pharmaceutical biotechnology concepts and applications

Pharmaceutical Biotechnology

Pharmaceutical Biotechnology
Concepts and Applications
Gary Walsh
University of Limerick, Republic of Ireland
Copyright © 2007 John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester,
West Sussex PO19 8SQ, England
Telephone (+44) 1243 779777
Email (for orders and customer service enquiries):
Visit our Home Page on www.wileyeurope.com or www.wiley.com
All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form
or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except under the terms of the
Copyright, Designs and Patents Act 1988 or under the terms of a licence issued by the Copyright Licensing Agency Ltd, 90
Tottenham Court Road, London W1T 4LP, UK, without the permission in writing of the Publisher. Requests to the Publisher
should be addressed to the Permissions Department, John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West
Sussex PO19 8SQ, England, or emailed to , or faxed to (ϩ44) 1243 770620.
Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product
names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The
Publisher is not associated with any product or vendor mentioned in this book.
This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is
sold on the understanding that the Publisher is not engaged in rendering professional services. If professional advice or other
expert assistance is required, the services of a competent professional should be sought.
Other Wiley Editorial Offi ces
John Wiley & Sons Inc., 111 River Street, Hoboken, NJ 07030, USA
Jossey-Bass, 989 Market Street, San Francisco, CA 94103-1741, USA
Wiley-VCH Verlag GmbH, Boschstr. 12, D-69469 Weinheim, Germany
John Wiley & Sons Australia Ltd, 42 McDougall Street, Milton, Queensland 4064, Australia
John Wiley & Sons (Asia) Pte Ltd, 2 Clementi Loop #02-01, Jin Xing Distripark, Singapore 129809
John Wiley & Sons Canada Ltd, 6045 Freemont Blvd, Mississauga, Ontario, L5R 4J3

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in
electronic books.
Anniversary Logo Design: Richard J. Pacifi co
Library of Congress Cataloging-in-Publication Data
Walsh, Gary, Dr.
Pharmaceutical biotechnology : concepts and applications / Gary Walsh.
p. ; cm.
Includes bibliographical references.
ISBN 978-0-470-01244-4 (cloth)
1. Pharmaceutical biotechnology. I. Title.
[DNLM: 1. Technology, Pharmaceutical. 2. Biotechnology. 3.
Pharmaceutical Preparations. QV 778 W224p 2007]
RS380.W35 2007
615Ј.19–dc22 2007017884
British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library
ISBN 978-0-470-01244-4 (HB)
ISBN 978-0-470-01245-1 (PB)
Typeset in 10.5/12.5 pt Times by Thomson Digital
Printed and bound in Great Britain by Antony Rowe Ltd., Chippenham, Wilts
This book is printed on acid-free paper responsibly manufactured from sustainable forestry
in which at least two trees are planted for each one used for paper production.
I dedicate this book to my beautiful daughter Alice.
To borrow a phrase:
‘without her help, it would have been written in half the time’!

Preface xv
Acronyms xvii
1 Pharmaceuticals, biologics and biopharmaceuticals 1
1.1 Introduction to pharmaceutical products 1

1.2 Biopharmaceuticals and pharmaceutical biotechnology 1
1.3 History of the pharmaceutical industry 2
1.4 The age of biopharmaceuticals 3
1.5 Biopharmaceuticals: current status and future prospects 8
Further reading 11
2 Protein structure 13
2.1 Introduction 13
2.2 Overview of protein structure 13
2.2.1 Primary structure 15
2.2.2 The peptide bond 18
2.2.3 Amino acid sequence determination 19
2.2.4 Polypeptide synthesis 22
2.3 Higher level structure 23
2.3.1 Secondary structure 23
2.3.2 Tertiary structure 26
2.3.3 Higher structure determination 26
2.4 Protein stability and folding 27
2.4.1 Structural prediction 28
2.5 Protein post-translational modifi cation 29
2.5.1 Glycosylation 29
2.5.2 Carboxylation and hydroxylation 33
2.5.3 Sulfation and amidation 34
Further reading 35
3 Gene manipulation and recombinant DNA technology 37
3.1 Introduction 37
3.2 Nucleic acids: function and structure 38
3.2.1 Genome and gene organization 41
3.2.2 Nucleic acid purifi cation 43
3.2.3 Nucleic acid sequencing 45
3.3 Recombinant production of therapeutic proteins 46

