Bioprocesses of Biopharmaceuticals: The Obligatory Role of Post Translational Modifications to Create Functional Bioactive Molecules

SUMMARY

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--1

Chapter 2: Biopharmaceuticals are Recombinant Products that Replicate Functions or Antibodies that are Inhibitory 2--1

• Reengineering Medicines 2--3
• Humanized Chimeric Antibodies 2--4Engineered Fusion Proteins--with and without Fc 2--6
• PEGylation 2--9
• Glycosylation 2--10
• Growth Hormones and Enzymes 2--11
• Human Growth Factors and Enzymes Approved by FDA in 2004 and 2005 2--12
• Categorization of Growth Factors and Hormones 2--13
• Therapeutic Antibodies 2--16
• Economic Considerations of the Commercial Manufacture of Antibodies 2--17
• Commercialization of Antibody Production 2--19
• Purification Considerations 2--21
• Monoclonal Antibody Successes in the Clinic 2--22
• Antibodies Entering the Clinic 2--26
• FDA Approval of 18 Therapeutic Antibodies 2--26
• Therapeutic Approved Antibodies--Outside the US 2--27
• Cytokines--A Potential $12--$15 Billion 2005 Market 2--28
• Survival Factors (SCF, CNTF, BDGF) 2--29
• FDA Approved Cytokines IL-2 and interferon-alfa 2b and GM-CSF 2--30
• Interleukin-2 2--31
• Granulocyte-Monocyte Colony Stimulating Factor 2--32
• Interleukin-12 2--32
• Vaccines 2--32
• Pasteur and the Discovery of Active Immunization 2--32
• Pediatric Vaccines 2--35
• Disease Eradication 2--35
• Cancer Vaccines 2--37
• Vaccines and Bioterrorism 2--38
• A Comparison of Pharmaceutical Expression Systems 2--40
• The Emerging Industry of Plant-Made Pharmaceuticals and Biopharming 2--43
• Farming Biopharma Drugs--Crops as Bioreactors 2--46
• Plant Cell Cultures Currently in Use for Commercial Recombinant Biopharmaceuticals 2--49
• Transgenic Animal Factories Cows, Chickens, and Rabbits 2--52
• Notable Companies Using Transgenics 2--54
• Process Development 2--55

Chapter 3: Proteins/Antibodies as Drugs 3--1

• Basic Protein Biochemistry 3--1
• The Amino Acids 3--1
• Chaperone Proteins 3--5
• Protein Structure 3--8
• The Ribosome 3--11
• Post-Translational Modification 3--12
• Proteolytic Cleavage 3--13
• The Coagulation Cascade 3--14
• Protein Cross Linking 3--15
• Glycosylation 3--16
• Biochemistry of Glycosylation 3--16
• Glycosylated Precursor in the Endoplasmic Reticulum and Golgi Apparatus 3--18
• Lysosomal Targeting of Enzymes 3--22
• Clinical Significances of Glycoproteins 3--22
• Vitamin C-Dependent Modifications 3--23
• Vitamin K-Dependent Modifications 3--24
• Acetylation 3--24
• Phosphorylation 3--25
• Kinases 3--26
• Sulfation 3--26
• Prenylation, Farnesylation, and Geranylgeranylation 3--28
• Protein Degradation: The Ubiquitin Pathway 3--30
• Ubiquitin Function 3--31
• The Ubiquitin-Proteasome Pathway 3--31
• What are the degradation signals? 3--31
• How Does Ubiquitination Lead to Protein Degradation? 3--32
• The Proteasome 3--33
• The 20S Proteasome Chamber 3--33
• The Function of the Proteasome 3--34
• S Proteasome 3--34
• Proteasomes and the Immune Response 3--34
• Comparing the Proteasome and Chaperon 3--35
• Bioreactors for Synthesis of Proteins 3--35
• Prokaryotic Expression Systems--The Earliest Bioreactor 3--36
• What is the Future for Bacterial Fermentation? 3--38
• Single Cell Eukaryotic Based Protein Synthesis 3--38
• Current Commercial Biopharmaceuticals Made in Yeast and Fungal Expression Systems 3--40
• Glycosylation of Therapeutic Proteins 3--41
• Baculovirus and Insect Cell-Based Protein Synthesis 3--42
• Mammalian Cell-Based Protein Expression 3--45
• Hybridomas and at the Production of Monoclonal Antibodies 3--45
• To Suppress the Immune System 3--45
• To Kill or Inhibit Malignant Cells 3--45
• To Block Angiogenesis 3--46
• Protein Expression in Mammalian Cells 3--46
• Mammalian Transfection Protocols 3--48
• Electroporation 3--49
• Lipofection 3--49
• Microinjection 3--49
• Viral Expression Systems 3--49
• Transient and Stable Transfection--Mammalian CHO cells 3--50
• The Basic Antibody Response 3--51
• Protein G Based Isolation of Monoclonal Antibody 3--55
• Uses for Monoclonal Antibodies in Pathology 3--55
• Transgenic Mice and Phage Display Libraries for Antibody Production 3--56
• Problems with Monoclonal Therapy 3--58
• The Science of Transgenes, using Plants and Animals as Drug Factories 3--61
• How to Make Transgenic Plants 3--61
• Potential Uses of the Transgenic Plants 3--62
• Ethical and Political Concerns with Genetically Modified Plants 3--63
• Transgenic Animals 3--64
• Nuclear Transfer and Animal Cloning 3--66
• Nuclear Transfer Technology 3--66
• Therapeutic Cloning 3--68

