2Pharmaceutical BiotechnologyAn Introduction for Pharmacists andPharmaceutical Scientists

Edited by

DaanJ.A. Crommelin (Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, The Netherlands)

and

Robert D. Sindelar (Department of Medicinal Chemistry, School of Pharmacy, University of Mississippi, USA)

harwood academic publishersAustralia • Canada • China • France • Germany • India • Japan • Luxembourg • MalaysiaThe Netherlands • Russia • Singapore • Switzerland • Thailand • United Kingdom

Table of Contents

Preface XIX

Contributors xxi

Acknowledgements XXIII

Abbreviations XXV

Chapter i: Molecular Biotechnology 1Wiel P.M. Hoekstra and SjefC.M. Smeekens

Introduction 1The Cell 1

The Prokaryotic Cell 1The Eukaryotic Cell 3

Gene Expression 3DNA Replication 5Transcription 5Translation 8

Recombinant DNA Technology 8DNA TransferDNA SourcesSynthetic DNAcDNA 13DNA Libraries

11

y

HProduction by Recombinant DNA Technology

Specific DNA Techniques 16DNA Sequencing 16DNA Hybridization 16PCR Technology lj

Cell Cultures 18Cultivation of Microbes 18Animal Cell Cultures 20Plant Cell Cultures 20

Concluding Remarks 21Further Reading 22Self-Assessment Questions 2}

CONTENTS

Chapter 2: Biophysical and Biochemical Analyses ofRecombinant Proteins

Structure and Analyses of Proteins 27William C. Kenney and Tsutomu Arakawa

Introduction 27Protein Structure 27

Primary Structure 27Secondary Structure 32

a-Helix 32fi-Sheet Structure 34Loops and Turns 35

Tertiary Structure 35Forces 37

Hydrophohic Interactions 37Hydrogen Bonds 37Electrostatic Interactions 37Van der Waals Interactions 37

Hydration 37Protein Folding 38

Techniques for Characterizing Folding 39Protein Stability 40Analytical Techniques 41

Blotting Techniques 41Transfer of Proteins 41Detection Systems 41

Immunoassays 43ELISA 43

Electrophoresis 44Polyacrylamide Gel Electrophoresis 45Isoelectric Focusing 452-Dimensional Gel Electrophoresis 45Detection of Proteins within Polyacrylamide Gels 45Capillary Electrophoresis 45

Chromatography 46Size Exclusion Chromatography 46Reversed-Phase High Performance Liquid

Chromatography 47Hydrophohic Interaction Chromatography 47Ion-Exchange Chromatography 48Other Chromatographic Techniques 48

Bioassays 48Mass Spectrometry 4$

Concluding Remarks 49Further Reading 50Self-Assessment Questions p

CONTENTS

Chapter 3: Production of Biotech CompoundsCultivation and Downstream Processing 53Farida Kadir

Introduction 53Cultivation 53

Expression Systems 53Cultivation Systems 53Medium 56Contaminants 36

Viruses 57Bacteria 58Cellular DNA 58Protein Contaminants 58

Downstream Processing $gIntroduction $gFiltration/Centrifugation 60

Filtration 60Centrifugation 61

Precipitation 61Chromatography 61

Introduction 61Chromatographic Stationary Phases 61Adsorption Chromatography 63Ion-Exchange Chromatography 63(Immuno)Affinity Chromatography 63Hydrophohic Interaction Chromatography 64Gel Permeation Chromatography 64Expanded Beds 65

Issues to Consider in Production and Purificationof Proteins 65N- and C-Terminal Heterogeneity 65Chemical Modification/Conformational Changes 66Glycosylation 66Proteolytic Processing 66Protein Inclusion Body Formation 66

References 67Self-Assessment Questions 70

Chapter 4: Formulation of Biotech Products, IncludingBiopharmaceutical Considerations 71Daan J.A. Crommelin

Introduction 71Microbiological Considerations 71

Sterility 71Viral Decontamination 71Pyrogen Removal 71

Excipients used in Parenteral Formulations ofBiotech Products 72Solubility Enhancers 72Anti-adsorption and Anti-aggregation Agents 72

CONTENTS

Buffer Components 73Preservatives and Anti-oxidants 73Osmotic Agents 74

Shelf Life of Protein Based Pharmaceuticals 74Freeze-Drying of Proteins 75Freezing 75Primary Drying 76Secondary Drying Stage 77Other Approaches to Stabilize Proteins 78

