFs, and other documents.
These studies are big and expensive. With studies of some rare diseases,
the numbers may be low because the number of patients and geographic
locations is limited. Large Phase 3 studies typically require an Operations
Manual to ensure that all aspects of the study are performed exactly the
same way at each clinical site. A manual also serves to resolve problems as
they arise and to facilitate communication and good medical and adminis-
trative practices.
An independent committee, referred to as the Data and Safety Monitoring
Board (DSMB), is included in the design of most Phase 3 studies. This board
of experts is unblinded to the treatment at established intervals. The DSMB
may do interim statistical analyses of the data to determine if the product
seems to be working and is safe. For example, the DSMB could, early in a
study, discover a distressingly large number of SAEs; in this case, it may ask
that the study be halted because the risks to patients outweigh the possible
benefits. In other instances, the DSMB might discover early in the study that
the biopharmaceutical is safe and quite efficacious, thus recommending that
it should not be withheld from the subjects in the placebo or control group.
Phase 4 Clinical Study and Risk Evaluation and Mitigation Strategy
When a biopharmaceutical has been approved for marketing by regulatory
authorities, it is not unusual for the agency to ask that an extended, open-
label study be performed in the postmarket approval period. Such a study,
sometimes referred to as therapeutic use, Phase 4, or postmarketing, is con-
ducted and financed by the sponsor. Biotechnology firms usually welcome
the suggestion of Phase 4 studies, because it means that their drug could be
approved in an expanded patient population without the need for additional
Phase 3 trials. This result is called conditional approval. The firm receives mar-
keting approval and may charge patients for the product, thus generating
income but with the understanding that one or more Phase 4 studies will be
conducted by the sponsor and in consideration of regulatory agency guide-
lines. Product safety and efficacy may be definitively demonstrated during
Phase 4. If product safety and efficacy are not demonstrated during this
period, the regulatory authorities have grounds to pull the market approval
and, hence, the derivation of conditional approval. In addition, extended test-
ing for drug-drug interactions, effects in special or high-risk populations,
and additional safety surveillance are considered for Phase 4.
Phase 4 studies are intended to reduce the risk to consumers from newly
marketed biopharmaceuticals. The FDA has the authority through the
FDA Amendments Act of 2007 to require risk evaluation and mitigation
strategy (REMS) for all newly licensed products. This is intended to ensure
that benefits to patients outweigh the risks after market approval under a

408 Biotechnology Operations
Biologics License Application or New Drug Application. Risk evaluation
and mitigation strategy considers patient population, condition severity,
benefit, duration of treatment, seriousness of AE, and historic safety record.
Although REMS does not involve additional Phase 4 studies, it does include
specific clinical guidelines that sponsors are required to generate based on
the clinical use of their product. These include a communication plan to edu-
cate, inform, and raise awareness of the associated risks. Some REMS require
elements to ensure safe use (ETASU), which also include a medication guide
for health care providers and information and instructions on safe use for
prescribers, dispensers (pharmacists), and users of selected products. If an
ETASU is required, it is dispensed with the product. Risk evaluation and
mitigation strategy thus represents a novel approach to use clinical informa-
tion to ensure safety of already licensed products by health care profession-
als and the general public. Establishment of REMS is issued outside of the
product label, which has helped to get and/or keep products on the market
by monitoring or mitigating the known product-associated risks. A list of
approved REMS is available on the FDA website. Again, the purpose and
directive of the REMS program is to ensure safe use of a product, which may
require follow-up testing or monitoring of the end user.
Clinical Trials for New Populations or Indications
Given the expense and complexity of performing any clinical study, it is
impossible to expect a firm to test, in the initial pivotal trial, every special
population that might benefit from the biopharmaceutical. Special popula-
tions may include the elderly, infants, children, adolescents, pregnant or lac-
tating women, and certain racial, ethnic, or geographic minorities. Yet, other
populations of individuals, notably those with an underlying disease such as
liver or lung disease or impaired kidney function, are also difficult to study
in initial Phase 2 and Phase 3 clinical studies. Some would argue that this dis-
criminates against such populations because they have no chance to benefit
from the product immediately after market approval. However, it is impos-
sible to study each group of individuals in the pivotal Phase 3, because of
resource and time constraints.
How does a biotechnology firm go about testing individuals of any popu-
lation when pursuing a new indication? The answer is to perform another
Phase 3 trial in that new population or with another indication. It is often
possible to begin these Phase 3 studies at Phase 2 or after having performed
small Phase 1 and Phase 2 studies. Assuming that the product has market
approval for at least one indication or one patient population (the first label-
ing claim), these postmarketing clinical trials may help the sponsor to market
a biotechnology product under a second labeling claim and thus bring benefit
to patients currently without access to that product. These Phase 3 studies,
focused on broadening the indication for the product by testing it in new
populations, for new indications, or, for example, applying novel methods

409Clinical Trials
or routes of administration, bring certain risk to the sponsor, as a study may
uncover previously unknown safety issues such as side effects of the biophar-
maceutical. This can lead to undesirable regulatory action, such as addition of
warnings to current labels or the need to begin more clinical trials.
Indeed, biotechnology firms are sometimes encouraged by a number of
programs, sponsored by FDA or other public health agencies such as the
National Institutes of Health (Chapter 4), to test a product as soon as pos-
sible, typically post licensure, for as many special populations as might ben-
efit from the product. Such studies are typically done post market approval,
not as Phase 4 studies but as Phase 2 or small Phase 3 studies in scope and
design. If they successfully demonstrate safety and efficacy in a special pop-
ulation, these studies, if adequate and well designed, may be the basis for
additional labeling claims for a biopharmaceutical product.
Global Clinical Trials
Many late-stage clinical trials are performed in countries distant from the
sponsor’s location. Indeed, today, it is common to place multicenter clini-
cal trials in numerous countries and to perform specialty studies and even
Phase 1 studies in a foreign country. The sponsor often finds such efforts
save significant time and money. However, there are caveats. Cultural and
regulatory differences can confound even the best planned global efforts.
In addition, there are the issues of different medical standards of care and
genetic differences in various populations. Some, but certainly not all, global
clinical trials are managed by large CROs, organizations that maintain clini-
cal trial facilities in many countries, and thus understand the language, cus-
toms, regulatory environment, medical practices, and population genetics
in many countries where they have offices and local national employees.
Foreign and multicenter global clinical trials are certainly possible and desir-
able but require considerable planning and assistance.
Quality Systems for Clinical Trials: Current Good
Clinical Practices
A quality system, cGCP, is applied throughout the clinical study process, from
preparation of a clinical plan to completion of the clinical study report. Why
must study integrity and quality be maintained at such high levels for clini-
cal trials? First and foremost, it is the right of each human subject. When a
volunteer enrolls in a study, he or she is subjected to a certain degree of risk
or potential risk. Owing to this, and with no guarantee of benefit, the subject
has the right to know that the study will, in the end, provide meaningful sci-
entific results, and certainly a correct answer, regarding the safety and efficacy

410 Biotechnology Operations
of a biopharmaceutical. Hence, maintaining high quality and integrity of the
study ensures that meaningful answers are achieved and that all rights of
human subjects are met. Financial responsibility, especially meeting the study
budget, is another reason to complete a study properly. Studies are expensive
and only few biotechnology firms have the resources to repeat a clinical trial.
Indeed, a single clinical study often means the difference between success and
failure of a biotechnology firm, and this alone is a compelling reason to get it
right the first time in a clinical research. An outline of the important elements
to achieve GCP in a Phase 1 human clinical study is provided in Box 9.8.
Under cGCP, certain systems and procedures are applied to clinical trials
to ensure the well-being of subjects, data integrity, overall quality, and suc-
cess of the trial. Some examples are as follows:
• Careful planning before the study and coordination during and after
the study
• Selecting proven clinical sites and investigators
• Training study staff to follow the protocol and other study docu-
ments and to accurately record and transfer data
• Ensuring quality and integrity of data by using time-proven methods
• One hundred percent internal audit of data sets and records
• Clean and screen all data entries on all documents to examine quality
and consistency of data, as it is transferred to a database
These and many other practices regarding the integrity of clinical trial data
are embodied in the principles and practices of cGCP. The remainder of this
section will focus on four clinical trial quality practices that are very impor-
tant to quality and compliance with cGCP.
Quality and cGCP in Clinical Trial Operations
Current Good Clinical Practice is further defined by ICH as “an international
ethical and scientific quality standard for designing, conduction, recording,
and reporting (clinical) trials that involve the participation of human sub-
jects” (ICH 1996). Compliance with cGCP provides assurance that the data
and reported results are credible and accurate and that the rights, integrity,
and confidentiality of human subjects are protected. The extensive ICH
(Chapter 4) guideline, “Good Clinical Practices,” is the international standard
for quality in clinical research. It is also adopted by most countries with a
developed regulatory agency, and most of these countries have supplemen-
tal regulations and guidance for conduct of studies in human subjects. The
cGCP in the U.S., outlined in Chapter 4, is further defined by several federal
regulations, notably 21 CFR, Parts 50, 54, 56, 312, 314, 812, and 814, which col-
lectively provide extensive guidance in this country. The U.S. government

411Clinical Trials
BOX 9.8 OUTLINE OF GOOD CLINICAL PRACTICE FOR
A PHASE 1 HUMAN CLINICAL STUDY—EXAMPLE
1. Subject
a. Well-being—should not only qualify for the study but also
be a good candidate and the study should not create undue
harm if the subject decided to participate in the study
b. Rights, integrity, and confidentiality—to participate or not
participate and to understand the study risks and poten-
tial benefits and alternatives to participation, shared result
information, compensation, consequences of withdrawing
from study, any additional costs resulting from participa-
tion, contact information of responsible individual in case of
any questions or concerns
c. Informed consent—need time to decide; understand study
purpose, duration, and procedures; ask questions; sign;
and receive a copy of the completed document.
2. IRB review and/or approval
3. Clinical investigator
a. Qualified to assume the responsibility of conducting clini-
cal study
b. Understands the investigational product and its appropri-
ate use
c. Willing to comply with GCP and applicable regulations
d. Willing to participate and prepare for audits and monitoring
e. Maintains a delegation of responsibilities log
f. Has adequate resources to recruit subjects, time to com-
plete the study, qualified staff, and facilities to conduct the
study
g. Obtains written approval from regulatory authorities (e.g.,
FDA and IRB)
h. Conducts the clinical research in compliance with the clinical
protocol
i. Documents and explains all/any deviations
j. Responsible for investigational product use, storage, and
disposal
4. Monitoring
(Continued)

412 Biotechnology Operations
has accepted the ICH guidelines and the U.S. and ICH systems are currently
harmonized.
Management responsibility, a critical hallmark of any quality system, is
clearly identified in cGCP. The ICH guideline and FDA regulations identify
responsibilities of the sponsor and the PI. In the United States, cGCP allows
a sponsor to transfer certain responsibilities to an investigator, an institution
(e.g., an university), or a commercial entity such as a CRO, but this trans-
fer must be made in writing and should be clearly described. Furthermore,
the guidelines state that any responsibilities not transferred in writing to an
investigator, institution, or CRO are assumed by the sponsor. Thus, cGCP
demands management responsibility and vendor and consultant control.
Control of the clinical trial process is clearly mandated by cGCP, and this is
done in a number of ways. As described earlier, written guidance, for exam-
ple, the clinical protocol or an Operations Manual, directs the clinical trial
processes. In addition, standard operating procedures are commonly applied
for routine tasks performed in support of a clinical trial. Procedures and data
are carefully documented in source documents (e.g., medical records), CRFs,
and electronic databases. These documents also contain the provision of a
proper environment for both investigational product and clinical processes.
For example, cGCP provides for product identification and traceability and for
BOX 9.8 (Continued) OUTLINE OF GOOD CLINICAL PRACTICE
FOR A PHASE 1 HUMAN CLINICAL STUDY—EXAMPLE
5. Reporting
a. Written annual reports (e.g., FDA and IRB)
b. Safety reporting
c. Result reporting
d. Study completion or termination
6. Documentation
a. Regulatory FDA/IRB/other local of federal regulatory
bodies
b. Informed consent and consent process
c. Clinical protocol
d. Study procedures
e. Product accountability log
f. Source documents and case report form
g. Record archival process and storage requirements
h. Training records, certificates, CVs, licenses, and so on
7. Written reports

413Clinical Trials
inspection or testing, thus meeting the cGCP requirement that investigational
product be clearly labeled and that the dosage form be clearly identified. The
sponsor typically assumes the responsibility for delivering a quality biophar-
maceutical to the clinical site and then the investigator assumes responsibility
for maintaining the integrity of that material and ensuring that each patient
receives the correct product (e.g., placebo or active product).
Current Good Clinical Practices apply to virtually every operational
aspect of a study. As noted earlier, responsibilities are clearly defined in
writing. A case in point is control of a nonconforming study. Clinical prac-
tices recognize that, despite the best of intentions and controls, there is a
high probability that mistakes are made over the course of the study. These
situations are referred to as noncomplaince with the protocol, the standard
operating procedures, cGCP, and/or applicable regulatory requirements.
Current Good Clinical Practices guidelines establish the need for self-
reporting, auditing, and careful review by several parties of all documents.
Most importantly, noncompliance must be reported and then corrective and
preventive action must be taken. With regards to preventive actions, GCPs
allow for changes in processes, as described in study documents, but they
also demand that change be controlled, reviewed, and approved by respon-
sible individuals.
The performance of clinical trials requires that all study staff have the
appropriate education and experience and are properly trained. Earlier, the
sponsor’s responsibility to select only qualified investigators and institutions
to perform clinical studies was mentioned. However, the quality require-
ment does not stop there. The sponsor is ultimately responsible for ensuring
proper education, experience, and training of individuals in the clinical labo-
ratory, in statistical group, and at any CRO. All professional staff must fully
understand the protocol and IB and know their respective professional roles
and responsibilities under the protocol.
Customer concerns and complaints, another hallmark of quality, focus on
the satisfaction of both human subjects and, in the case of contract studies,
the sponsor. As noted in cGCP, “the rights, safety and well-being of trial sub-
jects are the most important consideration and must prevail over interests
of science or the study staff, and society” (ICH, 1996). Everyone involved in
a clinical trial must, at all times, consider the rights and well-being of each
subject. Such consideration does not end with signing the CF but contin-
ues to the end of the study. Indeed, for some studies of novel biotechnology
products, responsibility for well-being of a subject extends through the life-
time of that person.
Integrity of Clinical Study Data and Documents
Data collection and control are important under cGCP. The data presented in
a CSR accurately reflect the information that was recorded in source docu-
ments during the study. The objective is the completeness and accuracy, that

414 Biotechnology Operations
is, all data points should be collected for every patient enrolled. Diligence is
taken by the investigator, sponsor, and others in handling, analyzing, and
reporting data.
Although 100% complete/0% errors is the goal, a number of seemingly
unavoidable problems can occur and data points may be corrupted, ques-
tionable, or missing from even the best designed and managed clinical study.
For example, patients may fail to meet appointments or they may drop out of
the study all together. This can be tolerated to some degree, but if too many
patients leave the study or fail to comply with follow-up visits, it may not
be adequate and well controlled and the overall study results are open to
question. In addition, the investigative staff will inevitably make errors when
entering data into source documents or transferring information from source
documents to CRFs or electronic databases. A high error rate can invalidate
a study. Serious violations of a protocol may occur if subjects are enrolled in
a study without meeting eligibility criteria or completing the IC process or if
there is insufficient documentation of the consent process. In such cases, the
data set may be considered incomplete. One patient missing a single dose of
product will not invalidate a complete data set, but when several patients
miss a dose or if a few patients miss several doses, the integrity of that study
will, at the very least, be flawed. These are but a few examples of why cGCP
stresses the importance of excellent study management and performance.
Monitoring and Auditing Clinical Trials
Clinical monitoring, not to be confused with the medical (safety) monitor, is
the process of overseeing all aspects of a clinical trial and is a responsibility
of the sponsor. Monitoring begins when the first subject is enrolled and ends
when the last subject is discharged from the study. Monitoring ensures that
the study is performed in accordance with cGCP, the study documents, nota-
bly the protocol, and other regulatory requirements. It is a big job to monitor
even a small clinical trial, and it is a tremendous effort to properly monitor a
Phase 3 study. However, monitoring is essential for every study.
Auditing, also described in Chapter 5, is a systematic examination of study
processes and documents. An important part of monitoring, it carries with
it a function of determining whether particular activities are being per-
formed correctly. Auditing normally involves the careful review of clinical
trial documents to ensure that they are correctly completed according to the
instructions in the protocol. Auditors, also referred to as clinical research
associates, are the individuals who perform the audits. The task of auditing a
clinical trial is very detail oriented and analytical. Auditors visit clinical trial
sites, where they review documents, speak with the investigational staff, and
identify issues or problems. In many cases, they assist in resolving those
issues by speaking with the PI and sometimes performing staff training.
Thus, auditors perform important roles in the overall monitoring process
and ensure the integrity of a study and compliance with cGCP.

