Pharmaceutical BiotechnologyConcepts and Applications

Gary WalshUniversity of Limerick, Republic of Ireland

Pharmaceutical Biotechnology

Pharmaceutical BiotechnologyConcepts and Applications

Gary WalshUniversity of Limerick, Republic of Ireland

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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 615�.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 DigitalPrinted and bound in Great Britain by Antony Rowe Ltd., Chippenham, WiltsThis book is printed on acid-free paper responsibly manufactured from sustainable forestryin 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 11.1 Introduction to pharmaceutical products 11.2 Biopharmaceuticals and pharmaceutical biotechnology 11.3 History of the pharmaceutical industry 21.4 The age of biopharmaceuticals 31.5 Biopharmaceuticals: current status and future prospects 8

Further reading 11

2 Protein structure 132.1 Introduction 132.2 Overview of protein structure 13

2.2.1 Primary structure 152.2.2 The peptide bond 182.2.3 Amino acid sequence determination 192.2.4 Polypeptide synthesis 22

2.3 Higher level structure 232.3.1 Secondary structure 232.3.2 Tertiary structure 262.3.3 Higher structure determination 26

2.4 Protein stability and folding 272.4.1 Structural prediction 28

2.5 Protein post-translational modifi cation 292.5.1 Glycosylation 292.5.2 Carboxylation and hydroxylation 332.5.3 Sulfation and amidation 34Further reading 35

3 Gene manipulation and recombinant DNA technology 373.1 Introduction 373.2 Nucleic acids: function and structure 38

3.2.1 Genome and gene organization 413.2.2 Nucleic acid purifi cation 433.2.3 Nucleic acid sequencing 45

3.3 Recombinant production of therapeutic proteins 463.4 Classical gene cloning and identifi cation 47

3.4.1 cDNA cloning 513.4.2 Cloning via polymerase chain reaction 51

Contents

viii CONTENTS

3.4.3 Expression vectors 533.4.4 Protein engineering 53

Further reading 54

4 The drug development process 574.1 Introduction 574.2 Discovery of biopharmaceuticals 584.3 The impact of genomics and related technologies upon drug discovery 594.4 Gene chips 614.5 Proteomics 624.6 Structural genomics 644.7 Pharmacogenetics 654.8 Initial product characterization 664.9 Patenting 67

4.9.1 What is a patent and what is patentable? 684.9.2 Patenting in biotechnology 68

4.10 Delivery of biopharmaceuticals 704.10.1 Oral delivery systems 704.10.2 Pulmonary delivery 714.10.3 Nasal, transmucosal and transdermal delivery systems 73

4.11 Preclinical studies 744.12 Pharmacokinetics and pharmacodynamics 74

4.12.1 Protein pharmacokinetics 754.12.2 Tailoring of pharmacokinetic profi le 774.12.3 Protein mode of action and pharmacodynamics 79

4.13 Toxicity studies 804.13.1 Reproductive toxicity and teratogenicity 824.13.2 Mutagenicity, carcinogenicity and other tests 834.13.3 Clinical trials 844.13.4 Clinical trial design 874.13.5 Trial size design and study population 87

4.14 The role and remit of regulatory authorities 894.14.1 The Food and Drug Administration 904.14.2 The investigational new drug application 924.14.3 The new drug application 944.14.4 European regulations 954.14.5 National regulatory authorities 964.14.6 The European Medicines Agency and the new EU drug

approval systems 964.14.7 The centralized procedure 984.14.8 Mutual recognition 1004.14.9 Drug registration in Japan 100

4.14.10 World harmonization of drug approvals 1014.15 Conclusion 101

Further reading 101

5 Sources and upstream processing 1055.1 Introduction 1055.2 Sources of biopharmaceuticals 105

5.2.1 Escherichia coli as a source of recombinant, therapeutic proteins 1055.2.2 Expression of recombinant proteins in animal cell culture systems 109

CONTENTS ix

5.2.3 Additional production systems 1105.2.3.1 Yeast 1105.2.3.2 Fungal production systems 1115.2.3.3 Transgenic animals 1115.2.3.4 Transgenic plants 1165.2.3.5 Insect cell-based systems 118