Delivery of Proteins: Routes of Administration andAbsorption Enhancement 78The Parenteral Route of Administration 78The Oral Route 79Alternative Routes of Administration 80Examples of Absorption Enhancing Effects 80

Delivery of Proteins: Approaches for Rate Controlled and

Target Site Specific Delivery by the Parenteral Route 82Approaches for Rate Controlled Delivery 84

Open Loop Systems: Mechanical Pumps 84Open Loop Systems: Osmotically Driven Systems 85Closed Loop Systems: Biosensor-Pump Combinations 86Protein Delivery by Self-Regulating Systems 86Protein Delivery by Microencapsulated Secretory Cells 87

Site Specific Delivery (Targeting) of Protein Drugs 88Anatomical, Physiological and Pathological Considerations

Relevant for Protein Targeting 89Soluble Carrier Systems for Targeted Delivery

of Proteins go(Monoclonal) Antibodies (MAh) as Targeted Therapeutic

Agents: Human and Humanized Antibodies goBispecijic Antibodies goImmunoconjugates: Combinations Between an Antibody

and an Active Compound goPotential Pitfalls in Tumor Targeting 91Colloidal Particulate Carrier Systems for Targeted Delivery

of Proteins g2Perspectives for Targeted Protein Delivery g$

References 95Self-Assessment Questions g8

Chapter 5: Pharmacokinetics and Pharmacodynamicsof Peptide and Protein Drugs 101

Rene Braeckman

Introduction 101Elimination of Protein Therapeutics 102

Proteolysis 102Renal Excretion and Metabolism 102Hepatic Metabolism 104Receptor-Mediated Elimination by Other Cells 104

Distribution of Protein Therapeutics 205

CONTENTS

Pharmacodynamics of Protein Therapeutics 106Direct Effects 107Indirect Effects 108PK/PD Link Models 108Indirect Effect Models 109Complex PK/PD Models 111Dose-Response and Concentration-Response Curves 111

Protein Binding of Protein Therapeutics 112Interspecies Scaling 124Heterogeneity of Protein Therapeutics 115Chemical Modifications of Protein Therapeutics 116Immunogenicity 117References 118Self-Assessment Questions 121

Chapter 6: Additional Biotechnology-Related Techniques 123Robert D. Sindelar

Introduction 123Polymerase Chain Reaction 123

Basic PCR Methodology 124Some Examples of Modified PCR and Related

Methodologies 124Genetically Engineered Animals 125

Transgenic Animals 125Production of Transgenic Animals by DNA

Microinjection and Random Gene Addition 126Production of Transgenic Animals by Homologous

Recombination in Embryonic Stem Cells I2gProtein Production in Transgenic Animals I2gExamples of Transgenic Animal Models of Human

Disease in Drug Discovery and Development 131Knockout Mice 131Transgenic Animal Patents 131Genetic Ablation 132

Protein Engineering 132Production of Engineered Proteins 132Site-Directed Mutagenesis 132Enzyme Engineering 133Fusion Proteins 133Antibody Engineering 1343-D Structures of Engineered Proteins: Protein X-Ray

Crystallography, Nuclear Magnetic Resonance Spectroscopyand Protein Modeling 134Protein X-ray Crystallography 135Nuclear Magnetic Resonance (NMR) Spectroscopy 133Protein Modeling 136

Peptide Chemistry and Peptidomimetics 136Peptidomimetics 137"Pseudopeptide" Peptidomimetic Approach 138Conformationally-Constrained Peptides 138Rational Design of Peptidomimetics 139

CONTENTS

Nucleic Acid Technologies 140Oligonucleotides 142

Biochemistry 142Physiochemical Properties of Oligonucleotides 143Chemistry and Modifications 143

Antisense Technology and Triplex Technology 143Normal Cell Activity: DNA Makes RNA Makes Protein 143Rationale for Antisense Technology 144Therapeutic Antisense Molecules 143Triplex Technology 146

Aptamer Technology 146Ribozymes 146

Catalytic Antibodies (Abzymes) 147Antibodies 147Catalysis 147Chemistry of Catalytic Antibodies 147Limitations 148Potential Pharmaceutical Uses i4g

Glycobiology i4gBasic Principles of Glycobiology I4gGlycosylation and Biological Activity 150The Role of Glycosylation in Disease 151Cell Adhesion Molecules 151