3.4 Classical gene cloning and identifi cation 47
3.4.1 cDNA cloning 51
3.4.2 Cloning via polymerase chain reaction 51
Contents
viii CONTENTS
3.4.3 Expression vectors 53
3.4.4 Protein engineering 53
Further reading 54
4 The drug development process 57
4.1 Introduction 57
4.2 Discovery of biopharmaceuticals 58
4.3 The impact of genomics and related technologies upon drug discovery 59
4.4 Gene chips 61
4.5 Proteomics 62
4.6 Structural genomics 64
4.7 Pharmacogenetics 65
4.8 Initial product characterization 66
4.9 Patenting 67
4.9.1 What is a patent and what is patentable? 68
4.9.2 Patenting in biotechnology 68
4.10 Delivery of biopharmaceuticals 70
4.10.1 Oral delivery systems 70
4.10.2 Pulmonary delivery 71
4.10.3 Nasal, transmucosal and transdermal delivery systems 73
4.11 Preclinical studies 74
4.12 Pharmacokinetics and pharmacodynamics 74
4.12.1 Protein pharmacokinetics 75
4.12.2 Tailoring of pharmacokinetic profi le 77
4.12.3 Protein mode of action and pharmacodynamics 79
4.13 Toxicity studies 80

4.13.1 Reproductive toxicity and teratogenicity 82
4.13.2 Mutagenicity, carcinogenicity and other tests 83
4.13.3 Clinical trials 84
4.13.4 Clinical trial design 87
4.13.5 Trial size design and study population 87
4.14 The role and remit of regulatory authorities 89
4.14.1 The Food and Drug Administration 90
4.14.2 The investigational new drug application 92
4.14.3 The new drug application 94
4.14.4 European regulations 95
4.14.5 National regulatory authorities 96
4.14.6 The European Medicines Agency and the new EU drug
approval systems 96
4.14.7 The centralized procedure 98
4.14.8 Mutual recognition 100
4.14.9 Drug registration in Japan 100
4.14.10 World harmonization of drug approvals 101
4.15 Conclusion 101
Further reading 101
5 Sources and upstream processing 105
5.1 Introduction 105
5.2 Sources of biopharmaceuticals 105
5.2.1 Escherichia coli as a source of recombinant, therapeutic proteins 105
5.2.2 Expression of recombinant proteins in animal cell culture systems 109
CONTENTS ix
5.2.3 Additional production systems 110
5.2.3.1 Yeast 110
5.2.3.2 Fungal production systems 111
5.2.3.3 Transgenic animals 111
5.2.3.4 Transgenic plants 116

5.2.3.5 Insect cell-based systems 118
5.3 Upstream processing 120
5.3.1 Cell banking systems 121
5.3.2 Microbial cell fermentation 124
5.3.3 Mammalian cell culture systems 127
Further reading 129
6 Downstream processing 131
6.1 Introduction 131
6.2 Initial product recovery 134
6.3 Cell disruption 134
6.4 Removal of nucleic acid 136
6.5 Initial product concentration 137
6.5.1 Ultrafi ltration 137
6.5.2 Diafi ltration 139
6.6 Chromatographic purifi cation 140
6.6.1 Size-exclusion chromatography (gel fi ltration) 142
6.6.2 Ion-exchange chromatography 142
6.6.3 Hydrophobic interaction chromatography 146
6.6.4 Affi nity chromatography 148
6.6.5 Immunoaffi nity purifi cations 150
6.6.6 Protein A chromatography 150
6.6.7 Lectin affi nity chromatography 150
6.6.8 Dye affi nity chromatography 152
6.6.9 Metal chelate affi nity chromatography 153
6.6.10 Chromatography on hydroxyapatite 154
6.6.11 Chromatofocusing 155
6.7 High-performance liquid chromatography of proteins 155
6.8 Purifi cation of recombinant proteins 157
6.9 Final product formulation 159
6.9.1 Some infl uences that can alter the biological activity of proteins 159

6.9.1.1 Proteolytic degradation and alteration of sugar side-chains 160
6.9.1.2 Protein deamidation 161
6.9.1.3 Oxidation and disulfi de exchange 162
6.9.2 Stabilizing excipients used in fi nal product formulations 164
6.9.3 Final product fi ll 166
6.9.4 Freeze-drying 168
6.9.5 Labelling and packing 169
Further reading 171
7 Product analysis 173
7.1 Introduction 173
7.2 Protein-based contaminants 173
7.3 Removal of altered forms of the protein of interest from the product stream 175
7.3.1 Product potency 175
x CONTENTS
7.3.2 Determination of protein concentration 179
7.4 Detection of protein-based product impurities 180
7.4.1 Capillary electrophoresis 182
7.4.2 High-performance liquid chromatography 183
7.4.3 Mass spectrometry 184
7.5 Immunological approaches to detection of contaminants 185
7.5.1 Amino acid analysis 185
7.5.2 Peptide mapping 186
7.5.3 N-terminal sequencing 188
7.5.4 Analysis of secondary and tertiary structure 188
7.6 Endotoxin and other pyrogenic contaminants 189
7.6.1 Endotoxin, the molecule 191
7.6.2 Pyrogen detection 191
7.6.3 DNA 195
7.6.4 Microbial and viral contaminants 196
7.6.5 Viral assays 198

