生物製藥的生物過程：為了創造機能性生物活性分子在轉換後所扮演的修飾角色

Abstract

The growing demands for existing biologics production are reflected in biopharmaceutical sales figures: The top 15 products in 2004 posted revenues of $38 billion. Although biologics manufacturing now is focused chiefly on a small number of blockbuster products, the future challenges will involve the ability to gear up for the production of a larger variety of products with lower sales volumes. D&MD's Bioprocesses of Biopharmaceuticals: The Obligatory Role of Post Translational Modifications to Create Functional Bioactive Molecules looks at protein drug manufacturing, starting with choice of methods and scale-up processes, highlighting important post translational modifications such as glycosylation.

Table of Contents

Chapter 1: Executive Summary 1�E

Chapter 2: Biopharmaceuticals are Recombinant Products that Replicate Functions or Antibodies that are Inhibitory 2�E

• Reengineering Medicines 2�E
• Humanized Chimeric Antibodies 2�EEngineered Fusion Proteins�with and without Fc 2�E
• PEGylation 2�E
• Glycosylation 2�E0
• Growth Hormones and Enzymes 2�E1
• Human Growth Factors and Enzymes Approved by FDA in 2004 and 2005 2�E2
• Categorization of Growth Factors and Hormones 2�E3
• Therapeutic Antibodies 2�E6
• Economic Considerations of the Commercial Manufacture of Antibodies 2�E7
• Commercialization of Antibody Production 2�E9
• Purification Considerations 2�E1
• Monoclonal Antibody Successes in the Clinic 2�E2
• Antibodies Entering the Clinic 2�E6
• FDA Approval of 18 Therapeutic Antibodies 2�E6
• Therapeutic Approved Antibodies�Outside the US 2�E7
• Cytokines�A Potential $12�E15 Billion 2005 Market 2�E8
• Survival Factors (SCF, CNTF, BDGF) 2�E9
• FDA Approved Cytokines IL-2 and interferon-alfa 2b and GM-CSF 2�E0
• Interleukin-2 2�E1
• Granulocyte-Monocyte Colony Stimulating Factor 2�E2
• Interleukin-12 2�E2
• Vaccines 2�E2
• Pasteur and the Discovery of Active Immunization 2�E2
• Pediatric Vaccines 2�E5
• Disease Eradication 2�E5
• Cancer Vaccines 2�E7
• Vaccines and Bioterrorism 2�E8
• A Comparison of Pharmaceutical Expression Systems 2�E0
• The Emerging Industry of Plant-Made Pharmaceuticals and Biopharming 2�E3
• Farming Biopharma Drugs�Crops as Bioreactors 2�E6
• Plant Cell Cultures Currently in Use for Commercial Recombinant Biopharmaceuticals 2�E9
• Transgenic Animal Factories Cows, Chickens, and Rabbits 2�E2
• Notable Companies Using Transgenics 2�E4
• Process Development 2�E5