5.3 Upstream processing 1205.3.1 Cell banking systems 1215.3.2 Microbial cell fermentation 1245.3.3 Mammalian cell culture systems 127

Further reading 129

6 Downstream processing 1316.1 Introduction 1316.2 Initial product recovery 1346.3 Cell disruption 1346.4 Removal of nucleic acid 1366.5 Initial product concentration 137

6.5.1 Ultrafi ltration 1376.5.2 Diafi ltration 139

6.6 Chromatographic purifi cation 1406.6.1 Size-exclusion chromatography (gel fi ltration) 1426.6.2 Ion-exchange chromatography 1426.6.3 Hydrophobic interaction chromatography 1466.6.4 Affi nity chromatography 1486.6.5 Immunoaffi nity purifi cations 1506.6.6 Protein A chromatography 1506.6.7 Lectin affi nity chromatography 1506.6.8 Dye affi nity chromatography 1526.6.9 Metal chelate affi nity chromatography 153

6.6.10 Chromatography on hydroxyapatite 1546.6.11 Chromatofocusing 155

6.7 High-performance liquid chromatography of proteins 1556.8 Purifi cation of recombinant proteins 1576.9 Final product formulation 159

6.9.1 Some infl uences that can alter the biological activity of proteins 1596.9.1.1 Proteolytic degradation and alteration of sugar side-chains 1606.9.1.2 Protein deamidation 1616.9.1.3 Oxidation and disulfi de exchange 162

6.9.2 Stabilizing excipients used in fi nal product formulations 1646.9.3 Final product fi ll 1666.9.4 Freeze-drying 1686.9.5 Labelling and packing 169

Further reading 171

7 Product analysis 1737.1 Introduction 1737.2 Protein-based contaminants 1737.3 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 1797.4 Detection of protein-based product impurities 180

7.4.1 Capillary electrophoresis 1827.4.2 High-performance liquid chromatography 1837.4.3 Mass spectrometry 184

7.5 Immunological approaches to detection of contaminants 1857.5.1 Amino acid analysis 1857.5.2 Peptide mapping 1867.5.3 N-terminal sequencing 1887.5.4 Analysis of secondary and tertiary structure 188

7.6 Endotoxin and other pyrogenic contaminants 1897.6.1 Endotoxin, the molecule 1917.6.2 Pyrogen detection 1917.6.3 DNA 1957.6.4 Microbial and viral contaminants 1967.6.5 Viral assays 1987.6.6 Miscellaneous contaminants 1997.6.7 Validation studies 199Further reading 202

8 The cytokines: The interferon family 2058.1 Cytokines 205

8.1.1 Cytokine receptors 2108.1.2 Cytokines as biopharmaceuticals 211

8.2 The interferons 2128.2.1 The biochemistry of interferon-α 2138.2.2 Interferon-β 2148.2.3 Interferon-γ 2148.2.4 Interferon signal transduction 2148.2.5 The interferon receptors 2158.2.6 The JAK–STAT pathway 2158.2.7 The interferon JAK–STAT pathway 2188.2.8 The biological effects of interferons 2198.2.9 The eIF-2α protein kinase system 221

8.3 Interferon biotechnology 2248.3.1 Production and medical uses of interferon-α 2268.3.2 Medical uses of interferon-β 2298.3.3 Medical applications of interferon-γ 2328.3.4 Interferon toxicity 2348.3.5 Additional interferons 235

8.4 Conclusion 236Further reading 237

9 Cytokines: Interleukins and tumour necrosis factor 2419.1 Introduction 2419.2 Interleukin-2 242

9.2.1 Interleukin-2 production 2469.2.2 Interleukin-2 and cancer treatment 2469.2.3 Interleukin-2 and infectious diseases 2489.2.4 Safety issues 2499.2.5 Inhibition of interleukin-2 activity 249

CONTENTS xi

9.3 Interleukin-1 2519.3.1 The biological activities of interleukin-1 2529.3.2 Interleukin-1 biotechnology 253

9.4 Interleukin-11 2549.5 Tumour necrosis factors 255

9.5.1 Tumour necrosis factor biochemistry 2559.5.2 Biological activities of tumour necrosis factor-α 2569.5.3 Immunity and infl ammation 2579.5.4 Tumour necrosis factor receptors 2589.5.5 Tumour necrosis factor: therapeutic aspects 260