7.6.6 Miscellaneous contaminants 199
7.6.7 Validation studies 199
Further reading 202
8 The cytokines: The interferon family 205
8.1 Cytokines 205
8.1.1 Cytokine receptors 210
8.1.2 Cytokines as biopharmaceuticals 211
8.2 The interferons 212
8.2.1 The biochemistry of interferon-α 213
8.2.2 Interferon-β 214
8.2.3 Interferon-γ 214
8.2.4 Interferon signal transduction 214
8.2.5 The interferon receptors 215
8.2.6 The JAK–STAT pathway 215
8.2.7 The interferon JAK–STAT pathway 218
8.2.8 The biological effects of interferons 219
8.2.9 The eIF-2α protein kinase system 221
8.3 Interferon biotechnology 224
8.3.1 Production and medical uses of interferon-α 226
8.3.2 Medical uses of interferon-β 229
8.3.3 Medical applications of interferon-γ 232
8.3.4 Interferon toxicity 234
8.3.5 Additional interferons 235
8.4 Conclusion 236
Further reading 237
9 Cytokines: Interleukins and tumour necrosis factor 241
9.1 Introduction 241
9.2 Interleukin-2 242
9.2.1 Interleukin-2 production 246
9.2.2 Interleukin-2 and cancer treatment 246

9.2.3 Interleukin-2 and infectious diseases 248
9.2.4 Safety issues 249
9.2.5 Inhibition of interleukin-2 activity 249
CONTENTS xi
9.3 Interleukin-1 251
9.3.1 The biological activities of interleukin-1 252
9.3.2 Interleukin-1 biotechnology 253
9.4 Interleukin-11 254
9.5 Tumour necrosis factors 255
9.5.1 Tumour necrosis factor biochemistry 255
9.5.2 Biological activities of tumour necrosis factor-α 256
9.5.3 Immunity and infl ammation 257
9.5.4 Tumour necrosis factor receptors 258
9.5.5 Tumour necrosis factor: therapeutic aspects 260
Further reading 262
10 Growth factors 265
10.1 Introduction 265
10.2 Haematopoietic growth factors 265
10.2.1 The interleukins as haemopoietic growth factors 268
10.2.2 Granulocyte colony-stimulating factor 269
10.2.3 Macrophage colony-stimulating factor 269
10.2.4 Granulocyte macrophage colony-stimulating factor 270
10.2.5 Clinical application of colony-stimulating factors 270
10.2.6 Erythropoietin 272
10.2.6.1 Therapeutic applications of erythropoietin 274
10.2.6.2 Chronic disease and cancer chemotherapy 278
10.2.7 Thrombopoietin 278
10.3 Growth factors and wound healing 279
10.3.1 Insulin-like growth factors 280
10.3.2 Insulin-like growth factor biological effects 281

10.3.3 Epidermal growth factor 282
10.3.4 Platelet-derived growth factor 283
10.3.5 Fibroblast growth factors 284
10.3.6 Transforming growth factors 284
10.3.7 Neurotrophic factors 286
Further reading 287
11 Therapeutic hormones 291
11.1 Introduction 291
11.2 Insulin 291
11.2.1 Diabetes mellitus 292
11.2.2 The insulin molecule 293
11.2.3 The insulin receptor and signal transduction 294
11.2.4 Insulin production 294
11.2.5 Production of human insulin by recombinant DNA technology 297
11.2.6 Formulation of insulin products 297
11.2.7 Engineered insulins 301
11.2.8 Additional means of insulin administration 304
11.3 Glucagon 305
11.4 Human growth hormone 307
11.4.1 The growth hormone receptor 307
11.4.2 Biological effects of growth hormone 308
11.4.3 Therapeutic uses of growth hormone 309
xii CONTENTS
11.5 The gonadotrophins 310
11.5.1 Follicle-stimulating hormone, luteinizing hormone
and human chorionic gonadotrophin 311
11.5.2 Pregnant mare serum gonadotrophin 315
11.5.3 The inhibins and activins 315
11.6 Medical and veterinary applications of gonadotrophins 319
11.6.1 Sources and medical uses of follicle-stimulating hormone,

