BIOPHARMACEUTICALS BIOCHEMISTRY AND BIOTECHNOLOGY - PART 1 potx

Chapter 1 Pharmaceuticals, biologics and biopharmaceuticals 1Introduction to pharmaceutical products 1Biopharmaceuticals and pharmaceutical biotechnology 1History of the pharmaceutical i

BIOPHARMACEUTICALS BIOCHEMISTRY AND BIOTECHNOLOGY

Second Edition

Gary Walsh Industrial Biochemistry Programme

CES Department University of Limerick, Ireland

BIOCHEMISTRY AND BIOTECHNOLOGY

Second Edition

BIOPHARMACEUTICALS BIOCHEMISTRY AND BIOTECHNOLOGY

Second Edition

Gary Walsh Industrial Biochemistry Programme

CES Department University of Limerick, Ireland

First Edition 1998u John Wiley & Sons, Ltd

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I dedicate this book to my beautiful son Shane, born during the revision of Chapter 6 I include his photograph in the hope that a Hollywood producer, looking for a child film star, will spot it and immediately offer to make us suitably rich In the future, I also hope

to use it to embarrass him during his teenage years, by showing it to

all his cool, sophisticated friends.

Chapter 1 Pharmaceuticals, biologics and biopharmaceuticals 1Introduction to pharmaceutical products 1Biopharmaceuticals and pharmaceutical biotechnology 1History of the pharmaceutical industry 3The age of biopharmaceuticals 5Biopharmaceuticals: current status and future prospects 8Traditional pharmaceuticals of biological origin 12Pharmaceuticals of animal origin 13The sex hormones 14The androgens 14Oestrogens 15Progesterone and progestogens 17Corticosteroids 19Catecholamines 21Prostaglandins 23Pharmaceutical substances of plant origin 27

Atropine and scopalamine 28Morphine and cocaine 29Additional plant alkaloids 30Ergot alkaloids 30Flavonoids, xanthines and terpenoids 30Cardiac glycosides and coumarins 33

Pharmaceutical substances of microbial origin 33The macrolides and ansamycins 38Peptide and other antibiotics 39Conclusion 39Further reading 40Chapter 2 The drug development process 43Drug discovery 44The impact of genomics and related technologies upon drug discovery 45

Gene chips 47Proteomics 49Structural genomics 50Pharmacogenetics 51Plants as a source of drugs 52Microbial drugs 53Rational drug design 54Combinatorial approaches to drug discovery 56Initial product characterization 57

What is a patent and what is patentable? 57Patent types 62The patent application 63Patenting in biotechnology 64Delivery of biopharmaceuticals 66Oral delivery systems 66Pulmonary delivery 67Nasal, transmucosal and transdermal delivery systems 68Pre-clinical trials 69Pharmacokinetics and pharmacodynamics 69Toxicity studies 71Reproductive toxicity and teratogenicity 71Mutagenicity, carcinogenicity and other tests 72Clinical trials 73Clinical trial design 75Trial size and study population 75Randomized control studies 76Additional trial designs 76The role and remit of regulatory authorities 78The Food and Drug Administration 78The investigational new drug application 80The new drug application 82European regulations 84National regulatory authorities 84The EMEA and the new EU drug approval systems 85The centralized procedure 86Mutual recognition 88Drug registration in Japan 88World harmonization of drug approvals 89Conclusion 89Further reading 89Chapter 3 The drug manufacturing process 93International pharmacopoeia 93Martindale, the Extra Pharmacopoeia 94Guides to good manufacturing practice 94

viii CONTENTS

The manufacturing facility 97Clean rooms 98Cleaning, decontamination and sanitation (CDS) 101CDS of the general manufacturing area 102CDS of process equipment 102Water for biopharmaceutical processing 104Generation of purified water and water for injections (WFI) 105Distribution system for WFI 107Documentation 109Specifications 110Manufacturing formulae, processing and packaging instructions 110

CONTENTS ix

Amino acid analysis 169Peptide mapping 170N-terminal sequencing 171Analysis of secondary and tertiary structure 173Endotoxin and other pyrogenic contaminants 173Endotoxin, the molecule 174Pyrogen detection 176

Microbial and viral contaminants 180Viral assays 181Miscellaneous contaminants 182Validation studies 183Further reading 185