415Clinical Trials
Ethical Behavior and the Well-Being of Clinical Trial Subjects
The Declaration of Helsinki, as noted earlier, is the foundation for protec-
tion of human volunteers in any study. It holds clinical research to an excep-
tionally high ethical standard, stating that, “Compliance provides public
assurance that the rights, well-being, and confidentiality of trial subjects
are protected and that the clinical trial data is credible” (ICH, 1996). This
is totally appropriate, and cGCP directly supports each principle in the
Declaration. Over the years, a number of human rights issues have arisen
in clinical studies, even as the clinical research community applied cGCP
to thousands of clinical research studies worldwide. The vast majority of
human research studies are, however, without breaches of ethical behavior,
suggesting that the clinical research community and cGCPs are doing an
outstanding job of maintaining the principles laid down in the Declaration.
Unfortunately, more ethical issues may arise in the future. Without further
delving into ethical behavior in clinical studies, some examples of common
ethical situations faced by biotechnology firms are worth mentioning as a
close to this chapter and, perhaps, as word of caution to those entering the
field of biopharmaceutical development.
Some years ago, as clinical research expanded to support development
of biotechnology products, clinical investigators in private practice or
associated with nonprofit institutions accepted stock options in return for
providing clinical investigative services for the biotechnology firm. This
seemed like a useful model in the beginning, as biotechnology firms essen-
tially deferred compensation, thus saving themselves considerable upfront
expenses. However, it was also felt that this practice was a potential conflict
of interest and regulatory agencies argued that at the least it should be fully
disclosed by both investigator and sponsor. Others asked how this differed
from the accepted practice of employees (of the sponsoring biotechnology
firm, including those staff members responsible for clinical monitoring)
accepting stock options from their employer. Although the issue has not
been fully resolved to everyone’s satisfaction, the consensus is that clinical
investigators must not hold significant interest in the sponsoring entity and
that any interest must be disclosed to regulatory authorities. Today, most
regulatory agencies demand full disclosure by outside investigators and the
indirect financial remuneration (e.g., stock options) of outside or independent
investigators is capped in some instances. This situation demonstrates how
important it is to ensure high ethical standards when dealing with human
subjects and the clinical study process.
In another example, regulatory authorities are authorized to blacklist
employees of the biopharmaceutical industries, preventing them from work-
ing in our industry if there is evidence that they egregiously or repeatedly
failed to comply with cGCP or other regulations. Although the practice of
individual sanctioning is applied by FDA to all areas of biotechnology devel-
opment, it is not uncommonly used to prevent certain clinical investigators,

416 Biotechnology Operations
those who repeatedly failed to adhere to cGCP or those who commit a major
infraction, from further participating in studies. Note that even though a
blacklisted investigator need not be convicted of a crime, his or her name
and affiliation still become a matter of public record. Although the clinical
research community may feel singled out by the practice of blacklisting, it
demonstrates how seriously we as a society take the rights of human subjects
and the sanctity of clinical trials as a means of ensuring a safe and effective
supply of biopharmaceuticals.
Another issue with ethical aspects is the problem of distinguishing clinical
research from medical treatment. The distinction between the two is often
blurred, and this challenges clinical researchers, worldwide, as they seek the
best treatment for patients. For example, a new biopharmaceutical product
to treat AIDS is taken to market after abbreviated clinical studies. On the
one hand, this is good, because it provides access to a seemingly promis-
ing treatment. On the other hand, it might also put users at potential risk
of using a product that has not been thoroughly tested for safety or efficacy
and this risk might have been mitigated if more extensive research had been
conducted.
Another example is the off-label use of a biopharmaceutical. Sometimes,
this occurs when it is quietly encouraged by biotechnology firms that wish
to increase sales. It also happens when well-meaning, but sometimes poorly
informed, physicians treat disease in an effort to save a patient from pain,
suffering, or even death. An important question is: in such cases, are the
patients being enrolled and treated in a clinical research study but without
full IC? If IC is the first principle of the Nuremberg Code, the Declaration of
Helsinki, and cGCP, then how can we justify such off-label use by individual
medical practitioners? Alternatively, does consent alone mean that a patient
is being treated ethically?
There is no simple answer to any of these examples. However, these types
of questions arise repeatedly in biotechnology and apply to many of today’s
most exciting biotechnology advances. There is no clear answer for every
type of clinical situation. Nonetheless, those in the biotechnology industry
face ethical issues, as they make difficult decisions on how to proceed into
clinical studies.
Summary on Clinical Trials
Clinical trials evaluate the safety and efficacy of a biopharmaceutical product
by testing that product in human subjects or volunteers. A clinical research
program—and every biopharmaceutical intended for use in humans must
have one—is based on the indication and patient population for the product
and requires careful planning and significant resources, both human and

417Clinical Trials
monetary. Importantly, clinical studies are observational or, more frequently,
experimental, and in either case, they are designed to test a hypothesis and
follow the scientific method. Clinical research of a biopharmaceutical is per-
formed in a series of three or four phases. Phase 1 is first time in human,
focusing on safety, that is, clinical toxicology and possible adverse reac-
tions, but yielding pharmacology and perhaps some efficacy data. Phase 2 is
designed to expand the dosing regimen, extend findings from the first phase,
and to establish a foundation for a pivotal or definitive study, Phase 3 is the
third phase and it confirms the safety profile and demonstrates therapeutic
or preventative efficacy. Postmarketing studies are considered Phase 4. Good
Clinical Practices are followed throughout clinical development; indeed, reg-
ulatory authorities will not approve data generated by a clinical trial unless
data are scientifically sound and in compliance with GCPs. Hence, clinical
development planning is a necessity from the outset, and counterintuitively
since clinical studies are in late stages of development, the clinical plan is
often the first step in an overall PDP. Clinical trials involve a number of indi-
viduals, each with unique skills and management to integrate their efforts,
and a large number of clinical trial documents, notably the protocol, must
be prepared. A PI is the key figure in a trial, but of course, there are clinical
support staff, the sponsor, and human subjects. The clinical protocol is an
instructive document, exactly and fully describing why and how a clinical
study is to be performed using these participants. Investigational product,
or a placebo, is provided by the sponsor and then given by the investigator
to each subject. Ethical behavior on the part of each participant is critical,
and the well-being of human subjects is the primary objective of any clini-
cal trial. Volunteers are enrolled in a study only after they give IC to par-
ticipate, and this process is reviewed and monitored by an IRB. Subjects are
followed throughout the trial, and AEs and detailed data about the condition
of each subject are recorded and, eventually, reported to the sponsor and to
regulatory agencies in clinical trial reports. The clinical program, which is
applied to the overall development program, is a large operation in and of
itself and must be fully managed and integrated into the overall develop-
ment program.
Reference
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Registration of Pharmaceuticals for Human Use (ICH). 1996. Guidance for
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419
Additional Readings
Chapter 1: Introduction to Biotechnology
Operations: Planning for Success
Billups NF. 2015. American Drug Index. Wolters Kluwer, St. Louis.
Dorland WAN. 2011. Dorland’s Illustrated Medical Dictionary. 32nd ed. Elsevier, New
York.
Food and Drug Administration (FDA). 2016. Approved Drug Products with Therapeutic
Equivalence Evaluation (The Orange Book). US Food and Drug Administration,
Rockville, MD; http://www.accessdata.fda.gov/scripts/cder/ob/ (accessed
May 31, 2016).
Walsh G. 2003. Biopharmaceuticals: Biochemistry and Biotechnology. John Wiley and
Sons, Chichester, UK.
FDA. 2016. National Drug Code Directory (NDC). US Food and Drug Administration,
Rockville, MD; http://www.fda.gov/Drugs/InformationOnDrugs/ucm142438.
htm (accessed May 31, 2016).
Ho RJY. 2013. Biotechnology and Biopharmaceuticals: Transforming Proteins and Genes
with Drugs. 2nd ed. Wiley-Blackwell, NY.
Jameel F, Hershenson S, Khan M, and Martin-Moe S. 2015. Quality by Design for
Biopharmaceutical Drug Product Development. Springer, NY.
Krinsky DL et al. (eds). 2015. Handbook of Nonprescription Drugs: An Interactive Approach
to Self-Care 18th ed. American Pharmacists Association, Washington DC.
The Merck Index. 2013. The Merck Index. 15th ed. Merck & Co., Whitehouse
Station, NJ.
Mosby. 2015. Mosby’s Drug Reference for Health Professionals. 5th ed. Elsevier Mosby,
St. Louis, MO.
Sobti RC. 2008. Essentials of Biotechnology. CRC Press, Boca Raton, FL.
Thompson Healthcare. 2010. PDR Drug Interactions and Side Effects. Thompson
Healthcare, Florence, KY.
Thompson Healthcare. 2015. PDR (Physician’s Desk Reference) for Nonprescription
Drugs, Dietary Supplements and Herbs. Definitions Guide to Over the Counter (OTC)
Medicines. Thompson Healthcare, Florence, KY.
Thompson Healthcare. 2016. Physician’s Desk Reference (PDR). Thompson Healthcare,
Florence, KY.
Thompson Healthcare. 2016. Physician’s Desk Reference (PDR) Generics. Thompson
Healthcare, Florence, MO.

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420 Additional Readings
Chapter 2: Project Management
Babler SD and Ekins S. 2010. Pharmaceutical and Biomedical Project Management in a
Changing Global Environment. Wiley, NY.
Brown L and Grundy T. 2011. Project Management for the Pharmaceutical Industry.
Gower Publishers, Aldershot, UK.
Harpum P. 2010. Portfolio, Program and Project Management in the Pharmaceutical and
Biotechnology Industries. Wiley, NY.
Project Management Institute. 2013. A Guide to the Project Management Body of
Knowledge (PMBoK Guide) 20. Project Management Institute, Newtown
Square, PA.
Wingate LM. 2014. Project Management for Research and Development. Auerbach
Publications, Boca Raton, FL.
Chapters 3 and 4: Regulatory Affairs
and Regulatory Compliance
Adams DT. et al. 2008. Food and Drug Law & Regulation. FDLI, Washington, DC.
Danzis SD and Flannery EJ (eds). 2010. In vitro Diagnostics: The Complete Regulatory
Guide. FDLI, Washington, DC.
Humi RA. 2012. Pharmaceutical Competitive Intelligence for the Regulatory Affairs
Professional. Springer-Werlag, NY.
Kahan JS. 2008. Medical Device Development: Regulation and Law. Parexel International
Corporation, Waltham, MA.
Klincewicz SL et al. 2009. Global Pharmacovigilance Laws and Regulations: The Essential
Reference. FDLI, Washington, DC.
Mathieu M. 2004. Biologics Development: A Regulatory Overview. 3rd ed. Parexel
International Corporation, Waltham, MA.
Pines WL. 2003. How to Work with the FDA. FDLI, Washington, DC.
Pisano DJ and Mantus D (eds). 2008. FDA Regulatory Affairs: A Guide for Prescription
Drugs, Medical Devices, and Biologics. CRC Press. Boca Raton, FL.
RAPS. 2008. Fundamentals of EU Regulatory Affairs. 4th ed. Regulatory Affairs
Professionals Society, Rockville, MD.
RAPS. 2009. Fundamentals of US Regulatory Affairs. 6th ed. Regulatory Affairs
Professionals Society (RAPS), Rockville, MD.
RAPS. 2010. Fundamentals of International Regulatory Affairs. Regulatory Affairs
Professionals Society, Rockville, MD.
RAPS. 2010. US Regulatory Acronyms & Definitions Pocket Guide. 4th ed. Regulatory
Affairs Professionals Society, Rockville, MD.
FDA. 2016. What does FDA inspect? US Food and Drug Administration; http://www.
fda.gov/aboutfda/transparency/basics/ucm194888.htm (accessed May 31,
2016).

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421Additional Readings
Chapter 5: Quality Assurance
Avis KE et al. (eds). 1998. Biotechnology: Quality Assurance and Validation. CRC Press,
Boca Raton, FL.
Haider SI and Asif ES. 2012. Quality Operations Procedures for Pharmaceuticals, API and
Technology. CRC Press, Boca Raton, FL.
Jameel F, Hershenson S, Khan M, and Martin-Moe S. 2015. Quality by Design for
Biopharmaceutical Drug Product Development. Springer, NY.
Ogg G. 2005. A Practical Guide to Quality Management in Clinical Trial Research. CRC
Press, Boca Raton, FL.
Sandle T. 2015. Pharmaceutical Microbiology: Essentials for Quality Assurance and Quality
Control. Woodhead Publishing, Cambridge, UK.
World Health Organization. 2007. Quality Assurance of Pharmaceuticals: A Compendium.
WHO, Geneva.
Chapter 6: Biomanufacturing
Avis KE. et al. 1996. Biotechnology and Biopharmaceutical Manufacturing, Processing and
Preservation. CRC Press, Boca Raton, FL.
Berry IR (ed). 2003. Pharmaceutical Process Validation. Marcel Dekker, NY.
Doble M. 2004. Biotransformations and Bioprocess. CRC Press, Boca Raton, FL.
FDA. 2009. Guidance for Industry: Assay Development for Immunogenicity Testing of
Therapeutic Proteins. US Food and Drug Administration; http://www.fda.gov/
downloads/drugs/guidancecomplianceregulatoryinformation/guidances/
ucm192750 (accessed May 31, 2016).
FDA. 2014. Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein
Products. US Food and Drug Administration; http://www.fda.gov/down-
loads/drugs/guidancecomplianceregulatoryinformation/guidances/
ucm338856 (accessed May 31, 2016).
FDA. 2015. Guidance for Industry: Cell-Based Products for Animal Use. US Food and Drug
Administration; http://www.fda.gov/downloads/animalveterinary/guidance-
complianceenforcement/guidanceforindustry/ucm405679 (accessed May 31,
2016).
Fink DW Jr. 2009. FDA regulation of stem cell-based products. Science, 324(5935):
1662–3.
Haider SI. 2009. Biotechnology. A Comprehensive Training Guide for the Biotechnology
Industry. CRC Press, Boca Raton, FL.
Haider SI. 2010. Cleaning Validation Manual. A Comprehensive Guide for the Pharmaceutical
and Biotechnology Industries. CRC Press, Boca Raton, FL.
Jamel F and Hershenson S. 2010. Formulation and Process Development. Strategies for
Manufacturing Biopharmaceuticals. Wiley, NY.
Ozturk S. 2005. Cell Culture Technology for Pharmaceutical and Cell-Based Therapies. CRC
Press, Boca Raton, FL.

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422 Additional Readings
Sofer G. 2005. Process Validation in Manufacturing of Biopharmaceuticals. CRC Press,
Boca Raton, FL.
Zhong J-J. 2004. Biomanufacturing: Advances in Biochemical Engineering/Biotechnology.
Springer-Verlag, Berlin, Germany.
Chapter 7: Quality Control
Chakraborty C and Jhingan R. 2015. Protein Based Drugs: A Techno-Commercial
Approach. Hindawi Publishing Corporation, London, UK.
Haider SI and Syed EA. 2011. Quality Control Training Manual: Comprehensive Training
Guide for API, Finished Pharmaceuticals and Biotechnology. CRC Press, Boca Raton,
FL.
Rodriguez-Diaz R, Wehr T, and Tuck S. 2005. Analytical Techniques for Biopharmaceutical
Development. Taylor & Francis Group/CRC Press, Boca Raton, FL.
Sandle T. 2015. Pharmaceutical Microbiology: Essentials for Quality Assurance and Quality
Control. Woodhead Publishing Co., Cambridge, UK.
Chapter 8: Nonclinical Studies
Cavagnaro JA. 2008. Preclinical Safety Evaluation of Biopharmaceuticals. John Wiley &
Sons, Hoboken, NJ.
Choy WN. 2007. Genetic Toxicology and Cancer Risk Assessment. Marcel Dekker, Inc., NY.
Descotes J. 2004. Principles and Methods of Immunotoxicology. Elsevier, NY.
FDA. 2006. Guidance For Industry: Considerations for Developmental Toxicity Studies
for Preventive and Therapeutic Vaccines for Infectious Disease Indications.
US Food and Drug Administration; http://www.fda.gov/downloads/
Biolog icsBloodVacci nes/Gu ida nceCompl ia nceReg u lator yI n for mat ion/
Guidances/Vaccines/ucm092170 (accessed May 31, 2016).
FDA. 2015. Guidance For Industry: Product Development Under the Animal Rule. US
Food and Drug Administration; http://www.fda.gov/downloads/Drugs/
GuidanceComplianceRegulatoryInformation/Guidances/UCM399217
(accessed May 31, 2016).
Horvanth CJ. 2009. TeGenero incident and the duff report conclusions: A series of
unfortunate events or an avoidable event? Toxicologic Pathology, 37: 372–383.
Kapp RW. 2013. Reproductive Toxicity. Taylor & Francis Group, NY.
Meibohm B. 2007. Pharmacokinetics and Pharmacodynamics of Biotechnology Drugs.
Principles and Case Studies on Drug Development. Wiley-VCH, NY.
Timbrell JA. 2007. Introduction to Toxicology. 3rd ed. Taylor & Francis Group/CRC
Press, Boac Raton, FL.
Weinberg S. 2003. Good Laboratory Practice Regulations. 4th ed. 2003. Marcel Dekker,
NY.
Zhang L. 2016. Nonclinical Statistics for Pharmaceutical and Biotechnology Industries
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423Additional Readings
Chapter 9: Clinical Trials
Chin R, Yoonsik C, and Lee BY. 2008. Principles and Practices of Clinical Trials Medicine.
Elsevier, NY.
Cook TD and DeMets DL. 2007. Introduction to Statistical Methods for Clinical Trials.
Chapman Hall & CRC Press, Boca Raton, FL.
Hackshaw AK. 2009. A Concise Guide to Clinical Trials. Wiley-Blackwell, Oxford.
Piantadosi S. 2005. Clinical Trials: A Methodologic Perspective. John Wiley & Sons,
Chicester, UK.
Rozovsky FA and Adams RK. 2003. Clinical Trials & Human Research: A Practical Guide
to Regulatory Compliance. John Wiley & Sons, NY.
Stone J. 2006. Conducting Clinical Research: A Practical Guide for Physicians, Nurses,
Study Coordinators and Investigators. Mountainside MD Press, Cumberland, MD.

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425
Glossary
21 CFR: part 21 of the U.S. Code of Federal Regulations, the part in which
most food and drug laws are located (FDA, 2016).
483: See Form 483.
510(k) premarket notification process: a regulatory route by which to seek
marketing approval from FDA for a medical device of low to moder-
ate risk and substantial equivalence to another device.
abbreviated new drug application (ANDA): an application submitted to
FDA for approval of marketing a generic drug.
absorption, distribution, metabolism, and excretion (ADME): measure-
ments of a biopharmaceutical in pharmacokinetic studies.
absorption phase: the pharmacokinetic phase during which a biopharma-
ceutical is absorbed into the body and, presumably, into the blood.
accuracy: the measure of an assay to agree with a known true value.
act: legislation that begins as a bill before congress, and once passed by
congress, becomes law.
active (pharmaceutical) ingredient (API): the part of a product that has the
desired biological activity, providing the primary therapeutic and
biological effects.
acute toxicity: an animal safety study that examines the toxicity of a bio-
pharmaceutical after a single dose with short-term follow-up.
adaptive (study design): a study design that allows changes to be made in
the protocol at a milestone, if data warrants.
ADE: See adverse (drug) event.
ADME: See absorption, distribution, metabolism, and excretion.
adequate and well-controlled study: a scientific study that is carefully
designed to test a hypothesis and has proper controls for the
intended purpose.
adulterated: a biopharmaceutical or drug that is putrid, filthy, or decom-
posed; lacks strength, purity, or quality; is not of cGMP nature; or is
contained in a deficient container.
adverse (drug) event (AE or ADE): a medical event in a human subject that
is undesirable and symptomatic of a physiological change or disease
and is due to a particular intervention or treatment, such as use of a
biopharmaceutical.
AE: See adverse (drug) event.
American type culture collection (ATCC): a nonprofit organization that col-
lects, stores, and distributes biological reference samples.
analyte: material or product that is being tested.
analytical method: laboratory procedure or test performed on a product to
measure an attribute.

426 Glossary
analytical tool: laboratory procedure or test performed on a product to mea-
sure an attribute.
analyze: in project management, it means an assessment of achievement
relative to the project plan. It may include evaluation of alternatives
and resource requirements and usage.
ANDA: See abbreviated new drug application.
APHIS: animal and plant health inspection service of the U.S. Department
of Agriculture.
API: See active pharmaceutical ingredient.
ascending dose study: an experimental design in which the dose of investi-
gational product is raised with each subsequent group of volunteers.
aseptic: used as a noun, it means without living organisms. As a verb or
adverb, aseptic describes processes that avoid to a great degree the
inclusion of or contact with microbes.
assay: laboratory procedure or test performed on a product to measure an
attribute.
ATCC: See American type culture collection.
attribute: a positive, desirable, or even necessary characteristic of a product
that lends itself to testing.
audit: a formal review of a process, study, or product by an auditor; it exam-
ines whether actual performance was conducted in accordance with
established instructions.
batch: an amount of product that is produced together as a single entity.
Batch usually refers to a defined amount of biopharmaceutical bulk
substance.
batch production record (BPR): a document used in manufacturing to both
guide a process and record critical information regarding perfor-
mance on a particular batch or lot of product.
BDS: See bulk (drug) substance.
bias: a predisposition or prejudice in scientific studies or a systemic distor-
tion of a statistical result (Oxford English Dictionary, 1997).
BIO: biotechnology industry organization.
bioavailability: the fraction of biopharmaceutical, of the total amount given,
available in the blood (or tissue) and its intended effect.
bioequivalence: assessment of the comparative activity and bioavailability
of two products after administration to animals or humans.
biologic or biological: historical terms used to describe the products that are
derived from or represent biological or living sources.
biologics license application (BLA): an application made to FDA for the
purpose of gaining marketing approval for a new biological (non-
therapeutic and nonpharmaceutical) in the U.S. This large document
provides complete information on development of the product and
its safety and efficacy.
biomanufacture: manufacture or production of biological molecules, cells,
tissues, or other products derived from biotechnology.