luteinizing hormone and human chorionic gonadotrophin 319
11.6.2 Recombinant gonadotrophins 320
11.6.3 Veterinary uses of gonadotrophins 321
11.7 Additional recombinant hormones now approved 323
11.8 Conclusion 325
Further reading 325
12 Recombinant blood products and therapeutic enzymes 329
12.1 Introduction 329
12.2 Haemostasis 329
12.2.1 The coagulation pathway 330
12.2.2 Terminal steps of coagulation pathway 332
12.2.3 Clotting disorders 334
12.2.4 Factor VIII and haemophilia 335
12.2.5 Production of factor VIII 336
12.2.6 Factors IX, IIVa and XIII 339
12.3 Anticoagulants 340
12.3.1 Hirudin 342
12.3.2 Antithrombin 344
12.4 Thrombolytic agents 345
12.4.1 Tissue plasminogen activator 346
12.4.2 First-generation tissue plasminogen activator 348
12.4.3 Engineered tissue plasminogen activator 348
12.4.4 Streptokinase 350
12.4.5 Urokinase 350
12.4.6 Staphylokinase 351
12.4.7 α
1
-Antitrypsin 353
12.4.8 Albumin 354
12.5 Enzymes of therapeutic value 355

12.5.1 Asparaginase 355
12.5.2 DNase 357
12.5.3 Glucocerebrosidase 359
12.5.4 α-Galactosidase, urate oxidase and laronidase 360
12.5.5 Superoxide dismutase 363
12.5.6 Debriding agents 364
12.5.7 Digestive aids 364
Further reading 366
13 Antibodies, vaccines and adjuvants 371
13.1 Introduction 371
13.2 Traditional polyclonal antibody preparations 371
13.3 Monoclonal antibodies 374
13.3.1 Antibody screening: phage display technology 376
13.3.2 Therapeutic application of monoclonal antibodies 378
CONTENTS xiii
13.3.3 Tumour immunology 379
13.3.3.1 Antibody-based strategies for tumour
detection/destruction 383
13.3.3.2 Drug-based tumour immunotherapy 386
13.3.3.3 First-generation anti-tumour antibodies:
clinical disappointment 388
13.3.4 Tumour-associated antigens 389
13.3.5 Antigenicity of murine monoclonals 391
13.3.6 Chimaeric and humanized antibodies 392
13.3.7 Antibody fragments 394
13.3.8 Additional therapeutic applications of monoclonal antibodies 395
13.4 Vaccine technology 396
13.4.1 Traditional vaccine preparations 396
13.4.1.1 Attenuated, dead or inactivated bacteria 398
13.4.1.2 Attenuated and inactivated viral vaccines 399

13.4.1.3 Toxoids and antigen-based vaccines 399
13.4.2 The impact of genetic engineering on vaccine technology 400
13.4.3 Peptide vaccines 402
13.4.4 Vaccine vectors 403
13.4.5 Development of an AIDS vaccine 407
13.4.6 Diffi culties associated with vaccine development 409
13.4.7 AIDS vaccines in clinical trials 409
13.4.8 Cancer vaccines 410
13.4.9 Recombinant veterinary vaccines 411
13.5 Adjuvant technology 412
13.5.1 Adjuvant mode of action 413
13.5.2 Mineral-based adjuvants 413
13.5.3 Oil-based emulsion adjuvants 414
13.5.4 Bacteria/bacterial products as adjuvants 414
13.5.5 Additional adjuvants 415
Further reading 416
14 Nucleic-acid- and cell-based therapeutics 419
14.1 Introduction 419
14.2 Gene therapy 419
14.2.1 Basic approach to gene therapy 420
14.2.2 Some additional questions 423
14.3 Vectors used in gene therapy 424
14.3.1 Retroviral vectors 424
14.3.2 Adenoviral and additional viral-based vectors 428
14.3.3 Manufacture of viral vectors 431
14.3.4 Non-viral vectors 432
14.3.5 Manufacture of plasmid DNA 436
14.4 Gene therapy and genetic disease 438
14.5 Gene therapy and cancer 441
14.6 Gene therapy and AIDS 444

14.6.1 Gene-based vaccines 444
14.6.2 Gene therapy: some additional considerations 445
14.7 Antisense technology 445
14.7.1 Antisense oligonucleotides and their mode of action 446
14.7.2 Uses, advantages and disadvantages of ‘oligos’ 448
xiv CONTENTS
14.8 Oligonucleotide pharmacokinetics and delivery 450
14.8.1 Manufacture of oligos 451
14.8.2 Additional antigene agents: RNA interference and ribozymes 451
14.9 Aptamers 453
14.10 Cell- and tissue-based therapies 453
14.10.1 Stem cells 457
14.10.2 Adult stem cells 459
14.11 Conclusion 460
Further reading 460
Index 465
Preface
This book has been written as a sister publication to Biopharmaceuticals: Biochemistry and
Biotechnology, a second edition of which was published by John Wiley and Sons in 2003. The
latter textbook caters mainly for advanced undergraduate/postgraduate students undertaking de-
gree programmes in biochemistry, biotechnology and related disciplines. Such students have
invariably pursued courses/modules in basic protein science and molecular biology in the earlier
parts of their degree programmes; hence, the basic principles of protein structure and molecular
biology were not considered as part of that publication. This current publication is specifi cally
tailored to meet the needs of a broader audience, particularly to include students undertaking pro-
grammes in pharmacy/pharmaceutical science, medicine and other branches of biomedical/clini-
cal sciences. Although evolving from Biopharmaceuticals: Biochemistry and Biotechnology,
its focus is somewhat different, refl ecting its broader intended readership. This text, therefore,
includes chapters detailing the basic principles of protein structure and molecular biology. It also
increases/extends the focus upon topics such as formulation and delivery of biopharmaceuticals,