Chapter 3: Proteins/Antibodies as Drugs 3�E

• Basic Protein Biochemistry 3�E
• The Amino Acids 3�E
• Chaperone Proteins 3�E
• Protein Structure 3�E
• The Ribosome 3�E1
• Post-Translational Modification 3�E2
• Proteolytic Cleavage 3�E3
• The Coagulation Cascade 3�E4
• Protein Cross Linking 3�E5
• Glycosylation 3�E6
• Biochemistry of Glycosylation 3�E6
• Glycosylated Precursor in the Endoplasmic Reticulum and Golgi Apparatus 3�E8
• Lysosomal Targeting of Enzymes 3�E2
• Clinical Significances of Glycoproteins 3�E2
• Vitamin C-Dependent Modifications 3�E3
• Vitamin K-Dependent Modifications 3�E4
• Acetylation 3�E4
• Phosphorylation 3�E5
• Kinases 3�E6
• Sulfation 3�E6
• Prenylation, Farnesylation, and Geranylgeranylation 3�E8
• Protein Degradation: The Ubiquitin Pathway 3�E0
• Ubiquitin Function 3�E1
• The Ubiquitin-Proteasome Pathway 3�E1
• What are the degradation signals? 3�E1
• How Does Ubiquitination Lead to Protein Degradation? 3�E2
• The Proteasome 3�E3
• The 20S Proteasome Chamber 3�E3
• The Function of the Proteasome 3�E4
• S Proteasome 3�E4
• Proteasomes and the Immune Response 3�E4
• Comparing the Proteasome and Chaperon 3�E5
• Bioreactors for Synthesis of Proteins 3�E5
• Prokaryotic Expression Systems�The Earliest Bioreactor 3�E6
• What is the Future for Bacterial Fermentation? 3�E8
• Single Cell Eukaryotic Based Protein Synthesis 3�E8
• Current Commercial Biopharmaceuticals Made in Yeast and Fungal Expression Systems 3�E0
• Glycosylation of Therapeutic Proteins 3�E1
• Baculovirus and Insect Cell-Based Protein Synthesis 3�E2
• Mammalian Cell-Based Protein Expression 3�E5
• Hybridomas and at the Production of Monoclonal Antibodies 3�E5
• To Suppress the Immune System 3�E5
• To Kill or Inhibit Malignant Cells 3�E5
• To Block Angiogenesis 3�E6
• Protein Expression in Mammalian Cells 3�E6
• Mammalian Transfection Protocols 3�E8
• Electroporation 3�E9
• Lipofection 3�E9
• Microinjection 3�E9
• Viral Expression Systems 3�E9
• Transient and Stable Transfection�Mammalian CHO cells 3�E0
• The Basic Antibody Response 3�E1
• Protein G Based Isolation of Monoclonal Antibody 3�E5
• Uses for Monoclonal Antibodies in Pathology 3�E5
• Transgenic Mice and Phage Display Libraries for Antibody Production 3�E6
• Problems with Monoclonal Therapy 3�E8
• The Science of Transgenes, using Plants and Animals as Drug Factories 3�E1
• How to Make Transgenic Plants 3�E1
• Potential Uses of the Transgenic Plants 3�E2
• Ethical and Political Concerns with Genetically Modified Plants 3�E3
• Transgenic Animals 3�E4
• Nuclear Transfer and Animal Cloning 3�E6
• Nuclear Transfer Technology 3�E6
• Therapeutic Cloning 3�E8

Chapter 4: Quality Control and Post-translational Modifications of Recombinant Drugs 4�E

• Biopharmaceutical Formulation: Potential Post-Translational Alterations 4�E
• Biotherapeutic Proteins versus Small Molecule Drugs 4�E
• Drug Development Factors for Recombinant Biologicals 4�E
• Critical Factors in Protein Analysis 4�E
• Biologic Stability 4�E0
• Process Development of Recombinant Biologicals 4�E2
• Recombinant Therapeutic Systems 4�E4
• Solubilization of Expressed Recombinants 4�E7
• Glycosylation and Microheterogeneity Affecting Recombinant Drugs 4�E8
• Erythropoietin 4�E0
• Follicular Stimulating Hormone 4�E1
• A Plant's N-glycans Contains �E1,3)-fucose and �E1,2)-xylose 4�E3
• Protein Immunogenicity�Neoepitopes 4�E4
• Prenylation and Myristoylation 4�E6
• Vaccine Development 4�E7
• Farensyl Transferase Inhibitors in Cancer 4�E8
• Myristoylated Recombinant Proteins 4�E8
• Phosphorylation of Biopharmaceuticals 4�E9
• Sulfation and Disulfide Bond Formation 4�E1
• Disulfide Bonds and Recombinant Protein Activity/Stability 4�E1
• Thiolation and Sulfation of Therapeutic Proteins 4�E3
• Metabolic Transformations of Biopharmaceuticals 4�E5
• Acetylation and Acylation 4�E6
• Myristyl Acylation 4�E6
• Ubiquitinylation and Proteolytic Processing 4�E7
• Proteolytic Processing of Biopharmaceuticals 4�E9
• Degradomics 4�E0
• Oxidation of Biopharmaceuticals 4�E1
• Deamidation 4�E3
• Glycation of Therapeutic Proteins 4�E6
• Glycomics and Proteomics 4�E7
• Changing Glycosylation in Protein Expression Systems 4�E9
• Glycan-like Formulation Strategies for Protein Pharmaceuticals 4�E0