Chapter 4: Quality Control and Post-translational Modifications of Recombinant Drugs 4--1

• Biopharmaceutical Formulation: Potential Post-Translational Alterations 4--1
• Biotherapeutic Proteins versus Small Molecule Drugs 4--4
• Drug Development Factors for Recombinant Biologicals 4--7
• Critical Factors in Protein Analysis 4--7
• Biologic Stability 4--10
• Process Development of Recombinant Biologicals 4--12
• Recombinant Therapeutic Systems 4--14
• Solubilization of Expressed Recombinants 4--17
• Glycosylation and Microheterogeneity Affecting Recombinant Drugs 4--18
• Erythropoietin 4--20
• Follicular Stimulating Hormone 4--21
• A Plant's N-glycans Contains á(1,3)-fucose and â(1,2)-xylose 4--23
• Protein Immunogenicity--Neoepitopes 4--24
• Prenylation and Myristoylation 4--26
• Vaccine Development 4--27
• Farensyl Transferase Inhibitors in Cancer 4--28
• Myristoylated Recombinant Proteins 4--28
• Phosphorylation of Biopharmaceuticals 4--29
• Sulfation and Disulfide Bond Formation 4--31
• Disulfide Bonds and Recombinant Protein Activity/Stability 4--31
• Thiolation and Sulfation of Therapeutic Proteins 4--33
• Metabolic Transformations of Biopharmaceuticals 4--35
• Acetylation and Acylation 4--36
• Myristyl Acylation 4--36
• Ubiquitinylation and Proteolytic Processing 4--37
• Proteolytic Processing of Biopharmaceuticals 4--39
• Degradomics 4--40
• Oxidation of Biopharmaceuticals 4--41
• Deamidation 4--43
• Glycation of Therapeutic Proteins 4--46
• Glycomics and Proteomics 4--47
• Changing Glycosylation in Protein Expression Systems 4--49
• Glycan-like Formulation Strategies for Protein Pharmaceuticals 4--50

Chapter 5: Protection of Biopharmaceutical Products 5--1

• Recent Problems with Counterfeited Drugs--National Association of Boards of Pharmacy Potential List of Counterfeit Medicines 5--3
• Anti-Counterfeiting Measures for Biopharmaceutical Brand Protection 5--5
• Financial Loss 5--6
• Brand Damage 5--6
• Organized Crime 5--6
• Terrorism 5--6
• Covert, Overt, and Forensic Solutions 5--7
• Nonprinted Security Features 5--8

Chapter 6: Products of the Leading Biopharmaceutical Companies 6--1

• Amgen Inc. 6--1
• Biogen Idec, Inc. 6--3
• Genentech, Inc. 6--5
• Serono, Inc. 6--8
• Eli Lilly and Company 6--10
• Roche 6--12
• Biogeneric--Biopharmaceutical Generics 6--13
• The Hatch Waxman Act 6--14
• US FDA Regulations 6--14
• Invalidation of Amgen Patent for EPO in UK 6--16
• Conclusions--2005 Sales Patterns 6--19
• Overview 6--21