and it contains numerous case studies in which both biotech and clinical aspects of a particular
approved product of pharmaceutical biotechnology are overviewed. The book, of course, should
also meet the needs of students undertaking programmes in core biochemistry, biotechnology or
related scientifi c areas and be of use as a broad reference source to those already working within
the pharmaceutical biotechnology sector.
As always, I owe a debt of gratitude to the various people who assisted in the completion of this
textbook. Thanks to Sandy for her help in preparing various fi gures, usually at ridiculously short
notice. To Gerard Wall, for all the laughs and for several useful discussions relating to molecular
biology. Thank you to Nancy, my beautiful wife, for accepting my urge to write (rather than to
change baby’s nappies) with good humour – most of the time anyway! I am also grateful to the
staff of John Wiley and Sons for their continued professionalism and patience with me when I
keep overrunning submission deadlines. Finally, I have a general word of appreciation to all my
colleagues at the University of Limerick for making this such an enjoyable place to work.
Gary Walsh
November 2006

Acronyms
ADCC antibody-dependent cell cytoxicity
BAC bacterial artifi cial chromosome
BHK baby hamster kidney
cDNA complementary DNA
CHO Chinese hamster ovary
CNTF ciliary neurotrophic factor
CSF colony-stimulating factor
dsRNA double-stranded RNA
EDTA ethylenediaminetetraacetic acid
ELISA enzyme-linked immunosorbent assay
EPO erythropoietin
FGF fi broblast growth factor
FSH follicle-stimulating hormone

GDNF glial cell-derived neurotrophic factor
GH growth hormone
hCG human chorionic gonadotrophin
HIV human immunodefi ciency virus
HPLC high-performance liquid chromatography
IGF insulin-like growth factor
ISRE interferon-stimulated response element
JAK Janus kinase
LAF lymphocyte activating factor
LIF leukaemia inhibitory factor
LPS lipopolysaccharide
MHC major histocompatibility complex
MPS mucopolysaccharidosis
mRNA messenger RNA
PDGF platelet-derived growth factor
PEG polyethylene glycol
xviii ACRONYMS
PTK protein tyrosine kinase
PTM post-translational modifi cation
rDNA recombinant DNA
RNAi RNA interference
rRNA ribosomal RNA
SDS sodium dodecyl sulfate
ssRNA single-stranded RNA
STATs signal transducers and activators of transcription
TNF tumour necrosis factor
tPA tissue plasminogen activator
tRNA transfer RNA
WAP whey acid protein
WFI water for injections

1
Pharmaceuticals, biologics
and biopharmaceuticals
1.1 Introduction to pharmaceutical products
Pharmaceutical substances form the backbone of modern medicinal therapy. Most traditional phar-
maceuticals are low molecular weight organic chemicals (Table 1.1). Although some (e.g. aspirin)
were originally isolated from biological sources, most are now manufactured by direct chemical
synthesis. Two types of manufacturing company thus comprise the ‘traditional’ pharmaceutical sec-
tor: the chemical synthesis plants, which manufacture the raw chemical ingredients in bulk quanti-
ties, and the fi nished product pharmaceutical facilities, which purchase these raw bulk ingredients,
formulate them into fi nal pharmaceutical products, and supply these products to the end user.
In addition to chemical-based drugs, a range of pharmaceutical substances (e.g. hormones and
blood products) are produced by/extracted from biological sources. Such products, some major
examples of which are listed in Table 1.2, may thus be described as products of biotechnology. In
some instances, categorizing pharmaceuticals as products of biotechnology or chemical synthe-
sis becomes somewhat artifi cial. For example, certain semi-synthetic antibiotics are produced by
chemical modifi cation of natural antibiotics produced by fermentation technology.
1.2 Biopharmaceuticals and pharmaceutical biotechnology
Terms such as ‘biologic’, ‘biopharmaceutical’ and ‘products of pharmaceutical biotechnology’ or ‘bio-
technology medicines’ have now become an accepted part of the pharmaceutical literature. However,
these terms are sometimes used interchangeably and can mean different things to different people.
Although it might be assumed that ‘biologic’ refers to any pharmaceutical product produced
by biotechnological endeavour, its defi nition is more limited. In pharmaceutical circles, ‘biologic’
generally refers to medicinal products derived from blood, as well as vaccines, toxins and allergen
products. ‘Biotechnology’ has a much broader and long-established meaning. Essentially, it refers
Pharmaceutical biotechnology: concepts and applications Gary Walsh
© 2007 John Wiley & Sons, Ltd ISBN 978 0 470 01244 4 (HB) 978 0 470 01245 1 (PB)
2 CH1 PHARMACEUTICALS, BIOLOGICS AND BIOPHARMACEUTICALS
to the use of biological systems (e.g. cells or tissues) or biological molecules (e.g. enzymes or
antibodies) for/in the manufacture of commercial products.