Integrins 153Selectins 152

Biosensors 152The Impact of Biotechnology on Drug Discovery 154

Overview 154In Vitro Screening 154

Contributions of Biotechnology 154High-Throughput Screening (HTS) 154Combinatorial Chemistry 154

Rational Drug Design 156Concluding Remarks 156Further Reading 157References 157Self-Assessment Questions 165

Chapter 7: Gene Therapy 167Abraham Bout

Introduction 167Ex Vivo versus In Vivo Gene Therapy 167

Ex Vivo Gene Therapy 167In Vivo Gene Therapy 167

Potential Target Diseases for Gene Therapy 167Inherited Disorders 167Cancer 168

Gene Transfer Methods 171Non-Viral Gene Transfer 171

Methods of Non-Viral Gene Transfer 171Application of Non-Viral Gene Transfer 171

CONTENTS

Gene Transfer Using Recombinant Viruses (Viral Vectors) 173General Requirements 173Retrovirus Vectors 173

Retrovirus Life Cycle 173Recombinant Retrovirus 174Application of Retroviral Vectors 175

Adenovirus Vectors 176Adenovirus Life Cycle 176Recombinant Adenoviruses 177Application of Adenoviral Vectors 178

Adeno-Associated Virus Vectors i7gReplication of Wild-Type AAV I7gRecombinant Adeno-Associated Virus 180Application of Adeno-Associated Viral Vectors 180

Clinical Studies 180Pharmaceutical Production and Regulation 181Concluding Remarks 181Further Reading 181References 182Self-Assessment Questions 183

Chapter 8: Hematopoietic Growth Factors 185Jeanne Flynn and Allen W. Rosman

Introduction 185Definitions 185

Chemical Description 186Chemical Properties of G-CSF and GM-CSF 186Chemical Properties of Erythropoietin (EPO) 187Chemical Properties of Other Hematopoietic Growth Factors 188

Pharmacology 188In Vitro Activity 188In Vivo Activity 191

Cellular Sources and Stimuli for Release ig2Physiologic Role of G-CSF and GM-CSF ig2Physiologic Role of EPO ig2Physiologic Role of Other Hematopoietic Growth Factors ig3

Pharmaceutical Concerns 193Status and Source of Hematopoietic Growth Factors ig3Storage and Stability 193Pharmacokinetics 193Pharmacodynamics 196

Clinical and Practice Aspects ig8Established Uses 198

Chemotherapy-Induced Neutropenia 200Bone Marrow Transplantation 201Peripheral Blood Progenitor Cell Transplantation 202Severe Chronic Neutropenia 203AIDS 203Anemia 203

Future Uses 205Disease-Related Cytopenia 205Treatment-Related Cytopenia, Transplantation and Transfusion 203Infectious Diseases 205

CONTENTS

Investigational Hematopoietic Growth Factors 206Toxicities 207

Recombinant G-CSF and GM-CSF 207Epoetin-alfa 208Investigational Hematopoietic Growth Factors 2og

Concluding Remarks 2ogReferences 2ogSelf-Assessment Questions 213

Chapter 9: Interleukins and Interferons 215Joseph Tami

Cytoldnes 215The Interleukins 215

Terminology 215Overview of the Interleukins 216

Interleukin-i 216Interleukin-2 217Interleukin-3 217Interleukin-4 217Interleukin-5 217Interleukin-6 217Interleukin-y 217Interleukin-8 217

Interleukin-g through Interleukin-16 218Interleukin-iy 218

Commercially Available Interleukins 218Introduction: Interleukin-2 218Chemical Description of IL-2 218Pharmacology of IL-2 218

Indication 218Mechanism of Action 218Biotransformation 218Elimination 218

Pharmaceutical Concerns of IL-2 218Clinical and Practice Aspects of IL-2 219

Interferons 219a Interferon 2ig

Chemical Description of a Interferon Products 220Pharmacology 220

Absorption 220Time to Peak Concentration 220Biotransformation 220Onset of Action 220Time to Peak Effect 220

Pharmaceutical Concerns of a Interferons 220Clinical and Practice Aspects of a Interferons 221Side Effects of a Interferons 221

(3 Interferon 222

Chemical Description of P Interferon Product 222Pharmacology of p Interferon 222Pharmaceutical Concerns of P Interferon 222Clinical and Practice Aspects of p Interferon 222

CONTENTS

y Interferon 223Chemical Description of 7 Interferon Product 223Pharmacology of 7 Interferon 224

