Protein Therapeutics E-Book
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- Herausgeber: Wiley-VCH Verlag GmbH & Co. KGaA
- Kategorie: Wissenschaft und neue Technologien
- Serie: Methods and Principles in Medicinal Chemistry
- Sprache: Englisch
In this practice-oriented two volume handbook, professionals from some of the largest biopharmaceutical companies and top academic researchers address the key concepts and challenges in the development of protein pharmaceuticals for medicinal chemists and drug developers of all trades.
Following an introduction tracing the rapid development of the protein therapeutics market over the last decade, all currently used therapeutic protein scaffolds are surveyed, from human and non-human antibodies to antibody mimetics, bispecific antibodies and antibody-drug conjugates. This ready reference then goes on to review other key aspects such as pharmacokinetics, safety and immunogenicity, manufacture, formulation and delivery. The handbook then takes a look at current key clinical applications for protein therapeutics, from respiratory and inflammation to oncology and immune-oncology, infectious diseases and rescue therapy. Finally, several exciting prospects for the future of protein therapeutics are highlighted and discussed.
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Veröffentlichungsjahr: 2017
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Table of Contents
Cover
Title Page
Copyright
Preface
References
A Personal Foreword
Acknowledgments
Part I: Introduction to Protein Therapeutics: Past and Present
Chapter 1: Early Recombinant Protein Therapeutics
1.1 Introduction
1.2 The Birth of Genetic Engineering
1.3 Recombinant Human Insulin
1.4 Recombinant Human Growth Hormone
1.5 Recombinant Human Interferons
1.6 Recombinant Human Erythropoietin
1.7 Recombinant Tissue – Type Plasminogen Activator
1.8 Recombinant Hepatitis B Virus (HBV) Vaccine
1.9 Postscript
Acknowledgments
References
Chapter 2: Evolution of Antibody Therapeutics
2.1 Overview of Antibody Therapeutics Development
2.2 Polyclonal Antibodies – Laying the Foundation for mAb Therapy
2.3 Evolution of Monoclonal Antibody Therapeutics
2.4 Fc Fusion Proteins
2.5 Evolution of Concepts in Antibody Pharmacology
2.6 Future Directions for Antibody Therapeutics Development
Acknowledgments
References
Part II: Antibodies: The Ultimate Scaffold for Protein Therapeutics
Chapter 3: Human Antibody Structure and Function
3.1 Introduction
3.2 General Sequence and Structural Features of Antibodies
3.3 Antibody Diversity
3.4 Canonical Structures of CDR Loops
3.5 Crystal Structures of Antibody–Antigen Interactions
3.6 Glycosylation
3.7 Role of the Fc and Fc Receptors
3.8 Conclusions and Outlook
Acknowledgments
References
Chapter 4: Antibodies from Other Species
4.1 Introduction
4.2 Mammals
4.3 Reptiles
4.4 Amphibians
4.5 Fish
4.6 Conclusions
References
Part III: Discovery and Engineering of Protein Therapeutics
Chapter 5: Human Antibody Discovery Platforms
5.1 Introduction to Therapeutic Human Antibody Platforms
5.2 Properties of Human Antibody Genes
5.3 New Technologies Driving Changes and Improvements in Human Antibody Discovery
5.4 Antigen-Specific Human mAbs from Human B Cells
5.5 Human Antibody Libraries
5.6 Human Antibodies from Transgenic Animals
5.7 Summary and Future Directions
References
Chapter 6: Beyond Antibodies: Engineered Protein Scaffolds for Therapeutic Development
6.1 Introduction
6.2 Motivation for Developing Antibody Alternatives
6.3 Non-antibody Scaffolds with Homogenous Secondary Structure
6.4 Non-antibody Scaffolds with Mixed Secondary Structure
6.5 Conclusions and Considerations
Acknowledgments
References
Further Reading
Chapter 7: Protein Engineering: Methods and Applications
7.1 Introduction
7.2 General Approaches for Protein Optimization
7.3 Engineering for Affinity and Specificity
7.4 Optimizing IgG Serum Half-Life
7.5 Engineering IgG Effector Function
7.6 Conclusion
Acknowledgments
References
Chapter 8: Bispecifics
8.1 Introduction: Continuing Evolution of Antibody-based Therapeutics
8.2 Enhancing Antibody Therapeutics by Addition of Functional Moieties
8.3 Format Selection – Pairing Function and Target Biology
8.4 Engineering Considerations
8.5 Conclusion
Disclosure of Potential Conflict of Interest
References
Chapter 9: Antibody–drug Conjugates (ADCs)
9.1 Introduction
9.2 General Mode of Action
9.3 The Components of an Antibody–Drug Conjugate