The term ‘biopharmaceutical’ was fi rst used in the 1980s and came to describe a class of thera-
peutic proteins produced by modern biotechnological techniques, specifi cally via genetic engineering
(Chapter 3) or, in the case of monoclonal antibodies, by hybridoma technology ( Chapter 13). Although
the majority of biopharmaceuticals or biotechnology products now approved or in development are
proteins produced via genetic engineering, these terms now also encompass nucleic-acid-based, i.e.
deoxyribonucleic acid (DNA)- or ribonucleic acid (RNA)-based products, and whole-cell-based products.
1.3 History of the pharmaceutical industry
The pharmaceutical industry, as we now know it, is barely 60 years old. From very modest beginnings, it
has grown rapidly, reaching an estimated value of US$100 billion by the mid 1980s. Its current value is
likely double or more this fi gure. There are well in excess of 10 000 pharmaceutical companies in exist-
ence, although only about 100 of these can claim to be of true international signifi cance. These compa-
nies manufacture in excess of 5000 individual pharmaceutical substances used routinely in medicine.
Table 1.1 Some traditional pharmaceutical substances that are generally produced by direct chemical
synthesis
Drug Molecular formula Molecular mass Therapeutic indication
Acetaminophen
(paracetamol)
C
8
H
9
NO
2
151.16 Analgesic
Ketamine C
13
H
16
C/NO 237.74 Anaesthetic
Levamisole C

11
H
12
N
2
S204.31Anthelmintic
Diazoxide C
8
H
7
C/N
2
O
2
S230.7Antihypertensive
Acyclovir C
8
H
11
N
5
O
3
225.2 Antiviral agent
Zidovudine C
10
H
13
N
5

O
4
267.2 Antiviral agent
Dexamethasone C
22
H
29
FO
5
392.5 Anti-infl ammatory and
immunosuppressive
agent
Misoprostol C
22
H
38
O
5
382.5 Anti-ulcer agent
Cimetidine C
10
H
16
N
6
252.3 Anti-ulcer agent
Table 1.2 Some pharmaceuticals that were traditionally obtained by direct extraction from biological source
material. Many of the protein-based pharmaceuticals mentioned are now also produced by genetic engineering
Substance Medical application
Blood products (e.g. coagulation factors) Treatment of blood disorders such as haemophilia

A or B
Vaccines Vaccination against various diseases
Antibodies Passive immunization against various diseases
Insulin Treatment of diabetes mellitus
Enzymes Thrombolytic agents, digestive aids, debriding agents
(i.e. cleansing of wounds)
Antibiotics Treatment against various infections agents
Plant extracts (e.g. alkaloids) Various, including pain relief
The fi rst stages of development of the modern pharmaceutical industry can be traced back to the
turn of the twentieth century. At that time (apart from folk cures), the medical community had at
their disposal only four drugs that were effective in treating specifi c diseases:
Digitalis (extracted from foxglove) was known to stimulate heart muscle and, hence, was used
to treat various heart conditions.
Quinine, obtained from the barks/roots of a plant (Cinchona genus), was used to treat malaria.
Pecacuanha (active ingredient is a mixture of alkaloids), used for treating dysentery, was ob-
tained from the bark/roots of the plant genus Cephaelis.
Mercury, for the treatment of syphilis.
This lack of appropriate, safe and effective medicines contributed in no small way to the low life
expectancy characteristic of those times.
Developments in biology (particularly the growing realization of the microbiological basis of
many diseases), as well as a developing appreciation of the principles of organic chemistry, helped
underpin future innovation in the fl edgling pharmaceutical industry. The successful synthesis of
various artifi cial dyes, which proved to be therapeutically useful, led to the formation of pharma-
ceutical/chemical companies such as Bayer and Hoechst in the late 1800s. Scientists at Bayer, for
example, succeeded in synthesizing aspirin in 1895.
Despite these early advances, it was not until the 1930s that the pharmaceutical industry
began to develop in earnest. The initial landmark discovery of this era was probably the
discovery, and chemical synthesis, of the sulfa drugs. These are a group of related molecules
derived from the red dye prontosil rubrum. These drugs proved effective in the treatment
of a wide variety of bacterial infections (Figure 1.1). Although it was first used therapeuti-