427Glossary
biopharmaceutical: a biological molecule, cell, tissue, or other material
of biological origin used in the treatment or prevention of disease
in humans. Biopharmaceuticals are complex in a molecular sense
and most have a biological origin.
bioreactor: a closed vessel designed to support the multiplication and
growth of eukaryotic cells for the purpose of expanding a cell line
or producing a biopharmaceutical.
Biotechnology Regulatory Services (BRS): a division of APHIS, the US
Department of Agriculture, that regulates certain genetically engi-
neered organisms.
BIS: Bureau of Industry and Security, the US Department of Commerce.
BLA: See biologics license application.
blinded study: a clinical study design in which certain individuals, usu-
ally the volunteers and investigative staff, are unaware of the treat-
ment (investigational product, placebo, or comparator) given to the
volunteer.
BPR: See batch production record.
BRS: See biotechnology regulatory services.
BS: See bulk substance.
bulk (drug) substance (BS or BDS): biopharmaceutical product that has
been produced and purified but has not yet been formulated or ali-
quoted into the final container. Also referred to as bulk drug sub-
stance or BDS.
campaign (manufacture): manufacture of more than one product in a facil-
ity. Each manufacturing area is used only for one product at any
given time, so projects are sequential.
cap: a stopper or other seal that is placed as a seal on the container once it
has been filled.
CAPA: corrective and preventive action; it is the process of investigating and
correcting a deficiency, deviation, or other problem or issue in the
manufacturing or quality control of product.
carcinogenicity: the ability or tendency to invoke cancer.
case report form (CRF): paper or electronic form on which the investigator
enters medical information gathered during a clinical trial.
CBER: Center for Biologics Evaluation and Research, the U.S. Food and Drug
Administration.
CBP: Customs and Border Protection of the U.S. Department of Homeland
Security.
CDC: See Center for Disease Control and Prevention.
CDER: Center for Drug Evaluation and Research, the U.S. Food and Drug
Administration.
CDRH: Center for Devices and Radiological Health, the U.S. Food and Drug
Administration.
cell: production cell that replicates and has particular traits. In biomanufac-
ture, this is often of bacterial, yeast, insect, or mammalian origin but

428 Glossary
may be derived from almost any species: plant or animal, eukaryotic
or prokaryotic.
cell bank: a source of live cells, derived from a clone or small number of
progenitor cells, that are kept in storage and then used as the seed or
source in biomanufacture.
Center for Disease Control and Prevention (CDC): a U.S. federal public
health agency under the Department of Health and Human Services.
Certificate of Analysis (CoA): a formal document used to identify attributes
or traits, quality control tests, specifications, and test results of a
product or raw material.
CF: See informed consent form (ICF).
CFR: See code of Federal Regulations.
cGCP: current Good Clinical Practices are the regulations promulgated by
FDA and international bodies and must be followed for conduct of
research in human subjects.
cGLP: current Good Laboratory Practices are the regulations promulgated
by FDA and international bodies and must be followed for nonclini-
cal safety studies of all biopharmaceuticals.
cGMP: current Good Manufacturing Practices are the regulations promul-
gated by FDA and international bodies and must be followed for the
production (manufacture) and distribution of all biopharmaceuticals.
change control: an active process under which proposed changes are intro-
duced, examined, and acted upon according to plan and with full
knowledge of everyone involved or impacted by the change.
charter (team): a mandate and authorization to achieve, as a team, an objec-
tive. A charter is bestowed by a higher authority, such as a stake-
holder or executive manager.
chemistry, manufacturing, and controls (CMC): the information related to
the production, testing, and distribution of a pharmaceutical prod-
uct is contained in the CMC (or Pharmaceutical Quality) section of
an IND application to FDA.
CHMP: committee for human medical products of the EMEA. Prepares
opinions on questions concerning medications for human use.
chronic toxicity: a safety study in animals that measures the toxicity of a
biopharmaceutical given in multiple doses with follow-up over a
long period of time (>6 months).
classified: a formal designation regarding the level of air quality in a clean
area or room.
clean room (area): an area or room in a biomanufacturing facility that is
controlled to reduce the change of microbial or particulate contami-
nation of product.
clinical (study) design: a brief description of a clinical study that includes
the scientific approach and hypothesis, as well as ensuring quality
elements.
clinical research: See clinical trial.

429Glossary
clinical research associate (CRA): also called a clinical monitor and is part
of a medical research team associated with human clinical trials.
clinical research unit (CRU): a human clinical research medical facility
staffed with CRAs, nurses, and physicians, all focused on conduct-
ing human clinical research.
clinical study: See clinical trial.
clinical summary report (CSR): a written report that fully summarizes the
performance and results of a clinical trial, including tabulated data
and statistical analyses.
clinical trial: a designed scientific study in which a principal investigator
evaluates an investigational biopharmaceutical product in human
volunteers.
Cmax: the maximum amount of biopharmaceutical that is available in blood
or tissue after delivery of a given dose.
CMC: See chemistry, manufacturing, and controls.
CMO: See contract manufacturing organization.
CoA: See certificate of analysis.
code of Federal Regulations (CFR): compilation of all current US federal
regulations.
cohort: group of human volunteers with common characteristics and treated
at the same time, although not necessarily in the same manner.
combination product: a product that combines two or three of the following:
biological, drug, and medical device.
commercial production: biomanufacture of a product at the final or commer-
cial scale. Typically happens just before or after marketing approval
by a regulatory agency.
common technical document (CTD): a format for preparing, organizing,
and writing market applications and investigational new drug
applications in many countries. The eCTD is the electronic version
of the CTD.
comparative clinical study: clinical research in which the investigational
product is compared with another product.
comparator: a control material, typically a product that is licensed for the
same indication, used in a clinical trial to compare against the use of
investigational product.
compendium/compendial: a reference book that provides product, process,
and test standards and specifications.
components: materials that are used in manufacture. Often include hard-
ware materials.
concentration-effect relationship: the relationship between the concentra-
tion of a biopharmaceutical in the blood (or tissue) and the desired
physiological effect it has on an animal or human.
concept protocol: a brief design of a nonclinical or clinical study used as the
basis for discussions between sponsor, investigator, and regulatory
authorities. It is the foundation for preparing a full study protocol.

430 Glossary
conditional approval: regulatory approval for a biopharmaceutical, in which
the regulatory agency stipulates that certain tasks, often Phase 4
clinical studies or follow-up of patients from Phase 3 studies, must
be performed as a condition to that approval.
conformance: this means that a product or, in a broader sense, a study report
or other document meets specifications and regulations.
consent form (CF): format used to inform a human subject and document
the process of informed consent. See informed consent.
construct (genetic): a biological material that is or has been derived from
genetic engineering of a molecule or a cell. It usually refers to a
plasmid.
container: the vial or other vessel that directly holds a final product.
contaminant: particle or chemical that is undesirable and has entered the
product stream during manufacture.
contract manufacturing organization (CMO): a manufacturing facility that
performs biomanufacturing on a contract basis.
contract research organization (CRO): a corporation or institute that pro-
vides contractual support to a biopharmaceutical sponsor in areas of
clinical or nonclinical studies or manufacture and control.
control: in quality control, a material that is used to ensure performance of
an assay. It may be a positive or negative control.
control: in project management, it means to use influence to follow the cur-
rent plan or improve it.
control article: the nonactive material that is given to experimental animals
as a control and in lieu of active ingredient during a nonclinical
study.
controlled clinical research: a clinical trial in which both an investigational
product and one or more control substances, such as a placebo or a
comparator, are given to patients randomized into groups.
CPMP: committee for proprietary medicinal products of the EMEA.
CRA: See clinical research associate.
CRF: See case report form.
crimp: process of sealing or closing a cap onto a container of final product.
This is often done with metal bands or covers.
critical pathway: the pathway in a project that is critical to achieving objec-
tives and schedules. It is also called the rate-limiting pathway, as it
determines the rate at which the product is going forward.
CRO: See contract research organization.
CSR: See clinical study report.
CTD or eCTD: See common technical document.
CVM: Center for Veterinary Medicine, the US Food and Drug Administration.
cycle or life cycle (project): the overall project, from beginning to end, with
all elements included.
Data and Safety Monitoring Board (DSMB): committee of independent
experts to evaluate the data of an ongoing clinical trial.

431Glossary
decision points: precise or particular moments in a project schedule that
require consensus on a particular management or technical matter.
Declaration of Helsinki: a series of ethical principles used to govern the
rights and well-being of human subjects in human clinical research.
design control: a formal and documented system of plans and procedures
that are used to ensure the quality development of products or
processes.
deviation: it is a situation when a value or process does not meet established
procedures, rules, or specifications. Deviations are discovered dur-
ing or after the fact and were not planned.
device: See medical device.
diary: the patient diary is a record kept by all clinical study volunteers to
record any medical conditions they might encounter after the treat-
ment and while not under direct medical supervision.
distribution phase: the pharmacokinetic phase during which a biopharma-
ceutical is distributed throughout the body, normally from blood to
tissues and organs.
DLT: See dose-limiting toxicity.
documentation: a formal process of a quality system in which all documents
for a product, process, or service are carefully and fully managed
from beginning to end.
dose: single delivery or application of a biopharmaceutical product.
dose-limiting toxicity: it is the toxicity associated with unwanted side
effects that are serious enough to prevent an increase in dose or
treatment level.
DOT: U.S. Department of Transportation.
downstream: the stage of manufacturing in which product, in a crude state,
is purified to bulk substance.
drug: a small molecule with pharmacological effects, usually of organic ori-
gin, and with a well-characterized chemical structure.
DSMB: See Data and Safety Monitoring Board.
early phase development: stage of biopharmaceutical development begin-
ning with the initiation of development efforts through the end of
Phase 1 clinical trials.
EEC: European Economic Community.
Efficacy: It refers to producing the desired effect, so that the therapeutic indi-
cation of the product is achieved.
EIR: See Establishment Inspection Report.
EMA: See European Medicines Agency.
end point: a measurable entity, such as weight or blood pressure, in a scien-
tific study.
enroll: to allow a volunteer to participate in a clinical study after that person
has completed the screening and informed consent processes and
has been found acceptable to participate.
EPA: U.S. Environmental Protection Agency.

432 Glossary
establishment inspection report: a document prepared by FDA inspectors
to note the findings made during an inspection.
European Medicines Agency: European agency responsible for evaluation
of medicines for use in the EEC.
excipient: a material that is added to a biopharmaceutical product and is not
an active ingredient; for example, a carrier or a preservative.
exclusion criterion: a medical characteristic of a potential volunteer that
requires the investigator to disallow that individual from enroll-
ment in a clinical study.
excretion phase: the pharmacokinetic phase during which a biopharmaceu-
tical is excreted from the body.
experimental clinical study: a clinical trial prospectively designed as an
experiment with active treatments, controls, and other methods of
making comparisons between treatment groups.
expression system: a biological construct that consists of a recombinant
gene stably inserted into a living cell.
FAO: Food and Agricultural Organization of the World Health Organization.
fast-track approval: an FDA review and approval process that is expedited
to treat serious or life-threatening disease with a current unmet
need.
FDA: See Food and Drug Administration.
FD&C Act: U.S. Food, Drug, and Cosmetics Act of 1937.
FDP: See final product
Federal Trade Commission (FTC): a US government agency regulating
commercial practices, including advertising, within the U.S., with
the exception of foods, drugs, and biopharmaceuticals.
feedback: a team member relating any aspect of the project to other team
members.
fermentation: process of growing bacterial or yeast cells in a closed ves-
sel under defined conditions for the purpose of manufacturing a
product.
FIFRA: Federal Insecticide, Fungicide, and Rodenticide Act.
fill: to actively place or aliquot a biopharmaceutical into a container.
final drug product (FDP): See final product.
final product (FP): it is the biopharmaceutical product once it has been for-
mulated, filled into a container, and finished. Also referred to as
final drug product or FDP.
finish: to place a cap onto a container, crimp or otherwise seal the container,
and, in some cases, label and package the product.
FOI or FOIA: See Freedom of Information (Act).
Food and Drug Administration (FDA): an administrative U.S. gov-
ernment agency under the Department of Health and Human
Services (DHHS). It is responsible for regulating many products
and their development, including food, drug, medical devices,
and biopharmaceuticals.

433Glossary
Form 483: FDA Inspectional Report, a form given to sponsors immediately
post inspection by an inspector, listing deficiencies or deviations
identified during the inspection.
formulation: addition of various solutions, buffers, excipients, or stabilizing
materials to a bulk product, so as to make a solution or powder that
is ready for fill into a container.
FP: See final product.
Freedom of Information (Act) (FOI or FOIA): a law that allows private citi-
zens to petition a government agency such as FDA to release infor-
mation that is not proprietary or confidential.
FTC: See Federal Trade Commission.
functional area: a particular scientific, management, or technical activity
and suborganization aimed at fulfilling an established purpose. As
regards biotechnology operations, seven functional areas are com-
monly listed: clinical, manufacture, nonclinical, project manage-
ment, quality assurance, quality control, and regulatory affairs.
FWS: fish and wildlife service of the US Department of Interior.
Gantt chart: a computer-generated rendering of a project, using narrative
and horizontal bars to show tasks, milestones, and their dependen-
cies and relationships.
genetically modified organism (GMO): an organism that has been changed
or modified (e.g., additions and deletions to the genetic makeup)
by genetic engineering, often referred to as recombinant DNA
technology.
GMO: See genetically modified organism.
GMP: See current Good Manufacturing Practices.
good tissue practices (GTP): a regulatory guideline from FDA for the pro-
cessing of tissues or cells for human use.
guideline: a public document written and promulgated by a government
agency, such as FDA, that recommends or suggests practices, both
administrative and technical, that would, if practiced, fulfill require-
ments given under regulations. However, guidelines do not have the
legal status of regulations.
hallmark of quality: one of the several operational quality criteria that com-
prise a quality system.
HEPA: high-efficiency particle air is a special filter that removes all but the
smallest particles, leaving the exiting air especially clean and >99%
free of bacteria and fungi.
hold (clinical and regulatory): a step taken either by FDA or by a sponsor to
stop or not begin a clinical study of an investigational product.
hold (biomanufacture): a step in biomanufacturing where product is kept in
a container, awaiting further processing.
host cell: a live cell that contains a biological molecule or microbe (not nor-
mally found in that cell).
HVAC: heating, ventilation, and air conditioning.

434 Glossary
IACUC: See Institutional Animal Care and Use Committee.
IATA: International Air Transport Association.
IB: See investigator’s brochure.
IBC: See Institutional Biosafety Committee.
IC: See informed consent.
ICF: See informed consent form.
ICH: See International Conference on Harmonization.
IDE: See investigational device exemption.
identity: individuality of a product and features that distinguish it from all
other products. In other words, it is the ability of a product to be of a
known and unique nature.
IEC: See Independent Ethics Committee.
IFPMA: International Federation of Pharmaceutical Manufacturers and
Associations.
impurity: undesirable material, usually macromolecular and submicro-
scopic, but may be visible, microscopic, or soluble organic or inor-
ganic, in a product or stream. It is often inherent to the process
itself, such as cell debris.
inclusion criterion: a medical characteristic of a potential volunteer that is
considered a positive trait for the enrollment of that individual into
a clinical study.
IND: See investigational new drug application.
Independent Ethics Committee (IEC): it serves the same function as an
Institutional Review Board, to review, approve, and monitor research
involving human subjects.
indication: a remedy, treatment, or prevention that is suggested by the
symptoms of the disease. For a biopharmaceutical, an indication is
a specific medical condition that may be treated or prevented by the
product.
induced pluripotent stem cells (iPSC): a pluripotent cell or cell line that has
been created from an adult cell. The adult cell has been reverted back
to a more stem-cell-like state using chemicals or recombinant DNA
technologies.
induction: biomanufacturing step in which a chemical is added to a fermen-
ter to induce or elicit the production of a product by an organism
that has been genetically engineered to respond to the chemical.
informed consent (IC): the process of informing a volunteer to a clinical
trial exactly on the nature of the trial and all possible risks and ben-
efits that the person might derive. To enroll, the volunteer must sign
the informed consent form.
informed consent form (ICF or CF): this clinical trials document explains
to a volunteer the potential risks and benefits of a clinical study. To
enroll in a study, a volunteer must understand and sign the CF.
in-process testing: testing that occurs on samples taken from the process
stream during the manufacturing process.

435Glossary
input: a component of design review during which the needs of the user are
considered and incorporated into the product or process design.
installation qualification (IQ): a step in validation of a biomanufacturing
facility, utility, or equipment in which the installation is demon-
strated to be according to specification.
Institutional Animal Care and Use Committee (IACUC): this institutional
committee reviews animal use and experimental protocols to ensure
ethical and proper use of laboratory animals.
Institutional Biosafety Committee (IBC): a committee of scientists, ethi-
cists, and laypersons established by an institution (e.g., a university)
to review the engineering, use, or transfer of genetically modified
organisms and related research and development.
Institutional Review Board (IRB): an institutionally based peer review com-
mittee that reviews all clinical research and protocols at the institu-
tion to ensure the proper treatment and well-being of volunteers.
International Conference on Harmonization (ICH): a nonprofit group,
supported by regulatory agencies and medical products industries,
dedicated to developing and disseminating medical product devel-
opment guidelines and pathways that are acceptable to regulatory
authorities in most countries and to ensuring the quality of those
products.
International Federation of Pharmaceutical and Manufacturers
Association (IFPMA): a trade organization that promotes harmoni-
zation of regulations at the international level.
International Standards Organization (ISO): an international organization
dedicated to quality through establishing requirements and speci-
fications for products, services, and processes. It is not a regulatory
agency but develops guidelines and provides ISO certification after
review and approval. A cornerstone guideline is ISO 9001.
investigational device exemption (IDE): an application submitted to FDA to
allow the human clinical study of a new device.
investigational new drug application (IND): formal application to the US
Food and Drug Administration to test in human volunteers a biophar-
maceutical that does not have marketing approval (investigational).
investigational product (or drug): a biopharmaceutical product that is tested
in human volunteers in clinical trials and under an IND and has not
received market approval.
investigator: individual leading the scientific and medical portion of a clini-
cal study. The principal investigator has the responsibility for the
study, whereas subinvestigators assume certain responsibilities
under the principal investigator.
investigator’s brochure (IB): an informative document that identifies for
each member of the investigative staff the information on the clini-
cal study, the product being tested in the study, and the possible
risks and benefits to volunteers enrolled in the study.