Chapter 4 The cytokines — the interferon family 189Cytokines 189Cytokine receptors 194Cytokines as biopharmaceuticals 195The interferons 196The biochemistry of interferon-a 197Interferon-b 198Interferon-g 198Interferon signal transduction 198The interferon receptors 199The JAK–STAT pathway 199The interferon JAK–STAT pathway 202The biological effects of interferons 203The eIF-2a protein kinase system 207Interferon biotechnology 207Production and medical uses of IFN-a 210Medical uses of IFN-b 213Medical applications of IFN-g 214Interferon toxicity 216Additional interferons 218Conclusion 219Further reading 219

Chapter 5 Cytokines: interleukins and tumour necrosis factor 223Interleukin-2 (IL-2) 225IL-2 production 228IL-2 and cancer treatment 228IL-2 and infectious diseases 230Safety issues 231Inhibition of IL-2 activity 231

x CONTENTS

Interleukin-1 (IL-1) 232The biological activities of IL-1 233IL-1 biotechnology 234Interleukin-3: biochemistry and biotechnology 235Interleukin-4 236Interleukin-6 238Interleukin-11 240Interleukin-5 241Interleukin-12 244Tumour necrosis factors (TNFs) 246TNF biochemistry 246Biological activities of TNF-a 247Immunity and inflammation 248TNF receptors 249TNF: therapeutic aspects 250Further reading 252Chapter 6 Haemopoietic growth factors 255The interleukins as haemopoietic growth factors 257Granulocyte colony stimulating factor (G-CSF) 258Macrophage colony-stimulating factor (M-CSF) 259Granulocyte-macrophage colony stimulating factor (GM-CSF) 259Clinical application of CSFs 261Leukaemia inhibitory factor (LIF) 263Erythropoietin (EPO) 264The EPO receptor and signal transduction 267Regulation of EPO production 267Therapeutic applications of EPO 268Chronic disease and cancer chemotherapy 271Additional non-renal applications 272Tolerability 273Thrombopoietin 273Further reading 275Chapter 7 Growth factors 277Growth factors and wound healing 277Insulin-like growth factors (IGFs) 279IGF biochemistry 280IGF receptors 280IGF-binding proteins 282Biological effects 282IGF and fetal development 283IGFs and growth 283Renal and reproductive effects 284Neuronal and other effects 285

CONTENTS xi

Epidermal growth factor (EGF) 285The EGF receptor 286Platelet-derived growth factor (PDGF) 287The PDGF receptor and signal transduction 288PDGF and wound healing 289Fibroblast growth factors (FGFs) 289Transforming growth factors (TGFs) 290

Neurotrophic factors 293The neurotrophins 294Neurotrophin receptors 296The neurotrophin low-affinity receptor 297Ciliary neurotrophic factor and glial cell line-derived neurotrophic factor 297Neurotrophic factors and neurodegenerative disease 298Amyotrophic lateral sclerosis (ALS) and peripheral neuropathy 298Neurotrophic factors and neurodegenerative diseases of the brain 298Further reading 300Chapter 8 Hormones of therapeutic interest 303

Diabetes mellitus 304The insulin molecule 304The insulin receptor and signal transduction 307Insulin production 307Enzymatic conversion of porcine insulin 311Production of human insulin by recombinant DNA technology 312Formulation of insulin products 314Engineered insulins 317Additional means of insulin administration 320Treating diabetics with insulin-producing cells 321

Human growth hormone (hGH) 324Growth hormone releasing factor (GHRF) and inhibitory factor (GHRIF) 325The GH receptor 325Biological effects of GH 327Therapeutic uses of GH 328Recombinant hGH (rhGH) and pituitary dwarfism 328Idiopathic short stature and Turner’s syndrome 330Metabolic effects of hGH 330

GH, lactation and ovulation 331The gonadotrophins 331Follicle stimulating hormone (FSH), luteinizing hormone (LH)

and human chorionic gonadotrophin (hCG) 331Pregnant mare serum gonadotrophin (PMSG) 335The inhibins and activins 337

xii CONTENTS

LHRH and regulation of gonadotrophin production 338Medical and veterinary applications of gonadotrophins 339Sources and medical uses of FSH, LH and hCG 340Recombinant gonadotrophins 342Veterinary uses of gonadotrophins 344Gonadotrophin releasing hormone (GnRH) 345Additional recombinant hormones now approved 345Conclusions 348Further reading 348