cally in the early 1920s, large-scale industrial production of insulin also commenced in the
1930s.
The medical success of these drugs gave new emphasis to the pharmaceutical industry, which
was boosted further by the commencement of industrial-scale penicillin manufacture in the early
1940s. Around this time, many of the current leading pharmaceutical companies (or their fore-
runners) were founded. Examples include Ciba Geigy, Eli Lilly, Wellcome, Glaxo and Roche.
Over the next two to three decades, these companies developed drugs such as tetracyclines, cor-
ticosteroids, oral contraceptives, antidepressants and many more. Most of these pharmaceutical
substances are manufactured by direct chemical synthesis.
1.4 The age of biopharmaceuticals
Biomedical research continues to broaden our understanding of the molecular mechanisms un-
derlining both health and disease. Research undertaken since the 1950s has pinpointed a host of
proteins produced naturally in the body that have obvious therapeutic applications. Examples in-
clude the interferons and interleukins (which regulate the immune response), growth factors, such
as erythropoietin (EPO; which stimulates red blood cell production), and neurotrophic factors
(which regulate the development and maintenance of neural tissue).
•
•
•
•
THE AGE OF BIOPHARMACEUTICALS 3
4 CH1 PHARMACEUTICALS, BIOLOGICS AND BIOPHARMACEUTICALS
Although the pharmaceutical potential of these regulatory molecules was generally appreciated,
their widespread medical application was in most cases rendered impractical due to the tiny quan-
tities in which they were naturally produced. The advent of recombinant DNA technology (genetic
engineering) and monoclonal antibody technology (hybridoma technology) overcame many such
diffi culties, and marked the beginning of a new era of the pharmaceutical sciences.
Recombinant DNA technology has had a fourfold positive impact upon the production of
pharmaceutically important proteins:
NH

2
N
H
2
N
N
O = S = O
NH
2
NH
2
O = S = O
NH
2
NH
2
COOH
N
N
N
N
OH H
H
H
2
N
CH
2
H
H

H
NH C NH CH COOH
O
(CH
2
)
2
COOH
Prontosil rubrum
(a)
Sulphanilamide
(b)
PABA
(c)
Pteridine
derivative
PABA
Glutamic acid
Tetrahydrofolic acid
(d)
Figure 1.1 Sulfa drugs and their mode of action. The fi rst sulfa drug to be used medically was the red dye
prontosil rubrum (a). In the early 1930s, experiments illustrated that the administration of this dye to mice
infected with haemolytic streptococci prevented the death of the mice. This drug, although effective in vivo,
was devoid of in vitro antibacterial activity. It was fi rst used clinically in 1935 under the name Streptozon. It
was subsequently shown that prontosil rubrum was enzymatically reduced by the liver, forming sulfanilamide,
the actual active antimicrobial agent (b). Sulfanilamide induces its effect by acting as an anti-metabolite
with respect to para-aminobenzoic acid (PABA) (c). PABA is an essential component of tetrahydrofolic acid
(THF) (d). THF serves as an essential cofactor for several cellular enzymes. Sulfanilamide (at suffi ciently high
concentrations) inhibits manufacture of THF by competing with PABA. This effectively inhibits essential
THF-dependent enzyme reactions within the cell. Unlike humans, who can derive folates from their diets, most

bacteria must synthesize it de novo, as they cannot absorb it intact from their surroundings
It overcomes the problem of source availability. Many proteins of therapeutic potential are
produced naturally in the body in minute quantities. Examples include interferons (Chapter 8),
interleukins (Chapter 9) and colony-stimulating factors (CSFs; Chapter 10). This rendered
impractical their direct extraction from native source material in quantities suffi cient to meet
likely clinical demand. Recombinant production (Chapters 3 and 5) allows the manufacture of
any protein in whatever quantity it is required.
It overcomes problems of product safety. Direct extraction of product from some native biological
sources has, in the past, led to the unwitting transmission of disease. Examples include the
transmission of blood-borne pathogens such as hepatitis B and C and human immunodefi ciency
virus (HIV) via infected blood products and the transmission of Creutzfeldt–Jakob disease to
persons receiving human growth hormone (GH) preparations derived from human pituitaries.
It provides an alternative to direct extraction from inappropriate/dangerous source material.
A number of therapeutic proteins have traditionally been extracted from human urine. Follicle-
stimulating hormone (FSH), the fertility hormone, for example, is obtained from the urine of post-
menopausal women, and a related hormone, human chorionic gonadotrophin (hCG), is extracted
from the urine of pregnant women (Chapter 11). Urine is not considered a particularly desirable
source of pharmaceutical products. Although several products obtained from this source remain on
the market, recombinant forms have now also been approved. Other potential biopharmaceuticals
are produced naturally in downright dangerous sources. Ancrod, for example, is a protein displaying
anti-coagulant activity (Chapter 12) and, hence, is of potential clinical use. It is, however, produced
naturally by the Malaysian pit viper. Although retrieval by milking snake venom is possible, and
indeed may be quite an exciting procedure, recombinant production in less dangerous organisms,
such as Escherichia coli or Saccharomycese cerevisiae, would be considered preferable by most.
It facilitates the generation of engineered therapeutic proteins displaying some clinical advantage
over the native protein product. Techniques such as site-directed mutagenesis facilitate the logi-
cal introduction of predefi ned changes in a protein’s amino acid sequence. Such changes can be as
minimal as the insertion, deletion or alteration of a single amino acid residue, or can be more sub-
stantial (e.g. the alteration/deletion of an entire domain, or the generation of a novel hybrid protein).
Such changes can be made for a number of reasons, and several engineered products have now gained