Absorption 224Time to Peak Plasma Concentration 224Peak Plasma Concentration 224

Pharmaceutical Concerns of 7 Interferon 224Clinical and Practice Aspects of 7 Interferon 224

References 224Self-Assessment Questions 226

Chapter 10: Insulin 22gJohn M. Beals and Paul M. Kovach

Introduction 22gChemical Description 22gPharmacology and Formulations 232

Regular and Rapid-Acting Soluble Preparations 232Intermediate-Acting Insulin Preparations 233Long-Acting Insulin Formulations 234

Pharmaceutical Concerns 234Chemical Stability of Insulin Formulations 234Physical Stability of Insulin Formulations 235

Clinical and Practice Aspects 235Vial Presentations 235Injectors 236Storage 236Usage 236

Resuspension 236Dosing 236Extemporaneous Mixing 236

Acknowledgements 236Further Reading 237References 237Self-Assessment Questions 23g

Chapter 11: Growth Hormones 241Melinda Marian

Introduction 241hGH Structure and Isohormones 241Pharmacology 241

Growth Hormone Secretion and Regulation 241Growth Hormone Biologic Actions 242hGH Receptor and Binding Proteins 242Molecular Endocrinology and Signal Transduction 242Dosing Schedules and Routes 242Pharmacokinetics and Metabolism 243

Protein Manufacture, Formulation and Stability 245Clinical Usage 247

Growth Hormone Deficiency/Idiopathic Short Staturein Children 247

Turner Syndrome 247

CONTENTS

Chronic Renal Insufficiency (CRI) 248Growth Hormone Deficient Adults and the Elderly 249Clinical Malnutrition and Wasting Syndromes 24gOther IndicationsSafety Concerns

Concluding RemarksFurther Reading 250References 250Self-Assessment Questions 233

Chapter 12: Vaccines 255Wim Jiskoot, Gideon F.A. Kersten and E. Coen Beuvery

Introduction 255 'Immunological Principles 255

Introduction 255Humoral and Cell-Mediated Immunity 257Vaccine Design in Relation with the Immune ResponseRoute of Administration 260

Conventional Vaccines 260Classification 260Live Attenuated Vaccines 260Non-Living Vaccines: Whole Organisms 262Non-Living Vaccines: Subunit Vaccines 262

Diphtheria and Tetanus Toxoids 262Acellular Pertussis Vaccines 262Polysaccharide Vaccines 262

Modern Vaccine Technologies 263Genetically Improved Live Vaccines 263

Genetically Attenuated Microorganisms 263Live Vectors 264

Genetically Improved Subunit Vaccines 264Genetically Detoxified Proteins 264Proteins Expressed in Host Cells 264Recombinant Peptide Vaccines 266

Anti-Idiotype Antibody Vaccines 266Synthetic Peptide-Based Vaccines 267Nucleic Acid Vaccines 268

Pharmaceutical Aspects 270Production 270Formulation 271

Additives 271Adjuvants and Delivery Systems 271Combination Vaccines 272

Characterization 272Storage 272

Regulatory and Clinical Aspects 272Further Reading 273References 274Self-Assessment Questions 276

CONTENTS

Chapter 13: Monoclonal Antibody-Based PharmaceuticalsJohn R. Adair, Robert A. Zivin, Norberto A. Guzman and Khurshid Iqbal

Introduction 279Antibody Structure 280Development of Antibody-Based Therapeutics 282

Antibody Binding Sites 282Antibody Humanization 282The Effector Element 283The Fc as Effector 283Non-Ig Effectors 284

Assembly and Production 284(Pre)Formulation of Monoclonal Antibody-Based Pharmaceuticals 285Appendices 285References 285Self-Assessment Questions 287

Chapter 13A: Monoclonal Antibody-Based PharmaceuticalsOKT3 Clinical Usage 288David S. Ziska

Indications 288Clinical Experience 288

Renal Allograft Acute Rejection Prophylaxis 288Renal Allograft Acute Rejection Treatment 288Other Applications 28g

Safety 28gConcluding Remarks 28gReferences 28g

Chapter 13B: The Pharmacodynamic Profile of Abciximab(ReoPro™) 291

Sven Warnaar and Robert Jordan

Introduction 2giMolecular Structure 291Mechanism of Action 291Clinical Pharmacology Studies 2g2Key Clinical Efficacy Studies 2g2Pharmacokinetics and Pharmacodynamics 2g3

Pharmacodynamics of Platelet-Bound Abciximab 2g3Pharmacokinetics of Free Plasma Abciximab