Further reading 262

10 Growth factors 26510.1 Introduction 26510.2 Haematopoietic growth factors 265

10.2.1 The interleukins as haemopoietic growth factors 26810.2.2 Granulocyte colony-stimulating factor 26910.2.3 Macrophage colony-stimulating factor 26910.2.4 Granulocyte macrophage colony-stimulating factor 27010.2.5 Clinical application of colony-stimulating factors 27010.2.6 Erythropoietin 272

10.2.6.1 Therapeutic applications of erythropoietin 27410.2.6.2 Chronic disease and cancer chemotherapy 278

10.2.7 Thrombopoietin 27810.3 Growth factors and wound healing 279

10.3.1 Insulin-like growth factors 28010.3.2 Insulin-like growth factor biological effects 28110.3.3 Epidermal growth factor 28210.3.4 Platelet-derived growth factor 28310.3.5 Fibroblast growth factors 28410.3.6 Transforming growth factors 28410.3.7 Neurotrophic factors 286Further reading 287

11 Therapeutic hormones 29111.1 Introduction 29111.2 Insulin 291

11.2.1 Diabetes mellitus 29211.2.2 The insulin molecule 29311.2.3 The insulin receptor and signal transduction 29411.2.4 Insulin production 29411.2.5 Production of human insulin by recombinant DNA technology 29711.2.6 Formulation of insulin products 29711.2.7 Engineered insulins 30111.2.8 Additional means of insulin administration 304

11.3 Glucagon 30511.4 Human growth hormone 307

11.4.1 The growth hormone receptor 30711.4.2 Biological effects of growth hormone 30811.4.3 Therapeutic uses of growth hormone 309

xii CONTENTS

11.5 The gonadotrophins 31011.5.1 Follicle-stimulating hormone, luteinizing hormone

and human chorionic gonadotrophin 31111.5.2 Pregnant mare serum gonadotrophin 31511.5.3 The inhibins and activins 315

11.6 Medical and veterinary applications of gonadotrophins 31911.6.1 Sources and medical uses of follicle-stimulating hormone,

luteinizing hormone and human chorionic gonadotrophin 31911.6.2 Recombinant gonadotrophins 32011.6.3 Veterinary uses of gonadotrophins 321

11.7 Additional recombinant hormones now approved 32311.8 Conclusion 325

Further reading 325

12 Recombinant blood products and therapeutic enzymes 32912.1 Introduction 32912.2 Haemostasis 329

12.2.1 The coagulation pathway 33012.2.2 Terminal steps of coagulation pathway 33212.2.3 Clotting disorders 33412.2.4 Factor VIII and haemophilia 33512.2.5 Production of factor VIII 33612.2.6 Factors IX, IIVa and XIII 339

12.3 Anticoagulants 34012.3.1 Hirudin 34212.3.2 Antithrombin 344

12.4 Thrombolytic agents 34512.4.1 Tissue plasminogen activator 34612.4.2 First-generation tissue plasminogen activator 34812.4.3 Engineered tissue plasminogen activator 34812.4.4 Streptokinase 35012.4.5 Urokinase 35012.4.6 Staphylokinase 35112.4.7 α1-Antitrypsin 35312.4.8 Albumin 354

12.5 Enzymes of therapeutic value 35512.5.1 Asparaginase 35512.5.2 DNase 35712.5.3 Glucocerebrosidase 35912.5.4 α-Galactosidase, urate oxidase and laronidase 36012.5.5 Superoxide dismutase 36312.5.6 Debriding agents 36412.5.7 Digestive aids 364Further reading 366

13 Antibodies, vaccines and adjuvants 37113.1 Introduction 37113.2 Traditional polyclonal antibody preparations 37113.3 Monoclonal antibodies 374