Chapter 9 Blood products and therapeutic enzymes 351Disease transmission 351Whole blood 353Platelets and red blood cells 353Blood substitutes 353

Vitamin K antimetabolites 375

Antithrombin 379Thrombolytic agents 380Tissue plasminogen activator (tPA) 381First-generation tPA 383Engineered tPA 383Streptokinase 385Urokinase 386Staphylokinase 386

a1-Antitrypsin 388Enzymes of therapeutic value 389Asparaginase 390

Glucocerebrosidase 393a-Galactosidase and urate oxidase 395Superoxide dismutase 397

CONTENTS xiii

Debriding agents 397Digestive aids 398

Further reading 400

Chapter 10 Antibodies, vaccines and adjuvants 403Polyclonal antibody preparations 403Anti-D immunoglobulin 406Normal immunoglobulins 407Hepatitis B and tetanus immunoglobulin 407Snake and spider antivenins 408Monoclonal antibodies 409Production of monoclonals via hybridoma technology 411Antibody screening: phage display technology 412Therapeutic application of monoclonal antibodies 414Tumour immunology 415Antibody-based strategies for tumour detection/destruction 417Drug-based tumour immunotherapy 424First-generation anti-tumour antibodies: clinical disappointment 426Tumour-associated antigens 426Antigenicity of murine monoclonals 428Chimaeric and humanized antibodies 429Antibody fragments 432Additional therapeutic applications of monoclonal antibodies 433Cardiovascular and related disease 433Infectious diseases 433Autoimmune disease 434Transplantation 434Vaccine technology 435Traditional vaccine preparations 436Attenuated, dead or inactivated bacteria 438Attenuated and inactivated viral vaccines 439Toxoids, antigen-based and other vaccine preparations 440The impact of genetic engineering on vaccine technology 441Peptide vaccines 444Vaccine vectors 445Development of an AIDS vaccine 447Difficulties associated with vaccine development 450AIDS vaccines in clinical trials 450Cancer vaccines 452Recombinant veterinary vaccines 452Adjuvant technology 453Adjuvant mode of action 455Mineral-based adjuvants 455Oil-based emulsion adjuvants 455

xiv CONTENTS

Bacteria/bacterial products as adjuvants 457Additional adjuvants 458Further reading 460Chapter 11 Nucleic acid therapeutics 463Gene therapy 463Basic approach to gene therapy 464Some additional questions 467Vectors used in gene therapy 468Retroviral vectors 468Additional viral-based vectors 472Manufacture of viral vectors 474Non-viral vectors 476Manufacture of plasmid DNA 480Gene therapy and genetic disease 482Gene therapy and cancer 485Gene therapy and AIDS 486Gene-based vaccines 488Gene therapy: some additional considerations 488Anti-sense technology 488Anti-sense oligonucleotides 490Uses, advantages and disadvantages of ‘oligos’ 491Delivery and cellular uptake of oligonucleotides 493Manufacture of oligonucleotides 493Vitravene, an approved antisense agent 494Antigene sequences and ribozymes 494Conclusion 495Further reading 496Appendix 1 Biopharmaceuticals thus far approved in the USA or European Union 499Appendix 2 Some Internet addresses relevant to the biopharmaceutical sector 509Appendix 3 Two selected monographs reproduced from the European Pharmacopoeia

with permission from the European Commission:

I Products of recombinant DNA technology 515

II Interferon a-2 concentrated solution 520Appendix 4 Manufacture of biological medicinal products for human use (Annex 2

from The Rules Governing Medicinal Products in the European Community,Vol 4, Good Manufacturing Practice for Medicinal Products) 527

CONTENTS xv

Advances in our understanding of the molecular principles underlining both health and diseasehas revealed the existence of many regulatory polypeptides of significant medical potential Thefact that such polypeptides are produced naturally within the body only in minute quantitiesinitially precluded their large-scale medical application The development in the 1970s of thetwin techniques of genetic engineering and hybridoma technology marked the birth of themodern biotech era These techniques facilitate the large-scale production of virtually anyprotein, and proteins of medical interest produced by these methodologies have been coined

‘biopharmaceuticals’ More recent developments in biomedical research highlights the clinicalpotential of nucleic acid-based therapeutic agents Gene therapy and anti-sense technology arelikely to become a medical reality within a decade The term ‘biopharmaceutical’ now alsoincorporates the polynucleotide sequences utilized for such purposes