TABLE OF EXHIBITS

• Exhibit 2.1 Biopharmaceuticals Approved and in the Market Through 2003 2--3
• Exhibit 2.2 Recent Chimeric Approved Antibodies To Date 2--5
• Exhibit 2.3 Humanized Antibodies in Clinical Trials in 2005 2--7
• Exhibit 2.4 FDA Approved Growth Hormones and Enzymes in 2004 and 2005 2--12
• Exhibit 2.5 Summary of Growth Factors in R&D Stages 2--13
• Exhibit 2.6 Flow Chart of Fundamental Steps in a Culture/bioreactor Product 2--21
• Exhibit 2.7 US and Europe Approved Therapeutic Antibodies Until 2005 2--23
• Exhibit 2.8 2004 and 2005 FDA-approved Antibodies 2--25
• Exhibit 2.9 Number of Therapeutic Monoclonal Antibodies Entering the Clinic from 1984 to 2004 2--26
• Exhibit 2.10 Bacterial/Viral Infections Targets for Vaccines 2--37
• Exhibit 2.11 Types of Cancer Vaccines in Development 2--38
• Exhibit 2.12 Companies Involved in Bioterror Vaccine Production 2--40
• Exhibit 2.13 Advantages and Disadvantages of Different Expression Systems 2--42
• Exhibit 2.14 Plant-derived Pharmaceuticals Close to Commercialization 2--44
• Exhibit 2.15 Biotech Companies Specializing in Plant Made Pharmaceuticals 2--48
• Exhibit 2.16 Expression Hosts and Yields of Recombinant Proteins in Production 2--50
• Exhibit 2.17 From a Transgenic Plants to a Commercial Product 2--52
• Exhibit 2.18 Companies Involved in the Manufacture of Biopharmaceuticals 2--55
• Exhibit 3.1 An Amino Acid 3--2
• Exhibit 3.2 The Peptide Bond in a Protein 3--2
• Exhibit 3.3 Amino Acids are Stereoisomers 3--3
• Exhibit 3.4 The 20 Naturally Occurring Amino Acids 3--4
• Exhibit 3.5 Hsp60 Chaperone Protein Crystal Structure 3--7
• Exhibit 3.6 Four Levels of Protein Structure 3--8
• Exhibit 3.7 Secondary Structure 3--9
• Exhibit 3.8 The Central Dogma 3--10
• Exhibit 3.9 Transfer RNA 3--10
• Exhibit 3.10 The Genetic Code 3--11
• Exhibit 3.11 Protein Synthesis on the Ribosome 3--12
• Exhibit 3.12 Common Post-Translational Modifications of Proteins 3--13
• Exhibit 3.13 Cleavage Sites of Common Proteases 3--14
• Exhibit 3.14 The Coagulation Cascade 3--15
• Exhibit 3.15 The Amino Acid Cysteine Creates Disulfide Linkages 3--16
• Exhibit 3.16 The Ring Structure of Monosaccharides 3--17
• Exhibit 3.17 Common Monosaccharides and Disaccharides 3--17
• Exhibit 3.18 The Glycosidic Bond 3--18
• Exhibit 3.19 O Linked Carbohydrates 3--20
• Exhibit 3.20 N Linked Carbohydrates 3--21
• Exhibit 3.21 Dolichol Structure 3--22
• Exhibit 3.22 Enzyme Defects in Degradation of Asn-GlcNAc Type Glycoproteins 3--23
• Exhibit 3.23 Structure of a Gla Residue 3--24
• Exhibit 3.24 The Universal PAPS Reaction 3--27
• Exhibit 3.25 Protein Prenylation 3--29
• Exhibit 3.26 The Proteasome 3--33
• Exhibit 3.27 Bioreactors for Protein Production 3--36
• Exhibit 3.28 Advantages of an Expression System Using Protozoa 3--39
• Exhibit 3.29 Yeasts S. Serevisiae and P. Pastoris Therapeutic Protein Production 3--42
• Exhibit 3.30 Baculovirus Expression System 3--44
• Exhibit 3.31 Mammalian Transfection and Expression Protocol 3--47
• Exhibit 3.32 Mammalian Expression Vector 3--48
• Exhibit 3.33 Basic Techniques for Creating Hybridomas 3--52
• Exhibit 3.34 Monoclonal Antibodies used in Pathology 3--55
• Exhibit 3.35 Panning of Phage Libraries 3--57
• Exhibit 3.36 Basic Antibody Structure 3--58
• Exhibit 3.37 Cancer Clinical Trails using Monoclonal Antibodies till June, 2005 3--60
• Exhibit 3.38 Current Genetically Modified Plant Studies 3--62
• Exhibit 3.39 Microinjection for Creating Transgenic Animals 3--64
• Exhibit 3.40 Microinjection Process 3--65
• Exhibit 3.41 Breeding Protocol to Produce Homozygote Transgenic Animals 3--65
• Exhibit 3.42 Drugs Currently Synthesized in Transgenic Animals 3--66
• Exhibit 3.43 Cloning of Livestock 3--67
• Exhibit 3.44 Therapeutic Cloning Protocol 3--69
• Exhibit 4.1 FDA's Division of Therapeutic Proteins (DTP) Product Diversity 4--5
• Exhibit 4.2 Recombinant Protein Analysis Solutions 4--8
• Exhibit 4.3 Successful Isolation of a Recombinant Antitumor Immunoreagent 4--10
• Exhibit 4.4 Shelf Life of Recombinant Protein Drugs 4--11
• Exhibit 4.5 Stability-Indicating Test Methods for Recombinant Proteins 4--11
• Exhibit 4.6 Solubilization Strategies for Expressed Recombinants 4--18
• Exhibit 4.7 Microheterogeneity of FSH 4--22
• Exhibit 4.8 Ubiquitinylation of Therapeutic Protein 4--38
• Exhibit 4.9 Protein Oxidation Reactions 4--42
• Exhibit 4.10 Protein Deamidation 4--43
• Exhibit 5.1 Counterfeited Nutropin Vials 5--2
• Exhibit 5.2 NABP's list of Susceptible Products 5--4
• Exhibit 5.3 Anticounterfeiting Security Measures 5--8
• Exhibit 6.1 Amgen's Lead Biopharmaceuticals 6--2
• Exhibit 6.2 Biogen's Lead Biopharmaceuticals 6--4
• Exhibit 6.3 Genentech's Lead Biopharmaceuticals 6--6
• Exhibit 6.4 Serono's Lead Biopharmaceuticals 6--9
• Exhibit 6.5 Lilly's Lead Biopharmaceuticals 6--11
• Exhibit 6.6 Top Biopharmaceuticals and Their Patent Positions in 2000 6--16
• Exhibit 6.7 Disease Targets for Biopharmaceuticals 6--21