Chapter 5: Protection of Biopharmaceutical Products 5�E

• Recent Problems with Counterfeited Drugs�National Association of Boards of Pharmacy Potential List of Counterfeit Medicines 5�E
• Anti-Counterfeiting Measures for Biopharmaceutical Brand Protection 5�E
• Financial Loss 5�E
• Brand Damage 5�E
• Organized Crime 5�E
• Terrorism 5�E
• Covert, Overt, and Forensic Solutions 5�E
• Nonprinted Security Features 5�E

Chapter 6: Products of the Leading Biopharmaceutical Companies 6�E

• Amgen Inc. 6�E
• Biogen Idec, Inc. 6�E
• Genentech, Inc. 6�E
• Serono, Inc. 6�E
• Eli Lilly and Company 6�E0
• Roche 6�E2
• Biogeneric�Biopharmaceutical Generics 6�E3
• The Hatch Waxman Act 6�E4
• US FDA Regulations 6�E4
• Invalidation of Amgen Patent for EPO in UK 6�E6
• Conclusions�E005 Sales Patterns 6�E9
• Overview 6�E1

TABLE OF EXHIBITS

• Exhibit 2.1 Biopharmaceuticals Approved and in the Market Through 2003 2�E
• Exhibit 2.2 Recent Chimeric Approved Antibodies To Date 2�E
• Exhibit 2.3 Humanized Antibodies in Clinical Trials in 2005 2�E
• Exhibit 2.4 FDA Approved Growth Hormones and Enzymes in 2004 and 2005 2�E2
• Exhibit 2.5 Summary of Growth Factors in R&D Stages 2�E3
• Exhibit 2.6 Flow Chart of Fundamental Steps in a Culture/bioreactor Product 2�E1
• Exhibit 2.7 US and Europe Approved Therapeutic Antibodies Until 2005 2�E3
• Exhibit 2.8 2004 and 2005 FDA-approved Antibodies 2�E5
• Exhibit 2.9 Number of Therapeutic Monoclonal Antibodies Entering the Clinic from 1984 to 2004 2�E6
• Exhibit 2.10 Bacterial/Viral Infections Targets for Vaccines 2�E7
• Exhibit 2.11 Types of Cancer Vaccines in Development 2�E8
• Exhibit 2.12 Companies Involved in Bioterror Vaccine Production 2�E0
• Exhibit 2.13 Advantages and Disadvantages of Different Expression Systems 2�E2
• Exhibit 2.14 Plant-derived Pharmaceuticals Close to Commercialization 2�E4
• Exhibit 2.15 Biotech Companies Specializing in Plant Made Pharmaceuticals 2�E8
• Exhibit 2.16 Expression Hosts and Yields of Recombinant Proteins in Production 2�E0
• Exhibit 2.17 From a Transgenic Plants to a Commercial Product 2�E2
• Exhibit 2.18 Companies Involved in the Manufacture of Biopharmaceuticals 2�E5
• Exhibit 3.1 An Amino Acid 3�E
• Exhibit 3.2 The Peptide Bond in a Protein 3�E
• Exhibit 3.3 Amino Acids are Stereoisomers 3�E
• Exhibit 3.4 The 20 Naturally Occurring Amino Acids 3�E
• Exhibit 3.5 Hsp60 Chaperone Protein Crystal Structure 3�E
• Exhibit 3.6 Four Levels of Protein Structure 3�E
• Exhibit 3.7 Secondary Structure 3�E
• Exhibit 3.8 The Central Dogma 3�E0
• Exhibit 3.9 Transfer RNA 3�E0
• Exhibit 3.10 The Genetic Code 3�E1
• Exhibit 3.11 Protein Synthesis on the Ribosome 3�E2
• Exhibit 3.12 Common Post-Translational Modifications of Proteins 3�E3
• Exhibit 3.13 Cleavage Sites of Common Proteases 3�E4
• Exhibit 3.14 The Coagulation Cascade 3�E5