This book attempts to provide a balanced overview of the biopharmaceutical industry, notonly in terms of categorizing the products currently available, but also illustrating how thesedrugs are produced and brought to market Chapter 1 serves as an introduction to the topic, andalso focuses upon several ‘traditional’ pharmaceutical substances isolated (initially at least) frombiological sources This serves as a backdrop for the remaining chapters, which focus almostexclusively upon recently developed biopharmaceutical products The major emphasis is placedupon polypeptide-based therapeutic agents, while the potential of nucleic acid-based drugs isdiscussed in the final chapter

In preparing the latest edition of this textbook, I highlight the latest developments within thesector, provide a greater focus upon actual commercial products thus far approved and howthey are manufactured, and I include substantial new sections detailing biopharmaceutical drugdelivery and how advances in genomics and proteomics will likely impact upon (bio)pharma-ceutical drug development

The major target audience is that of advanced undergraduates or postgraduate studentspursuing courses in relevant aspects of the biological sciences The book should proveparticularly interesting to students undertaking programmes in biotechnology, biochemistry, thepharmaceutical sciences, medicine or any related biomedical subject A significant additionaltarget audience are those already employed in the (bio)pharmaceutical sector, who wish to gain

a better overview of the industry in which they work

The successful completion of this text has been made possible by the assistance of severalpeople to whom I owe a depth of gratitude Chief amongst these is Sandy Lawson, who appears

to be able to read my mind as well as my handwriting Thank you to Nancy, my beautiful wife,who suffered most from my becoming a social recluse during the preparation of this text Thankyou, Nancy, for not carrying out your threat to burn the manuscript on various occasions, andfor helping with the proof-reading I am also very grateful to the staff of John Wiley and SonsLtd for the professionalism and efficiency they exhibited while bringing this book through the

publication process The assistance of companies who provided information and photographsfor inclusion in the text is also gratefully acknowledged, as is the cooperation of those publisherswho granted me permission to include certain copyrighted material Finally, a word ofappreciation to all my colleagues at Limerick, who continue to make our university such a greatplace to work.

Gary WalshLimerick, November 2002

xviii PREFACE

Chapter 1 Pharmaceuticals, biologics and biopharmaceuticals

INTRODUCTION TO PHARMACEUTICAL PRODUCTS

Pharmaceutical substances form the backbone of modern medicinal therapy Most traditionalpharmaceuticals are low molecular mass organic chemicals (Table 1.1) Although some (e.g.aspirin) were originally isolated from biological sources, most are now manufactured by directchemical synthesis Two types of manufacturing companies thus comprise the ‘traditional’pharmaceutical sector; the chemical synthesis plants, which manufacture the raw chemicalingredients in bulk quantities, and the finished product pharmaceutical facilities, which purchasethese raw bulk ingredients, formulate them into final pharmaceutical products, and supply theseproducts to the end-user

In addition to chemical-based drugs, a range of pharmaceutical substances (e.g hormonesand blood products) are produced by or extracted from biological sources Such products, somemajor examples of which are listed in Table 1.2, may thus be described as products ofbiotechnology In some instances, categorizing pharmaceuticals as products of biotechnology orchemical synthesis becomes somewhat artificial, e.g certain semi-synthetic antibiotics areproduced by chemical modification of natural antibiotics produced by fermentationtechnology

BIOPHARMACEUTICALS AND PHARMACEUTICAL

BIOTECHNOLOGY

Terms such as ‘biologic’, ‘biopharmaceutical’ and ‘products of pharmaceutical biotechnology’

or ‘biotechnology medicines’ have now become an accepted part of the pharmaceuticalliterature However, these terms are sometimes used interchangeably and can mean differentthings to different people

While it might be assumed that ‘biologic’ refers to any pharmaceutical product produced bybiotechnological endeavour, its definition is more limited In pharmaceutical circles, ‘biologic’

Biopharmaceuticals: Biochemistry and Biotechnology, Second Edition Gary Walsh

John Wiley & Sons Ltd: ISBN 0 470 84326 8 (ppc), ISBN 0 470 84327 6 (pbk)

generally refers to medicinal products derived from blood, as well as vaccines, toxins andallergen products Thus, some traditional biotechnology-derived pharmaceutical products (e.g.hormones, antibiotics and plant metabolites) fall outside the strict definition.