13.3.1 Antibody screening: phage display technology 37613.3.2 Therapeutic application of monoclonal antibodies 378

CONTENTS xiii

13.3.3 Tumour immunology 37913.3.3.1 Antibody-based strategies for tumour

detection/destruction 38313.3.3.2 Drug-based tumour immunotherapy 38613.3.3.3 First-generation anti-tumour antibodies:

clinical disappointment 38813.3.4 Tumour-associated antigens 38913.3.5 Antigenicity of murine monoclonals 39113.3.6 Chimaeric and humanized antibodies 39213.3.7 Antibody fragments 39413.3.8 Additional therapeutic applications of monoclonal antibodies 395

13.4 Vaccine technology 39613.4.1 Traditional vaccine preparations 396

13.4.1.1 Attenuated, dead or inactivated bacteria 39813.4.1.2 Attenuated and inactivated viral vaccines 39913.4.1.3 Toxoids and antigen-based vaccines 399

13.4.2 The impact of genetic engineering on vaccine technology 40013.4.3 Peptide vaccines 40213.4.4 Vaccine vectors 40313.4.5 Development of an AIDS vaccine 40713.4.6 Diffi culties associated with vaccine development 40913.4.7 AIDS vaccines in clinical trials 40913.4.8 Cancer vaccines 41013.4.9 Recombinant veterinary vaccines 411

13.5 Adjuvant technology 41213.5.1 Adjuvant mode of action 41313.5.2 Mineral-based adjuvants 41313.5.3 Oil-based emulsion adjuvants 41413.5.4 Bacteria/bacterial products as adjuvants 41413.5.5 Additional adjuvants 415Further reading 416

14 Nucleic-acid- and cell-based therapeutics 41914.1 Introduction 41914.2 Gene therapy 419

14.2.1 Basic approach to gene therapy 42014.2.2 Some additional questions 423

14.3 Vectors used in gene therapy 42414.3.1 Retroviral vectors 42414.3.2 Adenoviral and additional viral-based vectors 42814.3.3 Manufacture of viral vectors 43114.3.4 Non-viral vectors 43214.3.5 Manufacture of plasmid DNA 436

14.4 Gene therapy and genetic disease 43814.5 Gene therapy and cancer 44114.6 Gene therapy and AIDS 444

14.6.1 Gene-based vaccines 44414.6.2 Gene therapy: some additional considerations 445

14.7 Antisense technology 44514.7.1 Antisense oligonucleotides and their mode of action 44614.7.2 Uses, advantages and disadvantages of ‘oligos’ 448

xiv CONTENTS

14.8 Oligonucleotide pharmacokinetics and delivery 45014.8.1 Manufacture of oligos 45114.8.2 Additional antigene agents: RNA interference and ribozymes 451

14.9 Aptamers 45314.10 Cell- and tissue-based therapies 453

14.10.1 Stem cells 45714.10.2 Adult stem cells 459

14.11 Conclusion 460Further 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 WalshNovember 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

1Pharmaceuticals, 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)

C8H9NO2 151.16 Analgesic

Ketamine C13H16C/NO 237.74 AnaestheticLevamisole C11H12N2S 204.31 AnthelminticDiazoxide C8H7C/N2O2S 230.7 AntihypertensiveAcyclovir C8H11N5O3 225.2 Antiviral agentZidovudine C10H13N5O4 267.2 Antiviral agentDexamethasone C22H29FO5 392.5 Anti-infl ammatory and

immunosuppressive agent

Misoprostol C22H38O5 382.5 Anti-ulcer agentCimetidine C10H16N6 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 diseasesAntibodies Passive immunization against various diseasesInsulin Treatment of diabetes mellitusEnzymes Thrombolytic agents, digestive aids, debriding agents

(i.e. cleansing of wounds)Antibiotics Treatment against various infections agentsPlant 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:

NH2

N

H2N

N

O = S = O

NH2

NH2

O = S = O

NH2

NH2

COOH

N

N

N

N

OH H

H

H2N

CH2

H

HH

NH C NH CH COOH

O

(CH2)2

COOH

Prontosil rubrum(a)

Sulphanilamide(b)

PABA(c)