436 Glossary
in vitro diagnostic (IVD): a laboratory test used to diagnose disease and
that is regulated by FDA as a medical device.
iPSC: See induced pluripotent stem cells.
IQ: See installation qualification.
IRB: See institutional review board.
ISO: See International Standards Organization.
IVD: See in vitro diagnostic.
label: the printed identification for a product, usually on paper and held to
the final container or package of biopharmaceutical.
labeling: the sum total of printed materials, package insert, package print-
ing, and so on that accompany, or are adherent to, biopharmaceuti-
cal containers and packaging. Labeling, approved by FDA, provides
the approved indication, directions for use, dosage, and other critical
information provided by the sponsor to the user.
late-stage development: the final investigational stage of product develop-
ment and the activities associated with this stage and with Phase 3
clinical trials.
LD50: See lethal dose, 50%.
leachates: chemicals that are dissolved from a surface or other solid matrix
into the product stream during biomanufacture, thus becoming
contaminants.
lethal dose, 50% (LD50): the amount of an agent that causes death in 50% or
half of the population of animals over a defined study period.
letter of authorization (LOA): a formal letter submitted to FDA to allow an
independent investigator to reference confidential information cur-
rently on file at FDA. Also referred to as a letter of cross-reference.
limit of detection (LOD): the minimal amount of analyte that can be accu-
rately detected by a particular assay in a test substrate.
limit of quantitation (LOQ): the minimal amount of analyte that can be
reasonably measured, in a quantitative sense, by a particular assay.
linearity: for an analytical, quantitative measurement (test), it is the ability,
within a given range of analyte in a sample, to obtain test results that
are directly proportional to the concentration of the analyte.
LOA: See letter of authorization.
LOD: See limit of detection.
LOQ: See limit of quantitation.
lot: defined amount of manufactured final product that constitutes a legally
defined entity. A lot has unique character, quality, and source and is
a specifically identified amount, labeled as such.
lyophilize: a process in which a biological material in a solution is sub-
jected to freezing and drying simultaneously to preserve the cells
or molecules.
marketing application: application to a regulatory agency to market a prod-
uct in that country. See also Biologics License Application or New
Drug Application.

437Glossary
marketed product: a biopharmaceutical that has been approved for sale in
that country.
market approval: permission from the Food and Drug Administration to
market a biopharmaceutical in the U.S. for the indication and at the
dosage given in the approved labeling.
master cell bank (MCB): it is the ultimate source of any seed. A bank of cells,
usually derived from a single clone or source, is kept as a unique
resource for later expansion or use.
master file (MF): a regulatory document under which a sponsor may file
confidential information with FDA. Investigational use of a prod-
uct is not allowed under a Master File, as it is under an IND. Drug
Master Files (DMF) or Biologic Master Files (BMF) are used in bio-
pharmaceutical development.
matrix: the medium in which a product is disbursed or suspended to include
those of natural origin, for example, serum, or of synthetic origin, for
example, phosphate-buffered saline.
maximal efficacy: the greatest effect or response that is given by a biophar-
maceutical (and in the absence of toxicity).
maximum tolerated dose (MTD): this dose is the highest tested dose that
does not result in an unacceptable toxicity or adverse effect.
MCB: See master cell bank.
MDR: See medical device reporting.
measurement/measure: the act of or a system for determining, through
laboratory, clinical, or nonclinical testing and evaluation, the
quantity or quality of a particular end point or process. It refers
to the use of an assay or instrument or scientific and technical
skills to determine an unknown parameter. It may be qualita-
tive or quantitative and is usually determined in a stated unit or
capacity.
medical device (device): an object that may be any one of the many classes
of physical or engineered products. It achieves its intended primary
action in a manner other than pharmacological, biological, or meta-
bolic means. Medical devices are used to diagnose, prevent, monitor,
and treat disease.
medical device reporting (MDR): an FDA regulation aimed at ensuring that
manufacturers report defects in, or adverse events associated with,
medical devices.
medical monitor: also referred to as the medical safety Monitor, a medical
professional assigned to review AE or SAE or other matters relating
to safety of volunteers.
method (analytical): a test or analytical procedure that is used in a labora-
tory to measure quality.
metrics: in project management, it refers to measurement of progress against
established milestones, schedules, budgets, or other resources.
MF: See master file.

438 Glossary
middle-stage development: product development that occurs before, dur-
ing, and immediately after Phase 2 clinical trials.
milestone: it is a readily identified interim event or set of events and a major
waypoint in a project that demonstrates the achievement of a planned
outcome.
misbranding: it refers to labeling or branding falsely or in a misleading
manner or without supporting scientific data and in violation of
FDA regulations. It means not completely or legibly labeled or not
accurately reflecting the truth.
monitor: in a clinical trial, the medial monitor is either a safety monitor, that
is, a medical professional assigned to review AE or SAE or other
matters relating to safety, or a volunteer. The term monitor is also
used to indicate a sponsor’s monitor, an individual that reviews
activities and progress of a clinical study at the study site.
MTD: See maximum tolerated dose.
multiarm clinical study: a clinical study design that includes several treat-
ment groups, each group receiving a different treatment or dose.
multicenter clinical study: a trial that is performed in more than one medi-
cal center, but under the same protocol and for the same purpose.
Multiple centers are used because it is not possible to recruit all vol-
unteers at only one center.
NAI: See no action indicated.
national drug code (NDC): a unique product identifier for human drugs in
the U.S.
National Regulatory Authority (NRA): a regulatory body, such as FDA,
appointed by a national government in the area of food and drug
regulation.
National Science Advisory Board for Biosecurity (NSABB): consists of a
federal committee that addresses the issues related to biosecurity
and biological research.
NCIE: National Center for Import and Export (of animals), APHIS, the US
Department Agriculture.
NDA: See new drug application.
NDC: See national drug code.
neat: test article used in full strength or undiluted in a nonclinical study.
new drug application (NDA): an application made to FDA for the purpose
of gaining marketing approval for a new drug (pharmaceutical or
biological) in the U.S. An NDA also applies to certain therapeutic
biopharmaceuticals receiving review at CDER. This large document
provides complete information on development of the product and
its safety and efficacy.
NF: National Formulary of USP.
NIH: National Institutes of Health of the US Department of Health and
Human Services.
NIOSH: National Institute for Occupational Safety and Health, CDC.

439Glossary
no action indicated (NAI): it refers to findings from an inspection report or
EIR in which FDA states that no findings in an inspection warrant
further investigation or action.
NOAEL: See no observed adverse effect level.
NOEL: See no observable effect level.
nonclinical: studies, both in vitro and in vivo, that are performed outside of
man to define in the laboratory and in animals the pharmacology or
toxicity of a biopharmaceutical.
nonconformance: a product or, in a broader sense, a study report or other
document that fails to meet specifications after quality control test-
ing or quality review.
no observed adverse effect level: the highest dose tested in an animal spe-
cies that does not produce a statistically or biologically significant
increase in adverse effects in comparison to a control group.
no observable effect level: the highest dose tested in an animal species with
no detected effects.
NRA: See National Regulatory Authority.
NRC: Nuclear Regulatory Commission, the US Department of Energy.
NSABB: See National Science Advisory Board for Biosecurity.
Nuremburg code: it is a series of ethics principles established for conduct-
ing human research to protect the rights and well-being of research
subjects.
OAI: See official action indicated.
OBA: Office of Biotechnology Activities, Office of the Director, NIH.
OBP: Office of Biotechnology Products, CDER, FDA.
OBRR: Office Blood Research and Review, CBER, FDA.
observational clinical study: a study that observes patients for the dis-
tribution and incidence of disease, in the absence of specific treat-
ments and interventions. It is also referred to as epidemiological
study.
OCTGT: Office of Cellular, Tissue, and Gene Therapies, CBER, FDA.
off-track: this means that a project is not on schedule or budget.
official action indicated (OAI): it refers to findings on an EIR, the inspec-
tional report, in which FDA recommends that action be taken
immediately by a manufacturer and followed-up by FDA regarding
deficiencies or deviations noted during an inspection. It is the most
serious of the three EIR finding categories.
on-track: this means that a project is on schedule or budget.
open-label study: a type of clinical study in which the investigator and, in
some cases, the patient are aware of the treatment regimen (placebo
or investigational product). It is not a blinded study.
operational area: as regards biotechnology operations, an operational area is
one of the seven commonly listed (clinical, manufacture, nonclinical,
project management, quality assurance, quality control, and regula-
tory affairs) or other developmental specialty.

440 Glossary
operational management: it refers to managing technology development
under a product development plan in an operational area.
operational qualification (OQ): a stage of manufacturing facility validation
in which the operation of a utility or piece of equipment is shown to
meet specifications. It is performed before process qualification.
OQ: See Operational Qualification.
OSHA: Occupational Health and Safety Administration, the US Department
of Labor.
OTC: over-the-counter drugs.
outcome: it refers to broad results or visible effects that form the basis for a
study hypothesis. In clinical studies, these are often medical items.
output: a component of design control in which the process, service, or prod-
uct design, based on input and review by professionals, is proposed
and documented as the process and/or the product, in whole or in
part.
OVRR: Office of Vaccine Research and Review, CBER, FDA.
package: the inner (surrounds a primary product container) or outer (sur-
rounds multiple inner packages) material that is used to protect a
product from damage. Often, cardboard or plastic packaging mate-
rial is used for packaging.
package insert: the printed, extended instructions and information, approved
by both the manufacturer and a regulatory agency, for a product and
enclosed in the package. The package insert is an important part of
labeling and states indication, directions for use, warnings, and so
on. (See labeling.)
PAI: Preapproval Inspection by FDA.
parenteral: a route of delivery given beneath or through the epidermal layer
and is not oral, mucosal, or topical.
parenteral product: a product that is given beneath the skin or injected.
particle: it is a microscopic or visible piece of material, usually an undesir-
able contaminant in a product or stream.
pathway: in project management, it refers to a well-defined course of action
or sequence of events.
patients: individuals with a pre-existing medical condition enrolled in a
clinical trial for the purpose of testing a therapeutic product for that
condition.
PCB: production cell bank. (See working cell bank.)
PCR: See polymerase chain reaction.
PD: See pharmacodynamics.
PDP: See product development plan.
PDS: product development strategy. (See product development plan.)
PERT: it refers to the program evaluation and review technique, which is
actually an illustration of a project schedule to depict interrelation-
ships of various tasks and milestones in a project.

441Glossary
PhRMA: Pharmaceutical Research and Manufacturers of America.
pharmaceutical: a small molecule drug. (See drug.)
pharmacodynamics (PD): the study of how a biopharmaceutical interacts
with various tissues, fluids, or organs to achieve a therapeutic effect.
pharmacokinetics (PK): the study of how, when, and where a biopharmaceu-
tical gains access (e.g., absorption), is distributed, is metabolized, or is
excreted by the body.
pharmacology: the study of pharmacological agents (drugs or biopharmaceu-
ticals) and their mechanisms of action and effects on organisms.
pharmacopeia: a reference book that provides product, process, and test
standards and specifications.
phase 1: the first clinical phase and the early phase of product development.
phase 2: the second clinical phase and the mid phase of product development.
phase 3: the third clinical phase and the last phase of product development
before market approval.
phase 4: any development activities that occur after market approval of a
product.
phased manufacture: production of product over time by using phases of
development, going from simple systems to more complex, from pro-
ducing small to large batches, from small to larger clinical studies, and
so on. Phases numbered as 1, 2, 3, or 4 or as early, mid-, and late devel-
opment phases.
PI: See principal investigator.
pilot production: earliest production of a new product in the biomanufac-
turing cycle. It is usually done on a small scale and in an experimen-
tal mode.
pivotal: a clinical study, usually in Phase 3, designed to demonstrate or
confirm beyond reasonable doubt the safety and efficacy of a
biopharmaceutical.
PK: See pharmacokinetics.
Placebo: a sugar pill, containing any substance that is known to be safe and
not cause a direct physiological or therapeutic effect and is given to
volunteers assigned to a controlled clinical trial.
PMA: See premarket approval.
polymerase chain reaction: a powerful molecular biology technique used to
amplify DNA or RNA.
portfolio: a collection of projects or programs with common technologies or
goals.
potency: measurement, direct or indirect and generally quantitative, of a
product’s biological or therapeutic effect. It is established as a qual-
ity control test to evaluate BS or FD.
PQ: See process qualification.
precision: the ability of an assay to repeatedly produce the same or very
similar result on repeated testing when variables are held constant.

442 Glossary
preclinical: research and early development activities that occur before
Phase 1 clinical studies. It is most often used in reference to research
activities.
premarket approval (PMA): the regulatory process, and application docu-
ment, for marketing approval for a medical device in the higher-risk
classes.
preventive action: an activity that prevents a problem or issue from occur-
ring or recurring.
principal investigator (PI): the medical professional, usually a physician,
who is the responsible individual at a clinical study site for a par-
ticular clinical trial.
process: as a verb, a process refers to actively produce a product by using
defined technical skills. As a noun, a process refers to a defined por-
tion of biomanufacture.
process qualification (PQ): a stage of manufacturing validation in which a
process or part of a larger process is qualified by actual performance
against specifications.
product: a thing, substance, or material that is manufactured or produced
during biotechnology operations. It is a result of planning and
labor.
product development plan (PDP) or strategy (PDS): a document developed
early in the life cycle of a product’s development cycle that provides
a roadmap and specifications needed to conduct rational, compliant,
and resource-effective biopharmaceutical development from early to
late phases.
product life cycle: the manufacturing cycle involved in product develop-
ment, beginning with planning and continuing through all phase of
development and stages of manufacture.
product stream: See stream.
production: the act of biomanufacturing.
production cell bank (PCB): a working cell bank.
program: a group of related projects that are often coordinated and share a
common objective.
project: a distinct and planned enterprise that is identified by a unique
objective or goal, a beginning, an end, and a schedule.
project champion: individual serving as a stakeholder or on the project
team who has a strong personal and professional interest in achiev-
ing the objective or product. Champions argue and strongly sup-
port the objective.
project management: the function of planning, organizing, and managing
resources and schedules to bear on performance of a defined project.
project management plan: a written plan that outlines how a project will be
managed using modern project management processes and tools,
both technical and social.

443Glossary
project manager: a professional appointed to manage and lead a project
team and the processes, tasks, budget, schedule, and other activities
that fall within the scope of the project.
project schedule: a calendar with dates that demonstrates a project from
beginning to completion, with all major tasks and processes. It con-
tains start, milestone, and finish dates.
protocol: an instructive document that identifies exactly why and how a
study (e.g., clinical, nonclinical, or validation) will be performed and
provides schedules of events.
purification: it is a process used in biomanufacturing to remove contami-
nants and impurities while retaining the desired product.
purity: it is the amount of product in relation to impurities that might exist
in a product. It also refers to freedom from chemical or biological
contamination or impurities.
QAU: See quality assurance unit.
QbD: See quality by design.
QSR: See quality systems regulation.
qualification: it means to ensure that something, such as a test or a process,
is suitable for use. Qualification is typically less stringent than vali-
dation, and the process is often applied to critical laboratory tests or
manufacturing processes in early to mid-phase development.
quality: the degree of excellence of a thing; general excellence (Oxford English
Dictionary, 1997).
quality agreement: a contract between two parties, generally a contractor
and a client, that identifies quality aspects of a relationship, espe-
cially regarding compliance issues and conformance with a quality
system.
quality assurance: it is a function of planning, managing, operating, and
ensuring the performance of a quality system.
quality assurance unit (QAU): the functional area at a biotechnology firm
responsible for the overall quality operation and all aspects of qual-
ity assurance.
quality by design (QbD): a planning process in which a product and its
biomanufacture, are carefully described along with attributes and
specifications.
quality control: laboratory, test, or metrology function to ensure quality of
a product.
quality manual: a written document that proscribes the quality policies and,
in general, the criteria, operations, and organization for quality sys-
tems at a biotechnology firm.
quality plan: a written document that expands on the quality manual, and
describes the quality systems and plans to operate those systems at
a biotechnology firm. The quality plan is referenced in or is part of
each product development plan.

444 Glossary
quality system: a designated set of components, connected in a logical fash-
ion and focused on quality of a product or service. Quality systems
for biopharmaceutical development are codified in regulations,
guiding quality operations of a particular activity, such as manufac-
ture (e.g., cGMP), nonclinical studies (e.g., cGLP), or clinical studies
(e.g., cGCP).
quality systems regulations (QSR): an FDA guideline, based on a regu-
lation and focused on quality design and manufacture of medical
devices.
RAC: See Recombinant Advisory Committee.
randomize: the act of randomly placing a subject or an animal into one or
another treatment group in a controlled and blinded clinical or non-
clinical study.
range: the value defined by the upper and lower limits of a specification or
concentration of an analyte.
raw materials: items that are used to manufacture a product. The term gen-
erally refers to materials such as solutions, reagents, chemicals, and
biological substances.
Recombinant Advisory Committee (RAC): a committee established by the
director, NIH, to review and approve certain studies, in vitro or in
animals or man. It deals with genetic engineering or transfer or use
of genetically modified organisms.
recruiter: an individual employed by a clinical investigator or a sponsor for
the purpose of identifying individuals who might wish to volunteer
for a clinical trial.
reference standard: a well-characterized and known product or material
against which the attribute of a test material may be compared.
regulation: a rule that has the force of law. It is an interpretation of the law
by an administrative government agency in the executive branch
and is intended to carry out the intent of the law.
regulatory intelligence: the process of finding and analyzing publically
available regulatory information.
release: the action of allowing a product or study to be provided to the user,
customer, or client.
release testing: the panel of tests that are performed on a product before it
can be released.
REMS: See risk evaluation and mitigation strategy.
requirement: an attribute that a product possesses, usually defined in sci-
entific terms.
research seed: a microbial seed (e.g., clone) or a cell line derived from a
research laboratory.
risk evaluation and mitigation strategy (REMS): risk management plan-
ning and a plan to ensure that benefits of a product outweigh the
risk. It is performed by evaluation after marketing approval.

445Glossary
robust: a vigorous, strong, and sturdy manufacturing process or test method
in biopharmaceutical development. It is the overall reproducibility of
a process or test results when operational conditions are held within
established ranges.
root cause: the ultimate or original cause for an issue or a problem that
occurs. It can be clearly described in technical or scientific language
and hence is preventable in the future.
run: a single and clearly identifiable manufacturing process. A single run
produces an individual lot or batch or product.
SAE: See serious adverse event.
scale-up: increase in the amount of biomanufacturing for a product, so as to
increase the total amount of product in a single batch or lot of BS or
FP.
seed: a defined cell or viral particle from which other cells or particles may
be derived.
serious adverse event (SAE): an adverse event that is serious by medical
diagnosis or is life-threatening or causes death of the volunteer.
Six Sigma: a quality program aimed at reducing product or service failure
rates.
sociotechnical skills: practiced ability to integrate sociological or people
management expertise together with a technical knowledge and
capability, so as to lead a project team.
SOP: See standard operating procedure.
source document: any record, data, or other piece of information that is clos-
est to the source. These data are initial, original, or raw and on a
written or electronic document.
sparge: it means to move a gas into or through a liquid in a vessel such as a
fermenter.
specification: a stated value or range of values that are specific to a product
attribute and quality control test. Specifications are specific, strict,
and fully defined criteria based on which a product is found either
suitable or unsuitable for use.
specificity: the degree to which a measurement made by an analytical test is
due to the actual analyte of interest and not due to other materials in
the test matrix.
sponsor: the entity (institute, individual, or corporation) that is ultimately
responsible, in a scientific, business, and legal sense, to regulatory
agencies, the public, and the users for a product and its development.
stability: for a product, it means the trait of maintaining purity, potency, and
strength over time and in a given environment. It also refers to the
property of a biopharmaceutical or product to not degrade or break
down.
stability-indicating: a test that is capable of identifying when a product has
lost or is losing purity, potency, or strength.