The term ‘biopharmaceutical’ was first used in the 1980s and came to describe a class oftherapeutic protein produced by modern biotechnological techniques, specifically via geneticengineering or (in the case of monoclonal antibodies) by hybridoma technology This usageequated the term ‘biopharmaceutical’ with ‘therapeutic protein synthesized in engineered (non-naturally occurring) biological systems’ More recently, however, nucleic acids used forpurposes of gene therapy and antisense technology (Chapter 11) have come to the fore and theytoo are generally referred to as ‘biopharmaceuticals’ Moreover, several recently approvedproteins are used for in vivo diagnostic as opposed to therapeutic purposes Throughout thisbook therefore, the term ‘biopharmaceutical’ refers to protein or nucleic acid basedpharmaceutical substances used for therapeutic or in vivo diagnostic purposes, which areproduced by means other than direct extraction from natural (non-engineered) biologicalsources (Tables 1.3 and 1.4)

As used herein, ‘biotechnology medicines’ or ‘products of pharmaceutical biotechnology’ areafforded a much broader definition Unlike the term ‘biopharmaceutical’, the term

Levamisole C11H12N2S 204.31 Anthelmintic

Diazoxide C8H7ClN2O2S 230.7 Anti-hypertensive

Acyclovir C8H11N5O3 225.2 Anti-viral agent

Zidovudine C10H13N5O4 267.2 Anti-viral agent

Dexamethasone C22H29FO5 392.5 Anti-inflammatory and

immunosuppressive agentMisoprostol C22H38O5 382.5 Anti-ulcer agent

Cimetidine C10H16N6 252.3 Anti-ulcer agent

Table 1.2 Some pharmaceuticals which were traditionally obtained by direct extraction from biologicalsource material Many of the protein-based pharmaceuticals mentioned below are now also produced bygenetic engineering

Substance Medical application

Blood products (e.g coagulation factors) Treatment of blood disorders such as haemophilia A or BVaccines 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 infectious agents

Plant extracts (e.g alkaloids) Various, including pain relief

‘biotechnology’ has a much broader and long-established meaning Essentially, it refers to theuse of biological systems (e.g cells or tissues) or biological molecules (e.g enzymes orantibodies) for or in the manufacture of commercial products Therefore, the term is equallyapplicable to long-established biological processes, such as brewing, and more modernprocesses, such as genetic engineering As such, the term ‘biotechnology medicine’ is definedhere as ‘any pharmaceutical product used for a therapeutic or in vivo diagnostic purpose, which

is produced in full or in part by either traditional or modern biotechnological means’ Suchproducts encompass, for example, antibiotics extracted from fungi, therapeutic proteinsextracted from native source material (e.g insulin from pig pancreas) and products produced bygenetic engineering (e.g recombinant insulin) (Tables 1.3 and 1.4)

HISTORY OF THE PHARMACEUTICAL INDUSTRY

The pharmaceutical industry, as we now know it, is barely 60 years old From very modestbeginnings it has grown rapidly, reaching an estimated value of $100 billion by the mid-1980s.Its current value is likely double this figure or more There are well in excess of 10 000pharmaceutical companies in existence, although only about 100 of these can claim to be of trueinternational significance These companies manufacture in excess of 5000 individualpharmaceutical substances used routinely in medicine

The first stages of development of the modern pharmaceutical industry can be traced back tothe turn of the twentieth century At that time (apart from folk cures), the medical communityhad at their disposal only four drugs that were effective in treating specific 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 sp.), was used to treatmalaria

Pecacuanha (active ingredient is a mixture of alkaloids), used for treating dysentery, wasobtained from the bark/roots of the plant species Cephaelis

Mercury, for the treatment of syphilis

PHARMACEUTICALS, BIOLOGICS AND BIOPHARMACEUTICALS 3Table 1.3 A summary of the definition of the terms ‘biologic’, ‘biopharmaceutical’ and ‘biotechnologymedicine’ as used throughout this book Reprinted from European Journal of Pharmaceutical Sciences,vol 15, Walsh, Biopharmaceuticals and Biotechnology, p 135–138,s2002, with permission from ElsevierScience

Biopharmaceutical A protein or nucleic acid based pharmaceutical substance used for

therapeutic or in vivo diagnostic purposes, which is produced bymeans other than direct extraction from a native (non-engineered)biological source

Biologic A virus, therapeutic serum, toxin, antitoxin, vaccine, blood, blood

component or derivative, allergenic product or analogous product,

or arsphenamine or its derivatives or any other trivalent organicarsenic compound applicable to the prevention, cure or treatment ofdisease or conditions of human beings

The lack of appropriate safe and effective medicines contributed in no small way to the low lifeexpectancy characteristic of those times.