Pteridinederivative

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

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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 insulinSlow acting insulins (Chapter 11) Modifi ed amino acid sequence Generation of slow acting insulinModifi ed tissue plasminogen

activator (tPA; Chapter 12)Removal of three of the fi ve

native domains of tPAGeneration 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 AGBayer WyethNovo Nordisk GenzymeIsis Pharmaceuticals AbbottGenentech RocheCentocor NovartisBoehringer Manheim SeronoGalenus Manheim OrganonEli Lilly AmgenOrtho Biotech GlaxoSmithKlineSchering Plough CytogenHoffman-la-Roche ImmunomedicsChiron Biogen

established pharmaceutical company Eli Lilly. Genentech developed recombinant human insulin, which was then marketed by Eli Lilly under the trade name Humulin. The merger of biotech capabil-ity with pharmaceutical experience helped accelerate development of the biopharmaceutical sector.

Many of the earlier biopharmaceutical companies no longer exist. The overall level of specu-lative fi nance available was not suffi cient to sustain them all long term (it can take 6–10 years and US$800 million to develop a single drug; Chapter 4). Furthermore, the promise and hype of biotechnology sometimes exceeded its ability actually to deliver a fi nal product. Some biophar-maceutical substances showed little effi cacy in treating their target condition, and/or exhibited unacceptable side effects. Mergers and acquisitions also led to the disappearance of several biop-harmaceutical concerns. Table 1.4 lists many of the major pharmaceutical concerns which now manufacture/market biopharmaceuticals approved for general medical use. Box 1.1 provides a profi le of three well-established dedicated biopharmaceutical companies.

Box 1.1

Amgen, Biogen and Genentech

Amgen, Biogen and Genentech represent three pioneering biopharmaceutical companies that still remain in business.

Founded in the 1980s as AMGen (Applied Molecular Genetics), Amgen now employs over 9000 people worldwide, making it one of the largest dedicated biotechnology companies in existence. Its headquarters are situated in Thousand Oaks, California, although it has re-search, manufacturing, distribution and sales facilities worldwide. Company activities focus upon developing novel (mainly protein) therapeutics for application in oncology, infl ammation, bone disease, neurology, metabolism and nephrology. By mid 2006, seven of its recombinant products had been approved for general medical use (the EPO-based products ‘Aranesp’ and ‘Epogen’ (Chapter 10), the CSF-based products ‘Neupogen’ and ‘Neulasta’ (Chapter 10), as well as the interleukin-1 (IL-1) receptor antagonist ‘Kineret’, the anti-rheumatoid arthritis fu-sion protein Enbrel (Chapter 9) and the keratinocyte growth factor ‘Kepivance’, indicated for the treatment of severe oral mucositis. Total product sales for 2004 reached US$9.9 billion. In July 2002, Amgen acquired Immunex Corporation, another dedicated biopharmaceutical company founded in Seattle in the early 1980s.

Biogen was founded in Geneva, Switzerland, in 1978 by a group of leading molecular biologists. Currently, its global headquarters are located in Cambridge, MA, and it employs in excess of 2000 people worldwide. The company developed and directly markets the interferon-based product ‘Avonex’ (Chapter 8), but also generates revenues from sales of other Biogen-discovered products that are licensed to various other pharmaceutical companies. These include Schering Plough’s ‘Intron A’ (Chapter 8) and a number of hepatitis B-based vaccines sold by SmithKline Beecham (SKB) and Merck (Chapter 13).

Genentech was founded in 1976 by scientist Herbert Boyer and the venture capitalist Robert Swanson. Headquartered in San Francisco, it employs almost 5000 staff worldwide and has 10 protein-based products on the market. These include hGHs (Nutropin, Chapter 11), the anti-body-based products ‘Herceptin’ and ‘Rituxan’ (Chapter 13) and the thrombolytic agents ‘Ac-tivase’ and ‘TNKase’ (Chapter 12). The company also has 20 or so products in clinical trials. In 2004, it generated some US$4.6 billion in revenues.