446 Glossary
stability protocol: a document that designs and plans a stability testing pro-
gram for a specific product and applies various assays under an estab-
lished schedule.
stage: a major division of a biomanufacturing scheme, such as upstream
processing.
stakeholder: an individual who, by status or dominant position, is influ-
ential to a project team and, although not always serving directly
on the team, has a vested interest in the success of the team’s
efforts. Project teams serve, in part, to meet the expectations of a
stakeholder.
standard operating procedure (SOP): an instructive document that pro-
vides exact or detailed technical or administrative procedures. An
SOP may include forms to capture data during the performance of
that procedure.
steady state: pharmacokinetic phase during which the concentration of a
biopharmaceutical is maintained at a given level.
step: a small but important part of any stage of biomanufacture.
sterile fill: a process to fill final containers with product and in the absence
of microbial contamination. Aseptic methods are used throughout.
stop criterion: a medical situation that arises and, by definition, leads to ces-
sation in the enrollment and treatment of volunteers in a clinical
trial.
stopping rules: a prospectively defined plan, in a clinical study protocol, to
stop current treatment or additional enrolment, monitor potential
safety concerns, and evaluate potential risk before putting addi-
tional subjects at risk.
stream: it is the product stream, or main bulk of product, in process, during
biomanufacture. It is the material that is under production at a single
time in biomanufacture.
strength: measure of active ingredient in a product. It is typically measured
using an analytical method that does not measure biological activity
but instead measures the amount of chemical or biological substance
present.
study director: an individual responsible for overall design, performance,
and reporting of a nonclinical safety study.
subacute toxicity: a safety study that evaluates toxicity of a biopharmaceu-
tical, given in multiple doses, in animals over a brief period (e.g.,
30–60 days).
subchronic toxicity: a safety study that evaluates toxicity of a biophar-
maceutical, given in multiple doses, in animals over a moderate
period (e.g., 3–6 months).
subject: an individual without an underlying disease who volunteers for a
clinical trial.
synchronize (project): integrate and bring together the various parts under
a schedule and series of events.

447Glossary
system suitability: ability of an analytical test to achieve the objectives of
the assay. All components of the test are suitable for the intended
purpose.
T1/2: the time elapsed from when a biopharmaceutical reaches Cmax until it
reaches ½ the value of Cmax.
tags: molecular identifiers genetically engineered into a molecule. They may
be used for identification or affinity purification of that molecule.
tangential flow filtration (TFF): type of biomanufacturing preparative fil-
tration that allows filtration through a selective membrane as flow of
liquid sweeps the membrane surface to prevent clogging.
targeted product profile (TPP): a written document that prospectively
identifies the attributes and intended therapeutic indications for a
biopharmaceutical product. It is written in the format of product
labeling but is a planning tool and not a means of reporting results.
task: a piece of work included in a project that is exactly defined in technical
terms and has a beginning and end.
team (project): a group of professionals from different functional areas work-
ing together toward a common objective, each of them bringing a
specific expertise.
team dynamics: the sociological and psychological energies and motions
that affect the behavior and change for a project team.
team leader: an influential individual who serves on the project team but
typically not as a project manager. A team leader may be a chief sci-
entist or key executive or the founder or discoverer of the technology.
test: analytical method or laboratory procedure performed on a product to
measure an attribute.
test article: test product, or the biopharmaceutical in formulation, as given to
animals in a nonclinical study.
TFF: See tangential flow filtration.
timeline: a visualization of tasks or processes and milestones set against a
schedule.
Tmax: the elapsed time from when a biopharmaceutical is given until the
maximum concentration is seen in blood (or tissue).
tolerability: to determine in a clinical trial how well the subjects or patients
medically or physiologically accept or tolerate the investigational
product when it is given in measured dose.
tolerated: ability of an organism to be subjected to a biopharmaceutical over
a period of time without experiencing adverse effects or harm due
to that product.
total quality management (TQM): a management approach to quality that
aims to continuous satisfaction of the customer or client.
toxicology: the study of toxic effects of chemicals, a biological, or an ionizing
radiation on a living organism.
Toxic Substances Control Act (TSCA): a US law that deals with testing
before use or release to the environment of chemical substances.

448 Glossary
TPP: See targeted product profile.
TQM: See total quality management.
track: as a noun, it is the pathway of a project. The verb, to track, means to
monitor the processes and tasks within a project to ensure that the
processes and tasks are completed on schedule and budget.
tracking: the project management process of reviewing all aspects of the
project to ensure that tasks are completed on schedule and budget.
trait: a distinguishing feature or characteristic of a product. It is a specific
chemical or biological feature that can be measured with an analyti-
cal test.
transfection: placing a foreign or recombinant gene into a mammalian or
other cell derived from animals or plants.
transgenic: an organism, such as a plant or animal, that retains one or more
genes of another organism.
treatment group: in a scientific study design, it refers to a group of humans
or animals that receives the same treatment, such as either investiga-
tional product or placebo.
TSCA: See Toxic Substances Control Act.
U.S. Pharmacopeia (USP): a compendium or reference volume that pro-
vides information on biopharmaceutical raw materials, products,
processes, tests, and formulations.
upstream manufacture: biopharmaceutical production that yields the prod-
uct as a crude or unpurified material. The early stages of biomanu-
facturing from cell bank to crude cell paste.
USDA: U.S. Department of Agriculture.
USP: See U.S. Pharmacopeia.
VAI: See voluntary action indicated.
validate: to provide strong evidence, usually through experimentation, that
a piece of equipment, facility, utility, test, or process performs exactly
as intended and within established specifications.
validation: a process in which a test or process is demonstrated to perform
exactly as intended and planned and meets established specifications.
variance: a measurement, outcome, or part of a process or study that does not
meet established procedures, rules, or specifications but is known or
planned before it occurs in fact.
vector: a live organism or construct of DNA (e.g., plasmid) that contains
DNA or RNA of another organism, usually through recombinant
technology.
vehicle: a material, such as saline, in a formulation that serves to enhance
transfer, absorption, or distribution of a biopharmaceutical.
vendor: an entity that provides a material or service to a client.
verification: to demonstrate, with documented evidence, that something,
such as a piece of equipment, is what it is purported to be. It is the

449Glossary
specific act of verifying and documenting that a compendial test
performs as intended in a quality control laboratory.
viable particle: living contaminant (typically bacterial, fungal, or yeast)
found in a product or manufacturing stream. It is an undesirable
occupant of a manufacturing area.
virtual team: a project team that is separated by space and time but still
functions as an effective group of individuals working together
toward a common objective.
volume of distribution: the distribution of a biopharmaceutical, in quantita-
tive terms, between blood and other tissues of the body after dosing.
It measures the volume in which drug would be uniformly distrib-
uted at any point in time.
voluntary action indicated (VAI): refers to findings on an EIR in which
FDA recommends that action be taken by a manufacturer to correct
minor deficiencies or deviations noted during an inspection.
volunteer: any individual who requests to be enrolled in a clinical trial.
water for injection (WFI): highly purified water free of microbes, contami-
nants, or impurities and of a quality that can be injected into humans.
WCB: See working cell bank.
well-characterized: a product or material for which there is a significant
amount of scientific information, often chemical, biological, and
physical, that provides a high degree of understanding on the nature
and functional properties of the product.
WFI: See water for injection.
WHO: World Health Organization, United Nations.
WI: See work instruction.
withdrawl: the instance of a volunteer in a clinical study leaving that
study on his or her own initiative or on request of the principal
investigator.
work breakdown structure: a tool used in project management to identify
the various work pieces of a project, that is, the tasks, and place them
in a logical sequence of events or hierarchy. It is the basis for  project
planning—a visualization or narrative outline of project tasks,
as mapped to component parts.
working cell bank (WCB): Derived from an MCB, this is the source of cells
for production, also referred to as a production cell bank (PCB).
work instruction (WI): a document used in biomanufacturing to both
guide a process and record critical information regarding a partic-
ular batch or lot of product. Also called a batch production record
(BPR).
yield: the amount of product that results from a step or stage of biomanu-
facture. Often presented as a percentage, the amount obtained at the
end divided by the starting amount.

450 Glossary
References
FDA. 2016. U.S. Department of Health & Human Services: U.S. Food and Drug
Administration Home Page/Regulatory Information/Code of Federal Regulations/
CRF Title 21 – Food and Drugs: Parts 1 to 1499. http://www.accessdata.fda.gov/
scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm (accessed May 31, 2016).
Oxford English Dictionary. 1997. Oxford University Press. Oxford, UK.

http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm

http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm

451
Appendix
Overview
This addendum to this book provides an opportunity for readers to con-
sider situations that occur in biotechnology operations and to develop a
targeted product profile (TPP) and a product development plan for a bio-
pharmaceutical product directed at treating or preventing a disease. In the
first section, situations are posed. In each situation, a biopharmaceutical
product is to be developed, as a team project. A small amount of technical
background is provided and students are encouraged to make and state
assumptions regarding the research results, intended population, indica-
tion, and so on for each product. In the second section of this addendum,
questions are posed with regard to planning development for each prod-
uct. Each question on planning pertains to any of the six products.
Products and Projects
• Problem #1: The product to be developed is a monoclonal antibody.
Your firm, ABC Biologicals, Inc., (Osco, IL) is developing a propri-
etary monoclonal antibody to treat cutaneous T-cell lymphoma, a
cancer of white blood cells. This biopharmaceutical product is a
humanized monoclonal antibody that targets and binds specifically
to a cell surface molecule, CD545. The molecule, developed in your
laboratory, has been characterized in research and has been shown,
in a transgenic mouse model, to bind to cancer cells, leading to their
death. To be effective in mice, the product is given by subcutaneous
injection. Your firm now wishes to develop the monoclonal anti-
body product and apply for market approval.
• Problem #2: Your firm, EbolVac, Inc., has discovered and patented a
surface protein of Marburg virus, an organism that causes hemor-
rhagic fever in man. Ebola virus is considered a threat agent for bio-
terrorism, and so, governments are the potential customers. A gene
for an Ebola virus surface protein has been cloned and expressed in

452 Appendix
a host system as a 65 kDa glycoprotein. The product can be handled
safely in the laboratory. Using a model of Ebola virus in nonhu-
man primates kept in a high-containment facility, your firm has
demonstrated proof of principle by protecting monkeys by using a
nasal spray of the vaccine. You now wish to develop the product for
human use and obtain market approval.
• Problem #3: Your firm, GenTherLine, holds an exclusive license for a
novel gene therapy product composed of naked plasmid DNA. The
gene therapy is intended to resolve nevoid basal cell carcinoma, a
cancer of the skin, by replacing the gene for PTCH. The naked plas-
mid DNA is a vector that includes the PTCH gene. It must be deliv-
ered by a microneedle injection apparatus that exposes the DNA to
the cells of the basal epidermis. Unfortunately, there is no animal
model in which this mutation exists, as the mutation exists and is
expressed only in man. Once the PTCH gene enters the nucleus of
a cultured cancerous basal epidermal cell, it replaces the mutant
gene that causes this disease, but the concept has not been attempted
in man. You now wish to develop the product and obtain market
approval.
• Problem #4: Your firm, MalarTher, has discovered a 35 kDa protein,
MER24, that interrupts the life cycle of malaria parasites by blocking
their ability to further infect red blood cells. MER24 acts directly on
the merozoite stage of the malaria parasite, the erythrocytic or asex-
ual cycle of red blood cell infection. Indeed, MER24, mimics a red
blood cell receptor, thus binding to the parasite before it can bind to
the same molecule on the host’s erythrocyte. It is considered a poten-
tial therapeutic molecule to be given intravenously to individuals
infected with Plasmodium falciparum. You now wish to develop the
product and obtain market approval.
• Problem #5: Your firm, StemTechnolUS, develops human therapeutic
products from human pluripotent stem cells. You have discovered
and cloned a stem cell-derived cell line that differentiates into neu-
rons and is suitable for repair of uncomplicated spinal cord injuries
from blunt trauma. Cells can be given by surgical intervention. You
now wish to develop the product and obtain market approval.
• Problem #6: Your firm, TheraGentCure, has the patent for a gene
therapy construct. It is a retrovirus, specifically a lentivirus, that
carries the gene for a tissue inhibitor of a matrix metalloproteinase
that inhibits neuroblastoma tumor cell growth. It would be directly
injected into tumor mass or into blood vessels, feeding the mass in
an attempt to slow the growth of the tumor and perhaps cure the
cancer. You now wish to develop the product and obtain market
approval.

453Appendix
Questions by Chapter and Functional Area
Chapter 1: Background and Targeted Product Profile
• Discuss the class of biopharmaceutical represented by this product.
• Provide a name for the product.
• What competitive products, if any, are on the market, and how have
they been developed and marketed? Discuss market advantages
your product should possess.
• Describe the disease or condition (indication) and the population
that is subject to the disease.
• Develop a TPP for this product, with all elements of biopharmaceuti-
cal labeling.
• Define exactly the indication and population your product would
diagnose, treat, or prevent, as considered for the first market
approval. Discuss the rationale for choosing this indication and the
intended population.
• How, in general, will this TPP affect the plans for each functional
area?
• Provide a product design, to include input, design, and output, based
on the elements of the TPP.
Chapter 2: Project Management
• Identify the project and its purpose and provide an overview of the
project from the standpoint of the project manager. Include purpose,
scope, and technical and management objectives.
• Provide the composition of the project team enlisted for this project.
Identify team members, their affiliations and locations, and authori-
ties and responsibilities, and the rational for these choices. Identify
stakeholders to the project and define their roles, apart from the
team.
• Describe the team communication plan to include types of commu-
nication and frequency of meetings.
• Describe metrics that would be used to measure the progress of the
project.
• Describe the methods the project manager will apply for allocating,
tracking, and managing resources allocated to the project.
• Identify the 10 major risks this project is likely to encounter. Outline
the risk assessment and risk management plans for the project,

454 Appendix
focusing on risk mitigation early in the project. Describe the roles of
the project manager in risk management.
• Describe your plan for identifying actual problems or issues and
resolving them as a team.
• Establish a work breakdown structure, both as a narrative explana-
tion and as an illustration (e.g., Gantt or PERT chart).
Chapter 3 and 4: Regulatory Affairs and Regulatory Compliance
• Is there a regulatory precedent for the treatment of this indication
and population, and if so, are the predicate products approved by
FDA or any other regulatory agency for marketing? How might this
history of predicate products affect the regulatory development of
your product? Is there precedent for this type of product (e.g., molec-
ular or cellular nature) having received market approval or having
been tested as an investigational drug? If so, what were the outcomes
and how might this precedent affect the development of and regula-
tory activities for your product? What do you think is the status of
your product in the eyes of USFDA?
• Each product falls under one or the other office at FDA. Describe
which office or division at FDA is likely to review your product.
Explain how your product will fall into the scheme of FDA. Where
will you submit applications within FDA?
• Will market approval be sought, initially, in the United States alone
and/or in other countries? Describe your plans to submit marketing
applications in each chosen country.
• What types of investigational use and marketing applications will
be submitted to USFDA over the life of the product?
• For this product, outline the elements of the Investigational New
Drug application (IND), in either IND or Common Technical
Document format, as intended for FDA.
• Outline briefly an FDA marketing application in an acceptable
format. Highlight the key elements that must be achieved during
development, so that the application is complete. Will user fees be
necessary, and if so, how much are the fees in today’s dollars?
• What other types of applications might be submitted to FDA in an
effort to facilitate development, increase market share or exclusivity,
or speed the process of approval?
• What FDA guidance documents are most important for your firm to
consider for this product and indication?

455Appendix
• What is the nature and timing of meetings or teleconferences that
might be held with FDA during the development process and before
receiving market approval?
• As designed, could your product be considered in any way a com-
bination product? Explain the rationale for this conclusion. If it is
a combination product, then describe the impact on the regulatory
plan and development pathway.
• Describe the major risks and benefits for the product, as FDA might
perceive. Discuss the factors that will enter into a regulatory review
of the risk-to-benefit ratio for this product.
• Describe postmarketing activities that might be required by regula-
tory agencies for this type of product or indication?
• What non-FDA regulatory hurdles must be considered for this type
of product? Do these hurdles pose major obstacles to product devel-
opment, and if so, how will they be addressed and resolved?
• How might you add value or speed the development of this product
by using various opportunities provided by FDA? Describe these
programs the guidelines.
Chapter 5: Quality Systems and Quality Assurance
• Outline the contents of the quality manual that will serve your prod-
uct’s development operation. Cite specifically each quality system
that will be included and explain why and when in development it
will be applied. Include a brief quality policy and a brief statement
regarding management responsibility, as it would be approved by
upper management.
• Describe the need to design your product, and explain how this will
be accomplished. Provide product-related specifics regarding qual-
ity by design and design control.
• Review the elements of a design program specific for your prod-
uct, relating user needs, product attributes, and technical elements
of design. Is it possible that your product must be reworked in the
research laboratory before it begins development? If so or if not so,
explain the exact reason.
• Choosing from the list of hallmarks of quality, identify what you
consider to be the six most important with regard to the quality of
product. Do not choose hallmarks of design. Explain why each was
chosen and specifically elucidate how each will be applied to your
product during both development and marketing phases.

456 Appendix
• Describe the fully functional Quality Assurance Unit that will sup-
port the development of your product, and in your narrative, specifi-
cally identify the examples of how the unit will function in the areas
of audit, investigations, and change control.
• Describe the five most likely problems you will experience as you
establish quality systems and a Quality Assurance Unit for this par-
ticular product and project.
Chapter 6: Biomanufacturing
• Describe the biomanufacturing schemes that have been used to pro-
duce biopharmaceuticals of a similar molecular or cellular nature.
Identify regulatory guidelines that apply to the manufacture of such
products.
• Discuss the major risks associated with manufacturing this type of
product.
• Design a biomanufacturing plan for your product to include
objectives, input, process, equipment, facility considerations, out-
put, and review. Provide the process flow to include premanu-
facturing preparation of constructs, upstream and downstream
processing, holds, formulation, fill, finish, and raw material
requirements.
• Outline how and at what phase of development the biomanufactur-
ing process would be increased in scale. Provide plans to increase
facility size or to use contract manufacturing operations.
• Provide general plans to validate the process, facility, equipment,
and utilities.
Chapter 7: Quality Control
• Describe quality control test schemes that have been used to pro-
duce biopharmaceuticals of a similar molecular or cellular nature.
Identify regulatory guidelines that apply to the quality control of
such products.
• Define and justify the attributes of bulk substance as they are based
on its known nature and manufacturing scheme for bulk substance.
Once the attributes are listed, draft a Certificate of Analysis, adding
analytical methods and specifications to the attributes and consider-
ing testing for more than one parameter of key attributes.
• Define and justify the attributes of final product as they are based
on its known nature and the intended manufacturing scheme for
final product. Once the attributes are listed, draft a Certificate of

457Appendix
Analysis, adding analytical methods and specifications to the pro-
posed attributes and tests.
• Describe in-process samples that will be taken during the manufac-
turing process, and identify attributes, tests, and possible specifica-
tions for each sample.
• Briefly describe each test chosen for testing bulk substance and final
product, for both release and stability, and assays for in-process
samples. Explain why each test was chosen, based on performance,
intended use, and meaningful results. Describe what is known about
the specificity, accuracy, precision, range, and robustness of each
assay when applied to this or to other classes of product. If an assay
(e.g., potency) is to be developed just to test this product, justify the
need to develop the assay and present ideas on its nature. Discuss
the nature or need for control reagents and reference standards for
the assays.
• Develop stability protocols for drug substance (DS) and final prod-
uct (FP) and explain why each was chosen for the purpose of indicat-
ing stability. Describe any tests used to measure stability that were
not applied to product release.
• Consider attributes, analytical tests, and specifications for critical
raw materials used in the process, working closely with the manu-
facturing plan. Prepare a draft Certificate of Analysis for what you
consider to be the five most critical raw materials.
• Identify which analytical tests will be qualified, verified, and/or val-
idated, and mention the most likely point in the development cycle
of each activity. Highlight critical assays that might require special
attention in development, qualification, or validation.
Chapter 8: Nonclinical Studies
• Describe the nonclinical studies that are typically performed for this
class of product and for products used with this indication. Consider
both regulatory guidelines and precedent.
• What additional work must be performed to complete a pharmaco-
kinetic and pharmacodynamic profile for this product, if it is given
by the route and doses currently suggested in the clinical studies
plan. Outline the studies that must be performed, and describe how
and where these studies might be performed. Consider major design
criteria for these studies.
• Describe the nonclinical toxicology studies that have been per-
formed with other products in this class of product. Consider both
regulatory guidelines and precedent.