Formulation and Dosage Information 2g4References 2g$

Chapter 14: Recombinant Tissue-Type PlasminogenActivator and Factor VIII

Nishit B. Modi

Introduction 2g7Tissue-Type Plasminogen Activator

IntroductionStructure

CONTENTS

Recombinant t-PA (rt-PA) 299Pharmacology 2gg

Disposition of rt-PA 2ggPharmaceutical Considerations 300

Clinical Application 300Contraindications 301

Second Generation Thrombolytic Agents 301Factor VIII 302

Factor VIII Structure 302Recombinant Factor VIII 302Pharmacology 302

Disposition of Recombinant Factor VIII 302Pharmaceutical Considerations 302Clinical Application 302

Contraindications 303Concluding Remarks 303References 304Self-Assessment Questions 306

Chapter 15: Recombinant Human Deoxyribonuclease 307Melinda Marian and Sharon Baughman

Introduction 307Protein Chemistry and Structure 307Pharmacology 307

In Vitro Actions on Sputum 308In Vivo Actions of rhDNase on Sputum 3ogPharmacokinetics and Metabolism 310

Protein Manufacture and Formulation 310Clinical Usage 310

Indication and Clinical Dosage 310Clinical Experience 313

Cystic Fibrosis 311Other Studies 311

Safety Concerns 311Concluding Remarks 312Acknowledgements 312Further Reading 312References 312Self-Assessment Questions 314

Chapter 16: Follicle-Stimulating Hormone (FSH) 315Tom Sam and Willem de Boer

Introduction 315Biological Role 315Chemical Description 315Production of Recombinant FSH 316Isohormones 316

Structural Characteristics 316Biological Properties of Recombinant FSH Isohormones 317Pharmacokinetic Behavior of Recombinant FSH Isohormones 317

CONTENTS

Pharmaceutical Formulations 317Clinical Aspects 318

Differences with Urinary FSH Preparations 3igFurther Reading 3igReferences jigSelf-Assessment Questions 320

Chapter 17: Dispensing Biotechnology ProductsHandling, Professional Education and Product Information 321Gary H. Smith and Peggy Piascik

Introduction 321Pharmacist Reluctance 321Types of Information Needed by Pharmacists 321Sources of Information for Pharmacists 322The Pharmacist and Handling of Biotech Drugs 322

Storage 323Temperature Requirements 323Storage in Dosing and Administration Devices 324Storage in IV Solutions 325Light Protection 326

Handling 326Mixing and Shaking 326Travel Requirements 327

Preparation 327Administration 327

Routes of Administration 328Filtration 328Flushing Solutions 328

Outpatient/Home Care Issues 328Patient Assessment and Education 328Monitoring 328

Reimbursement 32gEducational Materials

Professional ServicesEducational Materials for Health Professionals 32gEducational Materials for Patients 331

Toll-Free Access to Manufacturers' Services 331The Internet and Biotech Information 332

Concluding Remarks 332Further Reading 332References 332Self-Assessment Questions 334

Chapter 18: Biotechnology Products in the Pipeline 337Ronald P. Evens and Robert D. Sindelar

Introduction 337Drug Development and Biotechnology 337

Time and Cost of Modern Drug Discovery 337Techniques of Biotech Drug Discovery 338Classes of Molecules Being Discovered and Studied

through Biotechnology 33g

CONTENTS

Some Protein Pharmaceuticals in Development 341Colony-Stimulating Factors, Interferons and Interleukins 341

Colony-Stimulating Factors 341Interferons 342Interleukins 342Liposomal IL-2 342Fusion Molecules 343

Enzymes 343Clotting Factors 344Superoxide Dismutase 344

Hormones, Erythropoietins and Growth Factors 344Thrombopoietin 344Neurotrophic Factors 345Other Growth Factors 347

Vaccines 347Recombinant Soluble Receptors 348Monoclonal Antibodies 34g

Therapeutic Monoclonal Antibodies 350Diagnostic Monoclonal Antibodies 350

Some Nucleic Acid Therapies in Development 350Gene Therapy 350Antisense Oligonucleotides 351

Development of Adhesion Molecules, Glycoproteins, Carbohydrate-Based Pharmaceuticals and Other Products of Glycobiology 351

Glycobiology 351Fibronectin and Fibronectin Receptor Antagonists 351

Concluding Remarks 351Further Reading 352References 352Self-Assessment Questions 354

Index 357