Contents

Preface xv

Acronyms xvii

1 Pharmaceuticals, biologies 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 modification 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 purification 43

3.2.3 Nucleic acid sequencing 45

3.3 Recombinant production of therapeutic proteins 46

3.4 Classical gene cloning and identification 47

3.4.1 cDNA cloning 51

3.4.2 Cloning via polymerase chain reaction 51

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 profile 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

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 Ultrafiltration 137

6.5.2 Diafiltration 139

6.6 Chromatographic purification 140

6.6.1 Size-exclusion chromatography (gel filtration) 142

6.6.2 Ion-exchange chromatography 142

6.6.3 Hydrophobic interaction chromatography 146

6.6.4 Affinity chromatography 148

6.6.5 Immunoaffinity purifications 150

6.6.6 Protein A chromatography 150

6.6.7 Lectin affinity chromatography 150

6.6.8 Dye affinity chromatography 152

6.6.9 Metal chelate affinity 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 Purification of recombinant proteins 157

6.9 Final product formulation 159

6.9.1 Some influences 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 disulfide exchange 162

6.9.2 Stabilizing excipients used in final product formulations 164

6.9.3 Final product fill 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

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

7.6.4 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-a 213

8.2.2 Interferon-P 214

8.2.3 Interferon-y 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-2a protein kinase system 221

8.3 Interferon biotechnology 224

8.3.1 Production and medical uses of interferon-a 226

8.3.2 Medical uses of interferon-P 229

8.3.3 Medical applications of interferon-y 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

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-a 256

9.5.3 Immunity and inflammation 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

11.5 The gonadotropins 310

11.5.1 Follicle-stimulating hormone, luteinizing hormone and human chorionic gonadotropin 311

11.5.2 Pregnant mare serum gonadotropin 315

11.5.3 The inhibins and activins 315

11.6 Medical and veterinary applications of gonadotropins 319

11.6.1 Sources and medical uses of follicle-stimulating hormone, luteinizing hormone and human chorionic gonadotropin 319

11.6.2 Recombinant gonadotropins 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 arAntitrypsin 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 a-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

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 Difficulties 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

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

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