458 Appendix
• Identify the nonclinical toxicology studies that should be performed
to ensure safety of this product. For each study, provide a brief pur-
pose and design and recommend when these studies might be per-
formed by phase of development.
• Describe how quality system, cGLP, will be ensured for the planned
studies.
Chapter 9: Clinical Studies
• Outline, in general, the clinical program that will be performed dur-
ing the course of development.
• Given the nature of the product and the indication, outline the piv-
otal or Phase 3 clinical study, and describe the objectives and indi-
cation and the population in which your product will be tested.
Describe the patient or subject population, approximate size, and
scope and critical elements of study design.
• Outline Phase 2 clinical study on which you will demonstrate proof
of principle and from which you intend to derive the information on
which to base Phase 3 clinical study.
• Outline Phase 1 clinical study or studies that will be the foundation
for Phase 2 study design.
• Present the requirements for choice of an investigational site and a
principal investigator for each phase of the clinical development you
outlined and in relation to your product and indication.
• Discuss the elements of current good clinical practices that would
be required for each phase of the clinical development you have
outlined.
• Describe any ethical considerations that might affect any one of your
clinical trials, given the nature of the product, the study design, and
the intended patient population.

459
Index
Note: Page numbers followed by f and t refer to figures and tables, respectively.
21 CFR, 71, 126, 180, 412
510(k) process, 77, 99–101
A
Abbreviated New Drug Application
(ANDA), 75–76, 104
Absorption phase, 328
Accelerated Approval, 102
Accelerated stability, 308
Acetaminophen, 75
Active pharmaceutical ingredients
(APIs), 122–123
Acute toxicity testing, 351–352, 354–355
Adaptive design, 406
Adenovirus, 74, 232
ADEs (adverse drug events), 107
The Administrative Procedures Act
(APA) of 1946, 71
Adsorption, Distribution, Elimination,
and Metabolism (ADME),
325–328, 325f
absorption, 325
on bioavailability, 329
distribution, 325–327
excretion, 328
metabolism and biotransformation, 327
Adulterated/misbranded product,
123–124
Adventitious agent testing, 297–298
Adverse drug events (ADEs), 107
Adverse events (AEs), 15, 288, 383–384,
387, 389
clinical, 364–365
report, 178
Advisory committees, 106
Affinity chromatography, 228–229
Agreement State Program, 141
American Type Culture Collection, 212
Ames test, 340
Analyte/test substrate, 266
Analytical chromatographic
technique, 275
Analytical methods, QC, 270, 286–295,
287f, 289f
attributes measurement, 266
controls and reference standards,
299–300
off-the-shelf, 266
qualification, validation, and
verification, 312–317
selection of, 270–277
tools and concepts, 295–297
traits, 267
Analytical precision, 313
ANDA. See Abbreviated New Drug
Application (ANDA)
Animal and Plant Health Inspection
Service (APHIS), 134–136, 143
Animal model, 333–334
development, 342–344
selection, 344, 356
system, 163
Antihypertensive peptide, 16
Antimalarial drug, 74
APA. See The Administrative
Procedures Act (APA) of 1946
APHIS. See Animal and Plant Health
Inspection Service (APHIS)
APIs (active pharmaceutical
ingredients), 122–123
Appearance test, 282, 288
Aseptic technique, 236, 251
Assay performance and validation,
application of statistics,
317–318, 318f
Assay verification, 316
Auditing/monitoring process, 389
Auditing process, 179, 185–188
performance, 186
quality, 187f
Autoinjector, 249

460 Index
B
Bacteria, 212, 232–234, 296
Bacterial cell expression systems,
212–213
Bacterial plasmid DNA, 231–232
Baculovirus, 216
Batch production records (BPRs), 172
Bibliography, 81
Bicinchoninic acid (BCA), 275, 283, 313
reagent-based test, 291
BIMO (biomonitoring), 98
BIO. See Biotechnology Industry
Organization (BIO)
Bioavailability, 329, 332–333
Bioequivalence, 332
Biological potency assays, 273, 285
Biologics, 72–75
follow-on, 104
The Biologics Control Act (BCA) of
1902, 66
Biologics License Application (BLA),
95–96, 98–99, 107, 142
Biomanufacture, 3, 309–310
compliance and quality in, 207–209
design, 196–200
facilities, utilities, and equipment,
253–257
clean work areas for, 255–257
controlled environment,
254–255
equipment, 257
facility design considerations,
253–254
validation, 259–261
for FP, 248–252, 250f, 252f
hypothetical scheme, 239f
life cycle, 201–205
overview, 195
planning, 23–24, 196, 248
process for biotechnology products
bacterial plasmid DNA, 231–232
biologically active peptides,
245–247
biological molecules production,
238–245
cellular products production,
236–238
combination products, 247–248
in-process testing and bulk
substance analysis, 230–231
lipids, glycolipids, and complex
carbohydrates (biologically
active), 245, 246f
live recombinant organisms
production, 232–234
mammalian somatic cell/tissue
products production, 234–236
of recombinant proteins, 217–230
recombinant proteins and nucleic
acids expression, 209–217
QbD in, 198–199
raw material considerations,
205–207
scale-up, 204–205
steps of, 196, 197f
technical considerations for, 200–201
Biomedical technology assessment, 143
Biomonitoring (BIMO), 98
Biopharmaceutical(s), 1, 72, 266
application, 284
bioavailability, 329
biodynamic experimentation, 333
biomanufacturing life cycle in,
201–205
early phase, 202
late-phase, 204
mid-phase, 202, 204
class of, 78f
compliance for
import, 119–120, 137
medical devices, 120
concentration
in blood over time, 328–329, 329f
effect curve, 331–332, 331f
delivery, 323–328
ADME, 325–328, 325f
product delivery to body, 323–325
designer molecule, 331
development, 11, 29, 165, 168, 266, 284,
371
biomanufacturing activities for,
203
Gantt chart format, 43f
life cycle, 87, 88f
PERT chart format for, 45f
timeline format, 44f

461Index
excipient, 345
fingerprint of, 274
FPs, 249, 283
immediate upregulation by, 361
immunological toxicity study, 362
label, 108
nonclinical activities in, 321–323,
322f, 337–339, 338f
nonclinical safety testing for
evaluation, 352–353
operation, 257
pharmacokinetics and
pharmacodynamics in,
333–335
phased scheme, 7, 8f
preventive and therapeutic, 7
problems, 175
processing, 196
promotional information, 109
safety assessment
acute toxicity testing, 351–352,
354–355
carcinogenicity testing, 360–361
genetic toxicology, 363, 366–367
immunotoxicology, 361–363
nonclinical safety testing,
351–353
nonclinical study design,
elements, 347–351
program, 337–340
protocols and performance,
346–347
reproductive, developmental,
and teratogenicity toxicity
testing, 359–360
subchronic and chronic toxicity
testing, 352, 356–359
tissue binding/local tissue
tolerance, 367–368
toxicology, 336–337
in vitro screening test, 340–342
in vivo safety testing, 342–346
treatment, 284
type of, 78f
Biopharming, 238, 244, 253
Bioreactor, 168, 220, 238
cell, 221
transgenic goat as, 242
Biotechnology, 32
development
ICH guidelines in, 127–128
program, 169–170
team, 13
environmental regulations in,
141–142
industry, biological materials,
132–133
material transportation, 133–136
microbial products, 142
operation, 1–3, 142, 259, 300
focal point, 2
functional areas, 2–3
international diligence, 144–146
metaphor, 5–6
project management in, 41–58
quality systems, 188–189
skills and backgrounds, 1–2
themes, 1
product(s), 4, 7, 13, 253
biomanufacturing processes, 209
development, 10
fermentation, 218
importation/exportation, 137, 139
with IVD, 100
medical, 126–132
nature, 198–199
and reviews FDA, 79–81
and project management, 29–31
project team, 37–38
QC, 265
quality in, 149–150
Biotechnology-derived products, 72, 79
Biotechnology Industry Organization
(BIO), 131, 145
Biotechnology Regulatory Services
(BRS), 134, 143
Biotransformation, 327
BLA. See Biologics License Application
(BLA)
Black box warning, 86
Blood-borne pathogen, 141
Blood products, 72–73
BPRs (batch production records), 172
Bradford test, 291
Brand name drug, 104
Breakthrough therapy, 103

462 Index
British Pharmacopoeia (BP), 271
BRS. See Biotechnology Regulatory
Services (BRS)
BS. See Bulk substance (BS)
Budgeting, 54
Budgeting monetary requirements, 55
Bulk API/final product, 123
Bulk substance (BS), 196, 217, 263, 265,
267, 282, 302
analysis, 230–231
batches, 281–282
biopharmaceutical
CoA, 268t–269t
stability protocol for, 305t, 307
potency assay, 281
sterile filtration, 249
Bureau of Industry and Security (BIS), 136
C
CAPA (corrective and preventive
action), 122
Carbohydrate analysis, 276
Carcinogenicity testing, 127, 340, 353,
360–361
Case report forms (CRFs), 380, 391,
395–396, 400
CBER. See Center for Biologics
Evaluation and Research
(CBER)
CBP. See Customs and Border Protection
(CBP)
CDC. See Centers for Disease Control
and Prevention (CDC)
CDER. See Center for Drug Evaluation
and Research (CDER)
CDRH. See Center for Devices and
Radiological Health (CDRH)
Cell banks
production, 211f, 216–217
QC of, 297–298
transgenic plant, 244
Cell-based assay, 276–277
Cell growth curve, 221
Cell karyotyping, 295
Cell phenotyping, 296
Center for Biologics Evaluation and
Research (CBER), 69–70, 72, 74,
76, 356
Center for Devices and Radiological
Health (CDRH), 70, 77, 100
Center for Drug Evaluation and
Research (CDER), 69–72, 74, 76
Center for Veterinary Medicine (CVM),
70, 79
Centers for Disease Control and
Prevention (CDC), 134, 136
Centrifugation, 222, 225, 234
Certificate of Analysis (CoA), 263,
266–267, 319
drug product, 282–285
QC, 267–270
vendor-supplied, 310
CF. See Consent form (CF)
CFR. See Code of Federal Regulations
(CFR)
cGCP. See Current Good Clinical
Practices (cGCP)
cGLP. See Current Good Laboratory
Practices (cGLP)
cGMP. See Current Good Manufacturing
Practices (cGMP)
CGTP (Current Good Tissue Practices), 73
Change control, 175, 184
Chemistry, Manufacturing, and
Controls (CMC), 129
Chinese hamster ovary (CHO), 214
CHMP (Committee for Human Medical
Products), 131
Chromatogram, 292, 307
Chromatography, 292
gel, 292
high-pressure liquid, 292–294,
293f–294f
preparative, 227–228
equipment, 227f
flow diagram, 226f
Chronic infection, 401
Chronic toxicity testing, 352, 356–359
Clean dose level, 356
Cleaning protocols, 257
Clearance, 328–329
Clinical data, 395–396
Clinical development, 371, 405
biopharmaceutical products, 375
key issues, 375
planning, 18–19, 377–378
Clinical laboratory testing, 248

463Index
Clinical pharmacology, 11, 15, 97, 375,
404–405
Clinical program, 376, 417
Clinical protocol, 89–90, 379, 382
clinical trials and, 378, 381–387
elements, 378, 381, 383
Clinical safety and toxicology, 375
Clinical study/research, 172, 174,
371, 376
associates, 414
organization, 375–376
cGCP, qualtiy in, 377
clinical development planning,
377–378
phases, 375–376
science, 376–377
overview, 373–374
Clinical summary report (CSR), 397, 400
Clinical testing laboratories, 396–397
Clinical trial(s), 371
documents, 379–380
historical information, 374–375
individuals and responsibilities, 379
infrastructure
clinical testing laboratories,
396–397
collection, 395–396
design of, 378–387
human subjects, patients, and
volunteers, 388
investigational product, 394–395
IRB, process of IC, and IC form,
392–394
PI, 391–392
results, 397
sponsor, 388–391
monitoring, 389
operations, 397–398
activities leading, 398–400
clinical pharmacology studies,
404–405
first-in-human study, 400–403,
402f, 403f
global, 409
new populations/indications,
408–409
proof-of-concept study, 405–406
REMS, 407–408
therapeutic confirmatory, 406–407
overview, 371–373, 372f
performance, 413
phases, 375–376
quality system, 409–412
clinical study data and
documents, 413–414
ethical behavior and well-being,
415–416
monitoring and auditing, 414
quality and cGCP, 412–413
scheme, 372f
WHO guidelines, 128, 131
CMC (Chemistry, Manufacturing, and
Controls), 129
CMO. See Contract Manufacturing
Options (CMO)
CoA. See Certificate of Analysis (CoA)
Code of Federal Regulations (CFR), 71,
128, 273, 393
Codex standards, 145
Committee for Human Medical
Products (CHMP), 131
Common Technical Document (CTD),
91–92, 92f
electronic submission of, 92–96
Communication, 37, 46, 50, 140
day-to-day, 48
direct, 38, 63, 107
electronic, 60
and feedback, 49–50
regulatory, 85–86
team, 48
Compendia, 82, 309
test, 271–273
Complex carbohydrates, 245
Computer software/program, 62
Concentration effect curve, 331–332, 331f
Concept protocol, 378–379
Conditional approval, 407
Consent form (CF), 379, 393
Contract Manufacturing Options
(CMO), 253, 257–259
Contractor, vendor, and consultant
control, quality system, 169–171
Contract research organization (CRO),
59, 117, 170–171, 273, 285, 388,
391
Control article, 345
Controlled bioavailability, 329

464 Index
Controlled studies, 374
Corrective and preventive action
(CAPA), 122
Corrective/preventive actions, change
control, 175–176, 177f
CRFs. See Case report forms (CRFs)
Criminal prosecution, FDA judicial
actions, 125
CRO. See Contract research organization
(CRO)
Cross-referencing information, 101
CSR (clinical summary report), 397, 400
CTD. See Common Technical Document
(CTD)
Current Good Clinical Practices (cGCP),
114, 154, 156, 373–374, 409–416
for clinical studies, 117
in clinical trial operations, 412–413
key components, 117, 119
quality in, 377
Current Good Laboratory Practices
(cGLP), 114, 154, 339, 347, 355,
368–369
elements of FDA, 117–118
for nonclinical laboratory studies, 117
Current Good Manufacturing Practices
(cGMP), 114, 154, 156, 170, 202,
207–209
elements, 114–117
for manufacture and quality control,
114–117
objective, 208
quality system, 157
Current Good Tissue Practices (CGTP), 73
Customs and Border Protection (CBP),
120, 136, 139
The Cutter Incident of 1955, 66
CVM. See Center for Veterinary
Medicine (CVM)
Cytogenetic analysis, 295
Cytokine storm, 363
D
Data and Safety Monitoring Board
(DSMB), 407
Day-to-day communication, 48
DBOP (Division of Biologic Oncology
Products), 76
Declaration of Helsinki, 392, 415–416
Degree of scatter, 314
Design change, quality system, 169
Design control, QbD, 163–169
change, 169
documents and records, 166–167
elements, 165–166, 165f
product specific, 166
Designer molecule biopharmaceutical, 331
Developmental toxicity testing, 359–360
Dietary supplements, 79–81
Direct communication, 38, 107
tools, 63
Division of Biologic Oncology Products
(DBOP), 76
Documentation system, 182–184
Document control, quality, 178, 179f
Downstream process, 196, 279
recombinant proteins, 222–230
Downstream purification, 222–230
material list, 207
Draft/concept product labeling, 11, 13
Drafting CoA, 267–270
BS, 268t
FP, 269t
Draft labeling, 13, 99
Draft project management plan, 17, 46
Draft TPP, 11
Draft WBS, 61
Drug(s), 75–76, 89, 102
CoA, product, 282–285
development, 7
generic, 104
interaction, 12, 327
laws, 67, 71
regulation/guidance, 71
nonclinical safety tests, 352–353
in vitro safety tests, 341
Drug-drug interactions, 407
Drug-receptor interaction, 330
DSMB (Data and Safety Monitoring
Board), 407
E
Early phase biomanufacturing
development, 202
Early phase dosing study, 401
Earned value management (EVM), 53, 54f

465Index
eCTD. See Electronic CTD (eCTD)
EEC (European Economic
Community), 131
Effective project tracking, PM, 53
Efficacy topics (E), 127–128
Electronic CTD (eCTD), 92–96, 98
Electrophoretic method, 290
Electrospray ionization-mass
spectrometry, 294
Elements to ensure safe use (ETASU), 408
Elimination phase, 328
Elution buffer, 228–229
EMA (European Medicines Agency), 131
Embryo-fetal development, 360
Endotoxin, 212–213
test, 271, 276, 288
End point, 354, 384, 387, 401
acute toxicity study, 355
immunological toxicity study, 362
subchronic/chronic toxicity study, 357
Engineered retrovirus, 247
Environmental assessments, 142
Environmental controls, 173
Environmental Protection Agency
(EPA), 142–143
Environmental release, 135
EPA (Environmental Protection
Agency), 142–143
Epidemiological, 373
Escherichia coli, 74, 212
Establishment Inspection Report (EIR), 122
ETASU (elements to ensure safe use), 408
European Economic Community
(EEC), 131
European Medicines Agency (EMA), 131
European Pharmacopoeia (EP), 271
EVM (earned value management), 53, 54f
Excretion, ADME, 328
Expression of recombinant proteins and
nucleic acids, 209–217
bacterial cell expression systems,
212–213
cell banks production, 211f, 216–217
genes, vectors, and host cells, 210–212
mammalian/insect cell expression
systems, 213–216
molecules production from
expression vectors, 209–210
yeast cell expression systems, 213
Expression vector, 209–210, 214
External audits, 180, 185
F
Face-to-face meetings, 61, 63
Failure mode and effects analysis
(FEMA), 52
Fast Track Designation, 103
Fault-tree analysis, 52
FDA. See Food and Drug
Administration (FDA)
The FDA Amendments Act of 2007, 407
FDA-regulated products, 71–81
biologics, 72–75
biotechnology products/reviews,
classes of, 79–81
combination products, 77–78, 78f
drugs, 75–76
medical devices, 76–77
FDA’s Inspection Operations Manual, 122
The Federal Food, Drug, and Cosmetic
(FD&C Act) Act of 1938, 65–66,
70, 77
The Federal Insecticide, Fungicide and
Rodenticide Act (FIFRA), 142
The Federal Plant Protection Act, 135
Federal trade commission (FTC), 65
FEMA (failure mode and effects
analysis), 52
Fermentation
microbial, 218
upstream, 206
vessel, 261
yeast cell, 218–220
FIFRA. See The Federal Insecticide,
Fungicide and Rodenticide Act
(FIFRA)
Final product (FP), 123, 196, 248–252, 263
active ingredient in, 283
CoA, 269t
manufacture, 250
problem, 283
QC tests, 282–283
stability protocol, 306t
strength, 283
First-in-human study, 400–403, 402f, 403f
FOIA. See The Freedom of Information
Act (FOIA)