Developments in biology (particularly the growing realization of the microbiological basis ofmany diseases), as well as a developing appreciation of the principles of organic chemistry,helped underpin future innovation in the fledgling pharmaceutical industry The successfulsynthesis of various artificial dyes, which proved to be therapeutically useful, led to theformation of pharmaceutical/chemical companies such as Bayer and Hoechst in the late 1800s,e.g scientists at Bayer succeeded in synthesizing aspirin in 1895

Despite these early advances, it was not until the 1930s that the pharmaceutical industrybegan to develop in earnest The initial landmark discovery of this era was probably thediscovery and chemical synthesis of the sulpha drugs These are a group of related moleculesderived from the red dye, Prontosil rubrum These drugs proved effective in the treatment of awide variety of bacterial infections (Figure 1.1) Although it was first used therapeutically in theearly 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

4 BIOPHARMACEUTICALS

Table 1.4 The categorization of pharmaceutically significant biological molecules using the indicateddefinitions as listed in Table 1.3 Reproduced in modified form from European Journal ofPharmaceutical Sciences, vol 15, Walsh, Biopharmaceuticals and Biotechnology, p 135–138, s2002,with permission from Elsevier Science

Pharmaceutical product Biopharmaceutical? Biotechnology medicine? Biologic?

Recombinant protein Yes Yes No

Monoclonal antibody Yes Yes No

Proteins obtained by direct

extraction from native

source (e.g blood derived

clotting factors)

No Yes Some (e.g blood

factors andpolyclonalantibodies)Gene therapy products Yes Yes No

direct extraction from

native producer source

Antibiotics obtained by direct

extraction from native

producer, or by

semi-synthesis

Plant-based products

obtained by direct extraction

from a native producer,

was boosted further by the commencement of industrial-scale penicillin manufacture in the early1940s Around this time, many of the current leading pharmaceutical companies (or theirforerunners) were founded Examples include Ciba Geigy, Eli Lilly, Wellcome, Glaxo andRoche Over the next two to three decades, these companies developed drugs such astetracyclines, corticosteroids, oral contraceptives, antidepressants and many more Most ofthese pharmaceutical substances are manufactured by direct chemical synthesis.

THE AGE OF BIOPHARMACEUTICALS

Biomedical research continues to broaden our understanding of the molecular mechanismsunderlining both health and disease Research undertaken since the 1950s has pinpointed a host

of proteins produced naturally in the body which have obvious therapeutic applications.Examples include the interferons, and interleukins, which regulate the immune response; growthfactors such as erythropoietin, which stimulates red blood cell production; and neurotrophicfactors, which regulate the development and maintenance of neural tissue

While the pharmaceutical potential of these regulatory molecules was generally appreciated,their widespread medical application was in most cases rendered impractical due to the tinyquantities in which they were naturally produced The advent of recombinant DNA technology(genetic engineering) and monoclonal antibody technology (hybridoma technology) overcamemany such difficulties, and marked the beginning of a new era of the pharmaceutical sciences.Recombinant DNA technology has had a four-fold positive impact upon the production ofpharmaceutically important proteins:

It overcomes the problem of source availability Many proteins of therapeutic potential areproduced naturally in the body in minute quantities Examples include interferons (Chapter4), interleukins (Chapter 5) and colony stimulating factors (Chapter 6) This renderedimpractical their direct extraction from native source material in quantities sufficient to meetlikely clinical demand Recombinant production (Chapter 3) allows the manufacture of anyprotein in whatever quantity it is required

It overcomes problems of product safety Direct extraction of product from some nativebiological sources has, in the past, led to the unwitting transmission of disease Examplesinclude the transmission of blood-borne pathogens such as hepatitis B, C and HIV viainfected blood products and the transmission of Creutzfeldt–Jakob disease to personsreceiving human growth hormone preparations derived from human pituitaries