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8 CH1 PHARMACEUTICALS, BIOLOGICS AND BIOPHARMACEUTICALS

1.5 Biopharmaceuticals: current status and future prospects

Approximately one in every four new drugs now coming on the market is a biopharmaceuti-cal. By mid 2006, some 160 biopharmaceutical products had gained marketing approval in the USA and/or EU. Collectively, these represent a global biopharmaceutical market in the region of US$35 billion (Table 1.5), and the market value is estimated to surpass US$50 billion by 2010. The products include a range of hormones, blood factors and thrombolytic agents, as well as vac-cines and monoclonal antibodies (Table 1.6). All but two are protein-based therapeutic agents. The exceptions are two nucleic-acid-based products: ‘Vitravene’, an antisense oligonucleotide, and ‘Macugen’, an aptamer (Chapter 14). Many additional nucleic-acid-based products for use in gene therapy or antisense technology are in clinical trials, although the range of technical diffi culties that still beset this class of therapeutics will ensure that protein-based products will overwhelm-ingly predominate for the foreseeable future (Chapter 14).

Many of the initial biopharmaceuticals approved were simple replacement proteins (e.g. blood factors and human insulin). The ability to alter the amino acid sequence of a protein logically coupled to an increased understanding of the relationship between protein structure and function (Chapters 2 and 3) has facilitated the more recent introduction of several engineered therapeutic proteins (Table 1.3). Thus far, the vast majority of approved recombinant proteins have been pro-duced in the bacterium E. coli, the yeast S. cerevisiae or in animal cell lines (most notably Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells. These production systems are discussed in Chapter 5.

Although most biopharmaceuticals approved to date are intended for human use, a number of products destined for veterinary application have also come on the market. One early such exam-ple is that of recombinant bovine GH (Somatotrophin), which was approved in the USA in the early 1990s and used to increase milk yields from dairy cattle. Additional examples of approved veterinary biopharmaceuticals include a range of recombinant vaccines and an interferon-based product (Table 1.7).

Table 1.5 Approximate annual market values of some leading approved biopharmaceutical products. Data gathered from various sources, including company home pages, annual reports and industry reports

Product (Company) Product description (use) Annual sales value (US$, billions)

Procrit (Amgen/Johnson & Johnson)

EPO (treatment of anaemia) 4.0

Epogen & Aranesp combined (Amgen)

EPO (treatment of anaemia) 4.0

Intron A (Schering Plough) IFN-α (treatment of leukaemia) 0.3Remicade (Johnson & Johnson) Monoclonal antibody based

(treatment of Crohn’s disease)1.7

Avonex (Biogen) Interferon-β (IFN-β; treatment of multiple sclerosis)

1.2

Embrel (Wyeth) Monoclonal antibody based (treatment of rheumatoid arthritis)

1.3

Rituxan (Genentech) Monoclonal antibody based (non-Hodgkin’s lymphoma)

1.5

Humulin (Eli Lilly) Insulin (diabetes) 1.0

At least 1000 potential biopharmaceuticals are currently being evaluated in clinical trials, al-though the majority of these are in early stage trials. Vaccines and monoclonal antibody-based products represent the two biggest product categories. Regulatory factors (e.g. hormones and

Table 1.6 Summary categorization of biopharmaceuticals approved for general medical use in the EU and/or USA by 2006

Product type Examples No. approved Refer to

Blood factors Factors VIII and IX 8 Chapter 12Thrombolytic agents tPA 6 Chapter 12Hormones Insulin, GH,

gonadotrophins33 Chapter 11

Haematopoietic growth factors

EPO, CSFs 8 Chapter 10

Interferons IFN-α, -β, -γ 16 Chapter 8Interleukin-based

productsIL-2 3 Chapter 9

Vaccines Hepatitis B-surface antigen

20 Chapter 13

Monoclonal antibodies Various 30 Chapter 13Nucleic acid based Antisense and aptamer 2 Chapter 14Additional products Tumour necrosis factor

(TNF), therapeutic enzymes

18 Various chapters

Table 1.7 Some recombinant (r) biopharmaceuticals recently approved for veterinary application in the EU

Product Company Indication

Vibragen Omega (r-feline interferon omega; IFN-ω)

Virbac Reduction of mortality/clinical symptoms associated with canine parvovirus

Fevaxyl Pentafel (combination vaccine containing r-feline leukaemia viral antigen as one component)