marketing approval. An overview summary of some engineered product types now on the market is
provided in Table 1.3. These and other examples will be discussed in subsequent chapters.
Despite the undoubted advantages of recombinant production, it remains the case that many
protein-based products extracted directly from native source material remain on the market. In
certain circumstances, direct extraction of native source material can prove equally/more attrac-
tive than recombinant production. This may be for an economic reason if, for example, the protein
is produced in very large quantities by the native source and is easy to extract/purify, e.g. human
serum albumin (HSA; Chapter 12). Also, some blood factor preparations purifi ed from donor
blood actually contain several different blood factors and, hence, can be used to treat several
haemophilia patient types. Recombinant blood factor preparations, on the other hand, contain but
a single blood factor and, hence, can be used to treat only one haemophilia type (Chapter 12).
The advent of genetic engineering and monoclonal antibody technology underpinned the
establishment of literally hundreds of start-up biopharmaceutical (biotechnology) companies in
•
•
•
•
THE AGE OF BIOPHARMACEUTICALS 5
6 CH1 PHARMACEUTICALS, BIOLOGICS AND BIOPHARMACEUTICALS
the late 1970s and early 1980s. The bulk of these companies were founded in the USA, with
smaller numbers of start-ups emanating from Europe and other world regions.
Many of these fl edgling companies were founded by academics/technical experts who sought to
take commercial advantage of developments in the biotechnological arena. These companies were
largely fi nanced by speculative monies attracted by the hype associated with the establishment of the
modern biotech era. Although most of these early companies displayed signifi cant technical expertise,
the vast majority lacked experience in the practicalities of the drug development process (Chapter 4).
Most of the well-established large pharmaceutical companies, on the other hand, were slow to invest
heavily in biotech research and development. However, as the actual and potential therapeutic signifi -
cance of biopharmaceuticals became evident, many of these companies did diversify into this area.
Most either purchased small, established biopharmaceutical concerns or formed strategic alliances

with them. An example was the long-term alliance formed by Genentech (see later) and the well-
Table 1.3 Selected engineered biopharmaceutical types/products that have now gained marketing
approval. These and additional such products will be discussed in detail in subsequent chapters
Product description/type Alteration introduced Rationale
Faster acting insulins (Chapter 11) Modifi ed amino acid sequence Generation of faster acting insulin
Slow acting insulins (Chapter 11) Modifi ed amino acid sequence Generation of slow acting insulin
Modifi ed tissue plasminogen
activator (tPA; Chapter 12)
Removal of three of the fi ve
native domains of tPA
Generation of a faster acting
thrombolytic (clot degrading)
agent
Modifi ed blood factor VIII
(Chapter 12)
Deletion of 1 domain of native
factor VIII
Production of a lower molecular
mass product
Chimaeric/humanized antibodies
(Chapter 13)
Replacement of most/virtually
all of the murine amino acid
sequences with sequences
found in human antibodies
Greatly reduced/eliminated
immunogenicity. Ability
to activate human effector
functions
‘Ontak’, a fusion protein (Chapter 9) Fusion protein consisting of the

diphtheria toxin linked to
interleukin-2 (IL-2)
Targets toxin selectively to cells
expressing an IL-2 receptor
Table 1.4 Pharmaceutical companies who manufacture and/or market
biopharmaceutical products approved for general medical use in the USA
and EU
Sanofi -Aventis Hoechst AG
Bayer Wyeth
Novo Nordisk Genzyme
Isis Pharmaceuticals Abbott
Genentech Roche
Centocor Novartis
Boehringer Manheim Serono
Galenus Manheim Organon
Eli Lilly Amgen
Ortho Biotech GlaxoSmithKline
Schering Plough Cytogen
Hoffman-la-Roche Immunomedics
Chiron Biogen