It provides an alternative to direct extraction from inappropriate/dangerous source material Anumber of therapeutic proteins have traditionally been extracted from human urine Thefertility hormone FSH, for example, is obtained from the urine of post-menopausal women,while a related hormone, hCG, is extracted from the urine of pregnant women (Chapter 8).Urine is not considered a particularly desirable source of pharmaceutical products Whileseveral products obtained from this source remain on the market, recombinant forms havenow also been approved Other potential biopharmaceuticals are produced naturally indownright dangerous sources Ancrod, for example, is a protein displaying anti-coagulantactivity (Chapter 9) and, hence, is of potential clinical use; however, it is produced naturally

by the Malaysian pit viper While retrieval by milking snake venom is possible, and indeedmay be quite an exciting procedure, recombinant production in less dangerous organisms,such as Escherichia coli or Saccharomyces cerevisiae, would be considered preferable by most It facilitates the generation of engineered therapeutic proteins displaying some clinical

PHARMACEUTICALS, BIOLOGICS AND BIOPHARMACEUTICALS 5

6 BIOPHARMACEUTICALS

Tetrahydrofolic acid

(d)

advantage over the native protein product Techniques such as site-directed mutagenesisfacilitate the logical introduction of pre-defined changes in a protein’s amino acid sequence.Such changes can be minimal, such as the insertion, deletion or alteration of a single aminoacid residue, or can be more substantial, e.g the alteration or deletion of an entire domain, orthe generation of a novel hybrid protein Such changes can be made for a number of reasonsand several engineered products have now gained marketing approval An overviewsummary of some engineered product types now on the market is provided in Table 1.5.These and other examples will be discussed in subsequent chapters.

Despite the undoubted advantages of recombinant production, it remains the case that manyprotein-based products extracted directly from native source material remain on the market.These products have proved safe and effective and selected examples are provided in Table 1.2

In certain circumstances, direct extraction of native source material can prove equally/moreattractive than recombinant production This may be for an economic reason, e.g if the protein

is produced in very large quantities by the native source and is easy to extract/purify, as is thecase for human serum albumin (Chapter 9) Also, some blood factor preparations purified from

PHARMACEUTICALS, BIOLOGICS AND BIOPHARMACEUTICALS 7Table 1.5 Selected engineered biopharmaceutical types/products, which have now gained marketingapproval These and additional such products will be discussed in detail in subsequent chaptersProduct description/type Alteration introduced Rationale

Faster-acting insulins (Chapter 8) Modified amino acid sequence Generation of faster-acting insulinSlow-acting insulins (Chapter 8) Modified amino acid sequence Generation of slow-acting insulinModified tissue plasminogen

activator (tPA: Chapter 9)

Removal of three of the fivenative domains of tPA

Generation of a faster-actingthrombolytic (clot degrading)agent

Modified blood factor VIII

Replacement of most/virtually all

of the murine amino acidsequences with sequences found

in human antibodies

Greatly reduced/eliminatedimmonogenicity Ability toactivate human effectorfunctions

‘Ontak’, a fusion protein

(Chapter 5)

Fusion protein consisting of thediphtheria toxin linked tointerleukin-2

Targets toxin selectively to cellsexpressing an IL-2 receptor

Figure 1.1 (Opposite) Sulpha drugs and their mode of action The first sulpha drug to be used medicallywas the red dye prontosil rubrum (a) In the early 1930s, experiments illustrated that the administration ofthis dye to mice infected with haemolytic streptococci prevented the death of the mice This drug, whileeffective in vivo, was devoid of in vitro antibacterial activity It was first used clinically in 1935 under thename Streptozon It was subsequently shown that prontosil rubrum was enzymatically reduced by the liver,forming sulphanilamide, the actual active antimicrobial agent (b) Sulphanilamide induces its effect byacting as an anti-metabolite with respect to para-aminobenzoic acid (PABA) (c) PABA is an essentialcomponent of tetrahydrofolic acid (THF) (d) THF serves as an essential co-factor for several cellularenzymes Sulphanilamide (at sufficiently high concentrations) inhibits manufacture of THF by competingwith PABA This effectively inhibits essential THF-dependent enzyme reactions within the cell Unlikehumans, who can derive folates from their diets, most bacteria must synthesize it de novo, as they cannotabsorb it intact from their surroundings