Fort Dodge Laboratories Immunization of cats against various feline pathogens

Porcilis porcoli (combination vaccine containing r-E. coli adhesins)

Intervet Active immunization of sows

Porcilis AR-T DF (combination vaccine containing a recombinant modifi ed toxin from Pasteurella multocida)

Intervet Reduction in clinical signs of progressive atrophic rhinitis in piglets

Porcilis pesti (combination vaccine containing r-classical swine fever virus E2 subunit antigen)

Intervet Immunization of pigs against classical swine fever

Bayovac CSF E2 (combination vaccine containing r-classical swine fever virus E2 subunit antigen)

Intervet Immunization of pigs against classical swine fever

BIOPHARMACEUTICALS: CURRENT STATUS AND FUTURE PROSPECTS 9

10 CH1 PHARMACEUTICALS, BIOLOGICS AND BIOPHARMACEUTICALS

cytokines) and gene therapy and antisense-based products also represent signifi cant groupings. Although most protein-based products likely to gain marketing approval over the next 2–3 years will be produced in engineered E. coli, S. cerevisiae or animal cell lines, some products now in clinical trials are being produced in the milk of transgenic animals (Chapter 5). Additionally, plant-based transgenic expression systems may potentially come to the fore, particularly for the production of oral vaccines (Chapter 5).

Interestingly, the fi rst generic biopharmaceuticals are already entering the market. Patent protection for many fi rst-generation biopharmaceuticals (including recombinant human GH (rhGH), insulin, EPO, interferon-α (IFN-α) and granulocyte-CSF (G-CSF)) has now/is now coming to an end. Most of these drugs command an overall annual market value in excess of US$1 billion, rendering them attractive potential products for many biotechnology/pharmaceutical companies. Companies already/soon producing generic biopharmaceuticals include Biopartners (Switzerland), Genemedix (UK), Sicor and Ivax (USA), Congene and Microbix (Canada) and BioGenerix (Germany). Genemedix, for example, secured approval for sale of a recombinant CSF in China in 2001 and is also commencing the manufacture of recombinant EPO. Sicor currently markets hGH and IFN-α in eastern Europe and various developing nations. A generic hGH also gained approval in both Europe and the USA in 2006.

To date (mid 2006), no gene-therapy-based product has thus far been approved for general medical use in the EU or USA, although one such product (‘Gendicine’; Chapter 14) has been ap-proved in China. Although gene therapy trials were initiated as far back as 1989, the results have been disappointing. Many technical diffi culties remain in relation to, for example, gene delivery and regulation of expression. Product effectiveness was not apparent in the majority of trials un-dertaken and safety concerns have been raised in several trials.

Only one antisense-based product has been approved to date (in 1998) and, although several such antisense agents continue to be clinically evaluated, it is unlikely that a large number of such products will be approved over the next 3–4 years. Aptamers represent an additional emerging class of nucleic-acid-based therapeutic. These are short DNA- or RNA-based sequences that adopt a specifi c three-dimensional structure, enabling them to bind (and thereby inhibit) specifi c target molecules. One such product (Macugen) has been approved to date. RNA interference (RNAi) rep-resents a yet additional mechanism of achieving downregulation of gene expression (Chapter 14). It shares many characteristics with antisense technology and, like antisense, provides a potential means of treating medical conditions triggered or exacerbated by the inappropriate overexpression of specifi c gene products. Despite the disappointing results thus far generated by nucleic-acid-based products, future technical advances will almost certainly ensure the approval of gene therapy and antisense-based products in the intermediate to longer term future.

Technological developments in areas such as genomics, proteomics and high-throughput screening are also beginning to impact signifi cantly upon the early stages of drug development (Chapter 4). By linking changes in gene/protein expression to various disease states, for example, these technologies will identify new drug targets for such diseases. Many/most such targets will themselves be proteins, and drugs will be designed/developed specifi cally to interact with. They may be protein based or (more often) low molecular mass ligands.

Additional future innovations likely to impact upon pharmaceutical biotechnology include the development of alternative product production systems, alternative methods of delivery and the development of engineered cell-based therapies, particularly stem cell therapy. As mentioned pre-viously, protein-based biotechnology products produced to date are produced in either microbial