• Exhibit 3.15 The Amino Acid Cysteine Creates Disulfide Linkages 3�E6
• Exhibit 3.16 The Ring Structure of Monosaccharides 3�E7
• Exhibit 3.17 Common Monosaccharides and Disaccharides 3�E7
• Exhibit 3.18 The Glycosidic Bond 3�E8
• Exhibit 3.19 O Linked Carbohydrates 3�E0
• Exhibit 3.20 N Linked Carbohydrates 3�E1
• Exhibit 3.21 Dolichol Structure 3�E2
• Exhibit 3.22 Enzyme Defects in Degradation of Asn-GlcNAc Type Glycoproteins 3�E3
• Exhibit 3.23 Structure of a Gla Residue 3�E4
• Exhibit 3.24 The Universal PAPS Reaction 3�E7
• Exhibit 3.25 Protein Prenylation 3�E9
• Exhibit 3.26 The Proteasome 3�E3
• Exhibit 3.27 Bioreactors for Protein Production 3�E6
• Exhibit 3.28 Advantages of an Expression System Using Protozoa 3�E9
• Exhibit 3.29 Yeasts S. Serevisiae and P. Pastoris Therapeutic Protein Production 3�E2
• Exhibit 3.30 Baculovirus Expression System 3�E4
• Exhibit 3.31 Mammalian Transfection and Expression Protocol 3�E7
• Exhibit 3.32 Mammalian Expression Vector 3�E8
• Exhibit 3.33 Basic Techniques for Creating Hybridomas 3�E2
• Exhibit 3.34 Monoclonal Antibodies used in Pathology 3�E5
• Exhibit 3.35 Panning of Phage Libraries 3�E7
• Exhibit 3.36 Basic Antibody Structure 3�E8
• Exhibit 3.37 Cancer Clinical Trails using Monoclonal Antibodies till June, 2005 3�E0
• Exhibit 3.38 Current Genetically Modified Plant Studies 3�E2
• Exhibit 3.39 Microinjection for Creating Transgenic Animals 3�E4
• Exhibit 3.40 Microinjection Process 3�E5
• Exhibit 3.41 Breeding Protocol to Produce Homozygote Transgenic Animals 3�E5
• Exhibit 3.42 Drugs Currently Synthesized in Transgenic Animals 3�E6
• Exhibit 3.43 Cloning of Livestock 3�E7
• Exhibit 3.44 Therapeutic Cloning Protocol 3�E9
• Exhibit 4.1 FDA's Division of Therapeutic Proteins (DTP) Product Diversity 4�E
• Exhibit 4.2 Recombinant Protein Analysis Solutions 4�E
• Exhibit 4.3 Successful Isolation of a Recombinant Antitumor Immunoreagent 4�E0
• Exhibit 4.4 Shelf Life of Recombinant Protein Drugs 4�E1
• Exhibit 4.5 Stability-Indicating Test Methods for Recombinant Proteins 4�E1
• Exhibit 4.6 Solubilization Strategies for Expressed Recombinants 4�E8
• Exhibit 4.7 Microheterogeneity of FSH 4�E2
• Exhibit 4.8 Ubiquitinylation of Therapeutic Protein 4�E8
• Exhibit 4.9 Protein Oxidation Reactions 4�E2
• Exhibit 4.10 Protein Deamidation 4�E3
• Exhibit 5.1 Counterfeited Nutropin Vials 5�E
• Exhibit 5.2 NABP's list of Susceptible Products 5�E
• Exhibit 5.3 Anticounterfeiting Security Measures 5�E
• Exhibit 6.1 Amgen's Lead Biopharmaceuticals 6�E
• Exhibit 6.2 Biogen's Lead Biopharmaceuticals 6�E
• Exhibit 6.3 Genentech's Lead Biopharmaceuticals 6�E
• Exhibit 6.4 Serono's Lead Biopharmaceuticals 6�E
• Exhibit 6.5 Lilly's Lead Biopharmaceuticals 6�E1
• Exhibit 6.6 Top Biopharmaceuticals and Their Patent Positions in 2000 6�E6
• Exhibit 6.7 Disease Targets for Biopharmaceuticals 6�E1