466 Index
Follow-on biologics, 104
Food and Agricultural Organization
(FAO), 145
The Food and Drug Act, 152
Food and Drug Administration (FDA),
6, 11, 65–71, 120–121, 129–131,
152, 161–162, 164, 186, 190
additional regulatory activities,
105–109
advisory committees, 106
applications, regulatory operations,
84–99
BLA/NDA, 96–99
environment, 84
IND application, 89–96
investigational use/marketing
approval, 87–88
planning, 84
risk vs. benefit, 84, 86–87
biotechnology products/reviews, 70,
79–81
CDER, 75
centers/offices, 69
enforcement actions, 123–125
form, 122–123
guidance documents, 129–131
clinical, 130
CMC, 129
preclinical, 131
quality, 130
inspection process, 120–123
law/regulations for
biopharmaceuticals
food and drug law, regulation,
and guidance, 71
historical basis for FDA
regulation, 65–66
regulatory organization of FDA,
66–71, 68f
letters to manufacturers, 108
product liability, 125
review staff, 94
user fee, 99
The Food and Drugs Act of 1906, 65
The Food, Drug, and Cosmetic Act of
1938, 121, 123
Formal clinical hold mechanism, 93
Formal meetings, 48
Forming stage, 46
Formulation–fill–finish production
methods, FP, 248–252, 250f, 252f
FP. See Final product (FP)
The Freedom of Information Act (FOIA),
107, 123
Freeze drying, 251
Funnel concept, 164
G
Gantt chart, 20, 42, 43f, 62–63
Gas chromatography, 292
Gel electrophoresis, 274
Generally Recognized As Safe (GRAS),
80
General safety test, 283, 288
Generic drugs, 75–76, 104
Generic tests, 273, 283
Genes, vectors, and host cells, 210–212
Gene therapy, 333, 401
Genetically modified organisms
(GMOs), 135, 142–144
Genetic engineering, 76, 135, 144, 200
Genetic toxicology, 363, 366–367
Global clinical trials, 409
GLP regulation, 368
Glycolipids, 245
Glycosylation, 212, 244
GMOs. See Genetically modified
organisms (GMOs)
Golden rule, 39
Gram-negative bacteria, 212–213, 288
GRAS (Generally Recognized As Safe), 80
H
Haemophilus influenzae type b, 245
Half-life value, 328, 404
Hallmarks, 159, 162, 230, 399
quality systems, 153, 159–180
Hamburg, Margaret A., 198
Health and safety plans, 140–141
Heat, ventilation, and air conditioning
(HVAC) system, 255, 257
High-pressure liquid chromatography
(HPLC), 292–294, 293f–294f,
307, 316
HIV test kits, 77
Homeopathic medicine, 81

467Index
Host cell, 210–212, 296–297
DNA, 291
protein, 291
RNA, 291
yeast, 213
HPLC. See High-pressure liquid
chromatography (HPLC)
Human clinical study, 410–411
Humanized monoclonal antibody,
166–167, 363
Human pharmacology, 375
Human subjects, patients, and
volunteers, clinical trial, 388
Hydrophobic interaction
chromatography, 228
Hypertension (high blood pressure),
14, 384
I
IATA (International Air Transportation
Association), 132
IB. See Investigator’s Brochure (IB)
IBC (Institutional Biosafety
Committees), 143–144
IC. See Informed consent (IC)
ICF. See Informed consent form (ICF)
ICH. See International Council for
Harmonization (ICH)
IDE (Investigational Device Exemption),
100
IFPMA. See International Federation of
Pharmaceutical Manufacturers
and Associations (IFPMA)
Immunoelectrophoresis, 290
Immunohistochemical methods, 290
Immunotoxicology, 361–363
Impurities-truncated peptides, 247
INAD (Investigational New Animal
Drug), 79
Inclusion and exclusion criteria, 384–386
IND. See Investigational New Drug
(IND) application
Individual effective concentration, 332
Induced pluripotent stem cell (iPSC),
236–237
Industry standard, 75, 208
Informal meetings, 48
Informal risk analysis, 52
Informed consent (IC), 379, 392, 410
elements, 393
process and form, 392–394
Informed consent form (ICF), 379, 393
Injunctions tools, 125
In-life
measures, 350–351
phase, 349
Inoculum, 219
Inorganic and organic chemicals, 310
In-process testing, 174, 223, 230–231,
285–286
Inspections, 98
FDA, 121–123, 147
quality systems, 173–174
Institutional Biosafety Committees
(IBC), 143–144
Institutional review board (IRB), 389,
391, 393–394, 399
Intergeneric microorganisms, 142–143
Internal audits, 180, 185
International Air Transportation
Association (IATA), 132
International Council for
Harmonization (ICH), 91,
126–128, 154, 164, 377, 412
International Federation of
Pharmaceutical Manufacturers
and Associations (IFPMA),
126, 131
International Standards Organization
(ISO) 9001, 154–156, 170
Intramuscular injection, 14
Investigational Device Exemption
(IDE), 100
Investigational New Animal Drug
(INAD), 79
Investigational New Drug (IND)
application, 89–96, 321, 377
contents, 90–91
CTD, 91–92
eCTD, 92–96
legal document, 93
regulatory operations, FDA
applications, 89–91
treatment, 102
Investigational product, 363, 392,
394–395
accountability log, 380

468 Index
Investigator’s Brochure (IB), 90, 380, 389
elements, 390
In vitro assay, 276–277
In vitro diagnostics (IVDs), 100
In vitro screening test, 340–342
In vivo safety testing, 341–346
animal model development, 342–344
test product formulations, delivery
routes, and dosing designs,
344–346
Ion-exchange chromatography, 228
Ionic strength, 288
iPSC (induced pluripotent stem cell),
236–237
IRB. See Institutional review board (IRB)
Isoelectric focus gels, 292
IVDs. See In vitro diagnostics (IVDs)
J
Jargon, 2, 174
The Jungle (book), 65
K
Kilodalton (kDa), 294
Knee joint cartilage replacement, 235
L
Label claim, 12, 13, 377
Labeling, FP, 251–252
The Lacey Act, 135
Late-phase biomanufacturing
development, 202, 204, 260
Lessons learned reports and meetings, 58
Lethality testing, 340
Lethal toxin, 279
Letter of authorization (LOA), 101
Limit of detection (LOD), 267, 313
Limit of quantitation (LOQ), 267, 313
Linearity, QC, 267, 313
Liquid chromatography-mass
spectrometry, 294
Live recombinant organisms, 232–234
Loading dose, 330
Local tissue tolerance, 367–368
Lymph amebocyte lysis (LAL), 288
Lyophilization, 251
M
Maintenance dose, 330
Mammalian/insect cell expression
systems, 213–216
Mammalian somatic cell/tissue
product, 234–236
Management reviews, 162
Market- and user-driven process, 248
Marketing
application, 96–99
approval, 87–88, 132
considerations for biotechnology
development, 4–7
Mass spectrometry-time of flight, 295
Mass spectroscopy, 276
Master cell banks (MCB), 216–217
Master File (MF), 101–102
Maximal efficacy, 332
Maximum concentration value, 328
MDR (medical device reporting), 120
Medical device, FDA
applications, 99–104
generic drugs and biosimiliar/
follow-on biologics, 104
special documents, pathways, or
exemptions, 101–104
-regulated products, 76–77
Medical device reporting (MDR), 120
MedWatch, 107
Merck Index (book), 271
Merck Manual (book), 271
Metabolism, 327
Microbes, 14, 255
Microbial enumeration test, 287
Microbial fermentation
equipment, 218f
phases of, 219, 220f
Microbial identification, 296
Microbial limits test (MLT), 287–288
Microbial load/bioburden, 287
Microbial product, 73, 296
Microprocessors, 61, 219
Microsoft Project®, 53–54, 62
Mid-phase biomanufacturing
development, 202, 204
Milestone-related task, 52
Misbranded labeling, 124
Monitoring and auditing clinical trials, 414

469Index
Monoclonal antibody, 76, 98,
163, 168, 170, 228, 290,
332–335, 367
Multidisciplinary topics, 127
Multiple potency assays, 284
Multiple rising dose design, 401, 403f
Mundane issues, 189
Mutagenicity testing, 340
N
NADA (New Animal Drug
Application), 79
National Center for Import and Export
(NCIE), 135
National Drug Code (NDC), 105
The National Environmental Policy Act
of 1969, 142
National Formulary (NF), 75, 272
National Institute for Occupational
Safety and Health
(NIOSH), 141
National Institutes of Health (NIH),
143–144
National regulatory authorities
(NRAs), 126
National Science Advisory Board for
Biosecurity (NSABB), 144
NCIE (National Center for Import
and Export), 135
NDC (National Drug Code), 105
Negative control antisera, 290
Network building, 46
Neupogen (filgrastim) drug, 104
Neural system, 3–5, 10
New Animal Drug Application
(NADA), 79
New drug application (NDA), 65, 96,
98–99, 107
NF. See National Formulary (NF)
NIH (National Institutes of Health),
143–144
NIOSH (National Institute for
Occupational Safety and
Health), 141
Nonclinical animal study, 345
Nonclinical planning, 22–23
Nonclinical safety testing, 339–340,
351–353
Nonclinical study, 172, 321–323, 322f
in biopharmaceutical development
project, 337, 338f
calculating dose, 346
design, 346
elements, 347–351
protocol, 175, 346–347
quality of, 368–369
route of delivery, 345
Nonclinical testing, 73, 332, 339
Nonclinical toxicology testing, 16
Nonconforming product, 175
Non-FDA regulations, 126
application of, 146
biotechnology
environmental regulations in,
141–142
importation/exportation, 137–139
importing, possessing/
transferring, 134–136
international and foreign NRAs,
126–132
international diligence in, 144–146
GMOs, 142–144
occupational health and safety,
140–141
The Public Health Security and
Bioterrorism Preparedness and
Response Act of 2002, 136–137
transporting infectious/hazardous
materials, 132–134
Nongovernmental organizations/
agencies (NGOs), 132
Normal saline, USP grade, 170
Novel biotechnology product, 1, 84, 401
NRAs (national regulatory
authorities), 126
NSABB (National Science Advisory
Board for Biosecurity), 144
N-terminal sequencing, 283, 289
Nuclear magnetic resonance, 276
Nuclear Regulatory Commission
(NRC), 141
Nuremberg Code, 392, 416
O
OAI (official action indicated), 122–124
Obligation log, 380

470 Index
Office of Biotechnology Activities
(OBA), 143–144
Office of Biotechnology Products
(OBP), 76
Office of Blood Research and Review
(OBRR), 72
Office of Cellular, Tissue and Gene
Therapies (OCTGT), 73
Office of Combination Products
(OCP), 77
Office of Vaccine Research and Review
(OVRR), 74
Official action indicated (OAI), 122–124
Off-label, 109
Operational documents, 161, 183
Operational research, 158
Ops Manual, 387
Optimized clinical formulation, 334
Oral-gastrointestinal absorption, 325
Oral ingestion, 324
Orphan Drug Exclusivity (ODE), 103
Orphan Products Development
(OPD), 103
OSHA (Occupational Health and Safety
Administration), 141
Osmolality, 219, 288
Osmometer, 288
Outsourcing models, PM, 59
Over-the-counter (OTC) drugs, 75, 108
P
Packaging
FP, 251
and labeling, 176
PAGE. See Polyacrylamide gel
electrophoresis (PAGE)
Pathogens, 133
PDP. See Product development plan
(PDP)
PDR. See Physician’s Desk Reference
(PDR)
PDS. See Product development strategy
(PDS)
Peptide, 14
antihypertensive, 15
biologically active, 245–247
mapping, 276, 292
therapeutic product, 13
Peptide-based biomolecules, 247
PERT chart, 20, 45f, 63
Pharmaceutical, 32, 75, 229
quality system (ICH Q10), 155
Pharmaceutical Research and
Manufacturers of America
(PhRMA), 131
Pharmacodynamic, 328–333
in biopharmaceutical development,
333–335
variability, 332
Pharmacokinetic (PK), 328–333
in biopharmaceutical development,
333–335
clearance aspect, 329
study, 376, 399, 404
Phased product biomanufacture, 202
pH, QC regulatory requirement, 289
PhRMA (Pharmaceutical Research and
Manufacturers of America), 131
PHS Act. See The Public Health Service
Act (PHS Act)
Physician’s Desk Reference (PDR), 96, 271
PI. See Principal investigator (PI)
Pichia pastoris, 212–213, 220
Pilot production, 217
Pivotal clinical trial, 376, 406
Placebo, 345, 374, 382, 387, 401, 405–406
Planning backward, 9f
Plant pest, 135
Plasmid DNA, 73
bacterial, 231–232
Platelet-rich plasma (PRP), 245
Pluripotent cells, 73
cellular product from, 236–238
PM. See Project manager (PM)
PMA. See Premarket approval (PMA)
PMP. See Project management plan (PMP)
Polyacrylamide gel electrophoresis
(PAGE), 273, 289–290, 289f
Polyhistidine tag chromatography, 229
Polymerase chain reaction, 210, 296
Polysaccharide-protein, 246f
Polyvalent antiserum, 290
Postmarketing requirements and
activities, 107–108
Potency, 270, 298, 331–332
assays, 276, 286
tests, 234, 266, 281, 284

471Index
Pre-approval Inspections (PAI), 98
Precision, 267, 313–314, 314f
Pre-IND communication, 95
Pre-IND meeting, 89, 94–95
Premarket approval (PMA), 77, 99–100
Preparative chromatography, 227–228
equipment, 227f
flow diagram, 226f
Preservation, storage, and handling, 176
Principal investigator (PI), 382, 388,
391–392
Process control, 172–173
Product attributes, QC, 265–266
Product design plan, 166
Product development
planning
PDP, 16–28
rationale, 7–10
TPP, 10–16, 9f
project team, skills, 38
quality systems approach to, 153–155
Product development plan (PDP),
16–29, 195
biomanufacturing, 23–24
clinical development, 18–19
nonclinical, 22–23
product planning, elements of, 26–28
project management, 19–20
quality control, 24–26
quality systems and assurance, 26
regulatory, 20–22
Product development strategy (PDS), 16,
84, 87
Product identification and traceability,
171–172
Product labeling, 96–99, 109, 176
concept, 11–12
Product liability, 125
Product stability testing, 302–308, 303f
Project champion, stakeholder, 38, 56
Project committee meetings, 158
Project leader, 38, 56
Project management, 17
background, 31–32
in biotechnology, 29–31, 30f
communication and feedback,
49–50
establishing, 41–42
human factors in, 55–57, 58f
metrics and tracking progress,
53–54, 54f
project completion, 57–58
project risk assessment, 51–52, 51f
project team/hands-on, 46
resources, 54–55
team dynamics, 46–49
WBS, 42–45
contracts and collaborations,
59–60
environment, 34–36
operational phase, 30
participants in, 37–41
planning, 19–20
project objectives and schedules,
36–37
sociotechnical considerations, 37
software, 42, 61, 63
stages, 30f
tools, 43f, 44f, 45f, 49
effective, 61–63
virtual teams, 60–61
Project management plan (PMP), 29–30,
32–34, 33f
elements, 33–34
environment, 34–36
participants in, 37–41
project objectives and schedules,
36–37
sociotechnical considerations, 37
Project manager (PM), 11, 17, 29, 37–39
attributes of effective, 40
diligent, 53
effective project tracking, 53
full-time, 41–42
human resource duties, 55–56
selection, 40
Project risk assessment and
management, 51–52, 51f
Project team, 6, 30–31, 52
biotechnology, 37–38
formation in biotechnology, 46
guidelines and elements of, 47
human sources conflict on, 57
types, 47
Project wrap, 58
Promotional labeling, 108
Proof-of-concept study, 405–406
Protein reference standards, 290

472 Index
The Public Health Security and
Bioterrorism Preparedness and
Response Act of 2002, 136–137
The Public Health Service Act (PHS
Act), 66
The Public Health Service Act (PHS Act)
defined, 72
Public law PL107-188, 138–139
Public regulatory information, 82
PubMed, 82
The Pure Food and Drugs Act in 1906, 65
Q
QA. See Quality assurance (QA)
QAU. See Quality assurance unit (QAU)
QbD. See Quality by design (QbD)
QC. See Quality control (QC)
QSR. See Quality systems regulation
(QSR)
Quality
agreements, 171
audits, 185
hallmark, 160
management
principles, 160, 160f
QAU/QA, 191
quality issues/problems, 192–193
quality systems for research,
191–192
risk-based approaches, 190
six sigma, 191
TQM, 190
unique and effective approaches,
190–193
manual, 156–157
plan, 157–159
professionals, 158, 167, 175, 180, 182,
192
steering committee, 158
Quality assurance (QA), 3, 175, 180, 192
function, 181–182
planning, 26
statistics, 191
Quality assurance unit (QAU), 159, 176,
180–181, 348
auditing, 185–188
control and manage documentation
system, 182–184
functions, 181–182
investigate situations, 184
major task, 183
qualified and trained staff, 184–185
report, 161
responsibilities of, 181f
staff members, 185
weak, 189
Quality by design (QbD), 149, 196, 198–199
design change, 169
and design control, 163–169
overview, 163–164
Quality control (QC), 263
additional analytical tools and
concepts, 295–297
analytical controls and reference
standards, 299–300
analytical methods, 286–295
qualification, validation, and
verification, 312–317
selection, 270–277
assays, 273
performance and validation,
317–318
cell banks, 297–298
CoA, 267–270
for drug product, 282–285
compendia and reference, 271
development cycle, 264–265, 264f
in-process testing, 285–286
laboratory, 270, 287f, 311
manufacturing environment, 310–311
objective, 263
overview, 263–265, 264f
plan, 265
planning, 24–26, 263–264
product attributes, 265–267
analytical methods, 266–267
product stability testing, 302–308
sampling methods, 298
specifications development, 277–281
test failures, out-of-specification
results, and retesting, 300–301
testing, 265, 282
of raw materials, 308–310
results, 282
Quality system(s)
in biotechnology, 149–150, 188–189
cGCP, 117, 119, 412–413

473Index
clinical study data and documents,
413–414
clinical trials, 409–416
defined, 162
ethical behavior and well-being,
415–416
evolution, 150–153, 151f
fundamental criteria for building
effective, 159–180
auditing, 179–180
contractor, vendor, and consultant
control, 169–171
control and corrective/preventive
actions, 175–176, 177f
customer concerns and adverse
event reports, 178
document control, 178, 179f
environmental control, 173
inspection/testing (quality
control), 173–174
management responsibility,
160–162, 160f, 161f
material, service, or product,
release of, 174
packaging and labeling, 176
preservation, storage, and
handling, 176–177
process control, 172–173
product identification and
traceability, 171–172
QbD and design control, 163–169
servicing, 178
training, 178–179
hallmarks of, 153
monitoring and auditing, 414
planning
objectives, 156
quality manual, 156–157
quality plan, 157–159
product development, 153–155
QAU, 180–188
regulatory compliance, 113–119
cGCP, 117, 119
cGLP, 117–118
cGMP, 114–117
for research, 191–192
Quality systems regulation (QSR), 99,
120, 155
Quality topics (Q), 127
R
RAC (Recombinant Advisory
Committee), 144
Randomization/blinding process,
374, 382
Recombinant Advisory Committee
(RAC), 144
Recombinant biological molecules, 158
Recombinant blood products, 72
Recombinant crop plant, 150
Recombinant DNA vaccine, 247
Recombinant microorganisms, 133
Recombinant protein (r-protein), 13, 173,
231, 265–266, 274–275, 277
analysis, 268t, 269t
biomanufacture of, 217–230
downstream process, 222–230
planning production, 217
upstream process, 218–222
CoA, 313
and nucleic acids expression See
Expression of recombinant
proteins and nucleic acids
production and control by transgenic
plant, 243f
Recombinant vaccine protein, 77
Record control, 178
Reference standards, 274–275,
299–300, 315
Regulatory compliance, 113
for biopharmaceuticals, 119–120
inspection and enforcement, 120–125
with non-FDA regulations, 126–146
quality systems to meet, 113–119
Regulatory environment, 84–85
Regulatory information and resources,
FDA, 81–83
Regulatory intelligence, 20–21, 81–82
Regulatory operations, FDA
applications, 84–99
CTD, 91–92, 92f
eCTD, 92–96
IND, 89–91
investigational use/marketing
approval, 87–88, 88f
marketing applications, 96–99
planning and environment, 84
risk vs. benefit, 84–87

474 Index
Regulatory planning, 20–22, 71–72, 84
elements, 85–86
Regulatory risk identification and
management, 86–87
Remedial bacterium, 15–16
REMS. See Risk Evaluation and
Mitigation Strategy (REMS)
Reproductive toxicity testing, 359–360
Review-to-revision process, 61
Risk assessment, 321–323, 322f
management and project, 51–52, 51f
Risk-based approach, quality
systems, 190
Risk Evaluation and Mitigation Strategy
(REMS), 108, 407–408
Risk management, 27, 49, 52
Risks impact product development, 49
Risk-to-benefit
balance, 86
evaluations, 27
r-protein. See Recombinant protein
(r-protein)
Rulemaking process, 71, 105
Rule of thumb, 159, 279
S
S-1, QC cycle drawing, 281
Saccharomyces cerevisiae, 213
SAEs. See Serious AEs (SAEs)
Safety assessment, biopharmaceutical
acute toxicity testing, 351–352,
354–355
carcinogenicity testing, 360–361
genetic toxicology, 363, 366–367
immunotoxicology, 361–363
nonclinical safety testing, 351–353
nonclinical study design, elements,
347–351
program, 337–340
protocols and performance, 346–347
subchronic and chronic toxicity
testing, 352, 356–359
tissue binding/local tissue tolerance,
367–368
toxicology, 336–337
in vitro screening test, 340–342
in vivo safety testing, 342–346
Safety topics (S), 127–128
Safety training program, 140
Sampling protocols, 287
Scale-up biomanufacturing, 204–205
Screening log, 380
SDS-PAGE. See Sodium dodecyl sulfate
PAGE (SDS-PAGE)
SEC. See Size exclusion chromatography
(SEC)
Seizures, FDA judicial actions, 125
The select agents and public health
security and bioterrorism act
of 2002, 138–139
Serious AEs (SAEs), 387, 389
Single rising dose study, 402f
Six sigma, 191
Size exclusion chromatography (SEC),
228, 275, 292
Skin tissue production, 235–236, 235f
Sociotechnical endeavor, 37
Sodium dodecyl sulfate PAGE
(SDS-PAGE), 275, 283, 289–290,
306–307
Somatic tissues/cells, 73
SOPs. See Standard operating
procedures (SOPs)
Source information/source
document, 395
Sponsor–FDA communication process,
87, 88f
Stability
protocol, 304, 305t, 306t
testing, 174
product, 302–308
Stakeholders, 37–39, 61
Standard operating procedures (SOPs),
172, 184, 274, 301
Statistical analysis, 191, 317, 318f
Statistical trend analysis, 152
Stem cell
cellular products production from,
236–238
development, 143
technology, 73
Sterile filtration, BS, 249
Sterility testing, 280, 286–287, 307
QC testing, 286–287
USP, 287

475Index
Stopping rules, 383–384, 401
Structured system, 190
Subacute toxicity testing, 356
Subchronic toxicity testing, 352,
356–359
T
Tangential flow filtration (TFF), 223,
224f, 225–226
Targeted product profile (TPP), 10–16,
9f, 29, 87, 99, 377
contraindication, 14
draft, 11
elements, 11–12
preparation, 16
warnings and precautions, 14
Team dynamics, 46–49, 48f
Teamwork, 11
TeGenero, 363
issues, challenges, and lessons,
364–366
Teratogenicity toxicity testing,
359–360
Terminal sterilization, 251
Test article, 345
TFF. See Tangential flow filtration (TFF)
Therapeutic
confirmatory, 376, 406–407
effect, 65, 79, 325, 330–331
exploratory, 376
food supplements as, 80
monoclonal antibodies, 76, 325, 359
protein, 229, 351, 360
recombinant protein, 76, 265, 359
use, 376
Threshold effect, 405
Tissue binding, 367–368
Tongue depressor, 77
Total protein assay, 275
Total quality management (TQM), 190
Toxicity testing, 339
acute, 351–352, 354–355
chronic, 356–359
developmental, 359–360
reproductive, 359–360
subacute, 356
subchronic, 352, 356–359
Toxicology, 336–337, 375–376
animal model selection for, 344
chronic, 359
developmental, 342
genetic, 363, 366–367
testing, 340
developmental, 353
reproductive, 352
The Toxic Substances Control Act
(TSCA), 142–143
TPP. See Targeted product profile (TPP)
TQM (total quality management), 190
Tracking, PM, 53, 62
Transfection method, 214, 215f
Transgenic animals/plants, 238–245
Transgenic goat, 240, 241f
as bioreactor, 242
Transportation community, 132
Treatment IND, FDA, 102
Trend analysis, 178, 311, 311f, 318, 318f
TSCA (the Toxic Substances Control
Act), 142–143
Tween 80, 345, 349
U
United States Pharmacopeia (USP), 75,
271–273
sterility test, 287
validation, 312
Upstream fermentation, material
list, 206
Upstream manufacturing, 278–279
Upstream process, 196
recombinant proteins, 218–222
by bacterial/yeast cell
fermentation, 218–220
by mammalian/insect cell
culture, 220–221
recovery, 221–222
Upstream production, 24, 205–206, 279
U.S. Department of Agriculture
(USDA), 79, 120, 134–135,
142–143, 152
U.S. Department of Health and Human
Services (DHHS), 67
U.S. Department of Transportation
(DOT), 132

476 Index
U.S. FDA regulations (21 CFR), 180
U.S. Pharmacopeia National Formulary
(USPNF), 82
V
VAI (voluntary action indicated),
122–124
Validation, 259–261, 312–317
assay, 312, 317–318
master plan, 260
protocols, 260
Vendor audit, 185
Virus, 232–234, 279, 296–297
production and preparation, 233f
Voluntary action indicated (VAI), 122–124
W
Water for injection (WFI), 257
Waxman–Hatch Act of 1984, 76
WBS. See Work breakdown structure
(WBS)
WCB. See Working cell banks (WCB)
Western blot test, 274, 283
WFI (Water for injection), 257
WHO. See World Health Organization
(WHO)
Woodcock, Janet, 196
Work breakdown structure (WBS), 37,
42–45
draft, 61
project management tool, 43f,
44f, 45f
Working cell banks (WCB),
216–217, 234
World Health Organization (WHO), 128,
132, 145
Y
Yeast cell expression systems, 213
Z
Zarxio (filgrastim-sndz) drug, 104

Cover
Half Title
Title Page
Copyright Page
Dedication
Table of Contents
Preface
Acknowledgments
Authors
1: Introduction to Biotechnology Operations: Planning for Success
Biotechnology Operations
Marketing, Financial, and Business Considerations for Development
Product Development Planning
Rationale for Product Development Planning
The Targeted Product Profile
The Product Development Plan
Clinical Development Planning
Project Management Planning
Regulatory Planning
Nonclinical Planning
Biomanufacturing Planning
Quality Control Planning
Quality Systems and Quality Assurance Planning
Additional Elements of Product Planning

Summary of Planning for Success
2: Project Management
Biotechnology and Project Management
Background of Project Management
Project Management Plan
The Project Management Environment
Project Objectives and Schedules
Sociotechnical Considerations
Participants in Project Management
Project Management in Biotechnology Operations
Establishing Project Management
The Work Breakdown Structure
Forming a Project Team and Hands-on Project Management
Team Dynamics
Communication and Feedback
Project Risk Assessment and Management
Metrics and Tracking Progress
Resources: Planning and Usage
Human Factors in Project Management
Project Completion
Project Management with Contracts and Collaborations
Virtual Teams
Tools for Effective Project Management
Summary of Project Management in Biotechnology Development
3: Regulatory Affairs
The U.S. Food and Drug Administration: Law and Regulations for Biopharmaceuticals
Historical Basis for FDA Regulation
Regulatory Organization of the FDA
Food and Drug Law, Regulation, and Guidance
FDA-Regulated Products
Biologics
Drugs
Medical Devices
Combination Products
Other Classes of Biotechnology Products and Their Review at the FDA
Products for Veterinary Use
Cosmetics, Food, Dietary Supplements, Homeopathic, or Nutritional Products

FDA Regulatory Information and Resources: Regulatory Intelligence
Regulatory Operations for FDA Applications
Regulatory Planning and the Regulatory Environment
Risk Versus Benefit
Applications Seeking FDA Investigational Use or Marketing Approval
Investigational Use Applications. The Investigational New Drug Application
Common Technical Document
Electronic Submission of a CTD
Marketing Applications: BLA and NDA
Medical Device Applications. 510(k) and PMA
Special Documents, Pathways, or Exemptions
Generic Drugs and Biosimiliar or Follow-on Biologics
Other Regulatory Activities
Public Meetings and Advisory Committees
Postmarketing Requirements and Activities
Advertising and Promotion
Summary of Regulatory Affair Activities in Biotechnology Operations
References
4: Regulatory Compliance
Regulatory Compliance
Quality Systems to Meet Regulatory Compliance
Compliance and Quality Systems
Current Good Manufacturing Practices for Manufacture and Quality Control
Current Good Laboratory Practices for Nonclinical Laboratory Studies
Current Good Clinical Practices for Clinical Studies
Compliance for Biopharmaceuticals: Other Regulations of Importance
Compliance for Import of Biopharmaceuticals into the United States
Compliance for Medical Devices
Inspection and Enforcement
Inspections
Enforcement Actions
Product Liability
Compliance with Non-FDA Regulations: International, National, State, and Local
International and Foreign National Regulatory Authorities for Medical Biotechnology Products
Transporting Infectious or Otherwise Hazardous Materials
Importing, Possessing, or Transferring Controlled Biotechnology Materials
The Public Health Security and Bioterrorism Preparedness and Response Act of 2002
Importation or Exportation of Biotechnology Products for the Purpose of Treatment of Diseases in Humans
Occupational Health and Safety
Environmental Regulations in Biotechnology
Genetically Modified Organisms or Molecules
International Diligence in Biotechnology Operations
Summary of Regulatory Compliance
Summary of Non-FDA Compliance
References
5: Quality Systems
Overview of Quality in Biotechnology
History: Evolution of Quality Concepts and Practices
Quality Systems Approach to Product Development
Planning a Quality System
Defining Objectives and Ensuring Management Support
The Quality Manual
The Quality Plan
Hallmarks of Quality: Fundamental Criteria for Building Effective Quality Systems
Management Responsibility
Defined Quality System
QbD and Design Control
Quality by Design
Design Control
Design Change
Contractor, Vendor, and Consultant Control
Product Identification and Traceability
Process Control
Environmental Controls
Inspection or Testing (Quality Control)
Release of Material, Service, or Product
Change Control and Corrective or Preventive Actions
Packaging and Labeling
Preservation, Storage, and Handling
Servicing
Customer Concerns and Adverse Event Reports
Document Control
Training
Auditing
The Quality Assurance Unit
Manage the Quality Assurance Function
Control Documents and Manage the Documentation System
Investigate Situations: Manage and Control Change
Ensure Qualified and Trained Staff
Perform Audits
Initiate a Quality System for a Biotechnology Operation
Unique and Effective Approaches to Quality Management
Risk-Based Approaches to Quality Systems
Total Quality Management
Six Sigma
Statistics in Quality Assurance
Quality Systems for Research
Resolving Quality Issues or Problems
Summary of Quality Systems
References
6: Biomanufacture
Overview of Biomanufacturing Requirements
Design in Biomanufacture
Technical Considerations for Biomanufacture
Phases and Scale-up: The Biomanufacturing Life Cycle
Raw Material Considerations
Compliance and Quality in Biomanufacture: Current Good Manufacturing Practices
Biomanufacturing Processes for Biotechnology Products
Expression of Recombinant Proteins and Nucleic Acids
Production of Recombinant Molecules from Expression Vectors
Genes, Vectors, and Host Cells
Bacterial Cell Expression Systems
Yeast Cell Expression Systems
Mammalian or Insect Cell Expression Systems
Production of Master Cell Banks and Working Cell Banks
Biomanufacture of Recombinant Proteins
Planning Production of a Recombinant Protein
Upstream Process: Production by Bacterial or Yeast Cell Fermentation
Upstream Process: Production by Mammalian or Insect Cell Culture
Upstream Process: Recovery
Downstream Process: Purification
In-Process Testing and Analysis of Bulk Substance
Production of Bacterial Plasmid DNA
Production of Live Recombinant Organisms: Bacteria and Virus
Production of Products Composed of Mammalian Somatic Cells or Tissues
Production of Cellular Products Derived from Pluripotent (Stem) Cells
Production of Biological Molecules by Transgenic Animals or Plants
Production of Biologically Active Lipids, Glycolipids, and Complex Carbohydrates
Production of Biologically Active Peptides
Production of Combination Products: Biopharmaceutical with a Drug or Medical Device
FP: Formulation, Fill, Finish, and Labeling
Biomanufacturing Facilities, Utilities, and Equipment
Facility Design Considerations
The Facility and Utilities: A Controlled Environment
Operation of Clean Work Areas for Biomanufacture
Biomanufacturing Equipment
Contract Manufacturing Options
Validation of Biomanufacturing Facilities, Utilities, Equipment, and Processes
Summary of Biomanufacture
References
7: Quality Control
Quality Control Overview
Definition of Product Attributes
Analytical Methods to Measure Attributes
Traits of Analytical Methods
Drafting a Certificate of Analysis (Bulk Substance)
Selection of Analytical Methods
Development of Specifications
Entering Test Results
Certificate of Analysis for Drug Product
In-Process Testing
Analytical Methods
Additional Analytical Tools and Concepts
Quality Control of Cell Banks
Samples and Sampling
Analytical Controls and Reference Standards
Test Failures, Out-of-Specification Results, and Retesting
Testing for Product Stability
Quality Control Testing of Raw Materials
Quality Control and the Manufacturing Environment
Qualification, Validation, and Verification of Analytical Methods
Application of Statistics in Assay Performance and Validation
Summary of Quality Control
Reference
8: Nonclinical Studies
Nonclinical Studies and Risk Assessment
Biopharmaceutical Delivery, Pharmacokinetics, and Pharmacodynamics
Product Delivery to the Body
Adsorption, Distribution, Elimination, and Metabolism (ADME)
Absorption
Distribution
Metabolism and Biotransformation
Excretion
Pharmacokinetics and Pharmacodynamics
Application of Pharmacokinetics and Pharmacodynamics in Biopharmaceutical Development
Safety Assessment of Biopharmaceuticals
Toxicology
Design of a Safety Assessment Program
In Vitro Screens: Surrogate Measures of Toxicity
In Vivo Safety Testing of Biopharmaceuticals
Animal Model Development
Test Product Formulations, Routes of Delivery, and Dosing Designs
Protocols and Performance of Biopharmaceutical Safety Studies in Animals
Elements of a Nonclinical Study Design
Nonclinical Safety Testing
Acute Toxicity Testing
Subchronic and Chronic Toxicity Testing
Reproductive, Developmental, and Teratogenicity Toxicity Testing
Carcinogenicity Testing
Immunotoxicology
Genetic Toxicology
Tissue Binding or Local Tissue Tolerance
Quality of Nonclinical Studies: Current Good Laboratory Practices
Summary of Nonclinical Studies
Reference
9: Clinical Trials
Introduction to Clinical Trials
Background of Clinical Research
Introduction
Historical Information on Clinical Trials
Organization of Clinical Research
Phases of Clinical Trials
The Science of Clinical Research
Quality in Clinical Research and Current Good Clinical Practices
Clinical Development Planning
Infrastructure for a Clinical Trial: Individuals, Documents, and Investigational Product
Design of Clinical Trials and the Clinical Protocol
Human Subjects, Patients, and Volunteers
The Sponsor
The Principal Investigator and His or Her Study Staff
Institutional Review Boards, the Process of IC, and IC Form
Investigational Product
Collection of Clinical Data: Case Report Forms and the Patient Diary
Clinical Testing Laboratories
Reporting Results of Clinical Trials: Clinical Summary Reports
Clinical Trial Operations
Activities Leading to a Clinical Trial
Phase 1 Clinical Trial: First-In-Human Study
Clinical Pharmacology Studies of Biopharmaceuticals in Human
Phase 2 Clinical Trial: Proof-of-Concept Study
Phase 3 Clinical Trial: Therapeutic Confirmatory
Phase 4 Clinical Study and Risk Evaluation and Mitigation Strategy
Clinical Trials for New Populations or Indications
Global Clinical Trials
Quality Systems for Clinical Trials: Current Good Clinical Practices
Quality and cGCP in Clinical Trial Operations
Integrity of Clinical Study Data and Documents
Monitoring and Auditing Clinical Trials
Ethical Behavior and the Well-Being of Clinical Trial Subjects
Summary on Clinical Trials
Reference
Additional Readings
Glossary
Appendix
Index