9.4 Assembling the ADC
9.5 Approved ADCs: Adcetris and Kadcyla
9.6 Developing the ADC Platform
9.7 New Warheads and Payloads with a Novel Mechanism of Action
9.8 Conclusion
References
Chapter 10: Pharmacokinetics of Therapeutic Proteins
10.1 Absorption
10.2 Distribution
10.3 Metabolism and Elimination
10.4 Pharmacokinetic Modeling of Therapeutic Proteins
References
Part IV: Physiological and Manufacturing Considerations for Biologics
Chapter 11: Safety Considerations for Biologics
11.1 Introduction
11.2 Small Molecules versus Large Molecules – A Comparison
11.3 Toxicity Related to Exaggerated Pharmacology – Importance of Species Selection
11.4 Toxicity Unrelated to Exaggerated Pharmacology
11.5 Regulatory Guidance
11.6 Development Considerations Due to Biological Characteristics
11.7 First in Human (FIH) to Registration
11.8 Selection of a Safe Starting Dose for First Time in Human Clinical Study
11.9 Summary
References
Chapter 12: Immunogenicity of Biologics
12.1 Introduction
12.2 Mechanistic View of Immunogenicity: Innate and Adaptive Immunity
12.3 Immunogenicity of Protein Therapeutics in Autologous Cell Therapies
12.4 Regulatory Context
12.5 Application of the “Risk-Based Approach” for Undesirable Immunogenicity
12.6 Clinical Proof of Concept and Beyond
12.7 Future Perspectives
References
Chapter 13: Expression Systems for Recombinant Biopharmaceutical Production by Mammalian Cells in Culture
13.1 Introduction
13.2 Host Cell Systems
13.3 Mammalian Cell Transfection
13.4 Controlling Recombinant Gene Expression
13.5 Selection and Amplification Systems
13.6 Transient Production Systems
13.7 Protein Purification
13.8 CHO Cell Engineering for Enhanced Bioprocessing Properties
Abbreviations
References
Chapter 14: Stability, Formulation, and Delivery of Biopharmaceuticals
14.1 Introduction
14.2 Stability
14.3 Drug Product Development
14.4 Handling and Administration Considerations
14.5 Summary and Conclusion
References
Chapter 15: Protein Therapeutics in Autoimmune and Inflammatory Diseases
15.1 Introduction
15.2 Rheumatoid Arthritis
15.3 Psoriasis
15.4 TNF-α Antagonist Therapy
15.5 Anti-IL-12/IL-23 Therapies
15.6 Anti-IL-17 Therapies
15.7 Atopic Dermatitis
15.8 Inflammatory Bowel Disease (IBD)
15.9 Systemic Lupus Erythematosus
15.10 Conclusions
References
Part V: Clinical Applications
Chapter 16: Antibody-Based Therapeutics in Oncology
16.1 Introduction
16.2 Targeting Cell-Surface Signaling Pathways in Solid Tumors
16.3 Targeting of Immune Modulators
16.4 Bispecific Antibodies
16.5 Conclusions and Future Directions
References
Chapter 17: Protein Therapeutics in Respiratory Medicine
17.1 Introduction
17.2 Asthma
17.3 Th2-Targeted Therapies
17.4 Mepolizumab
17.5 Other Anti-IL-5-Targeted Treatments
17.6 Other Respiratory Uses of Monoclonal Antibodies
17.7 Summary
References
Chapter 18: Antibodies for the Prevention, Treatment, and Preemption of Infectious Diseases
18.1 Prophylaxis and Precision Medicine
18.2 Antibacterial Immune Therapy – A Nineteenth Century Breakthrough
18.3 Other Potential Anti-infective mAbs
References
Chapter 19: Rescue Therapies
19.1 Introduction
19.2 Antidotes/Reversal Agents
19.3 Antivenoms and Antitoxins
19.4 Conclusion
References
Chapter 20: Biosimilars
20.1 Introduction
20.2 Concept and Definition of Biosimilars
20.3 Rationale and Significance of Biosimilars
20.4 Current Approvals and Trends
20.5 Challenges and Future Trends
References
Part VI: Future Horizons
Chapter 21: Future Horizons and New Target Class Opportunities
21.1 Introduction
21.2 Targeting the Central Nervous System
21.3 Intracellular Biologics
21.4 Building on the Success of Traditional Monoclonal Antibodies
References
Index
End User License Agreement
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Guide
Cover
Table of Contents
Preface
Part I: Introduction to Protein Therapeutics: Past and Present
Begin Reading
List of Illustrations
Chapter 1: Early Recombinant Protein Therapeutics
Figure 1.1 Paul Berg's construction of hybrid genome.
Figure 1.2 From left to right: Keiichi Itakura, Art Riggs, David Goeddel, and Roberto Crea.
Figure 1.3 Production of insulin from separate genes encoding the A and B chains of human insulin.
Figure 1.4 Zinc crystals of recombinant human insulin produced from the separate chains recombination process.
Figure 1.5 Yeast expression plasmid for dibasic insulin precursors.
Figure 1.6 Assembly of the plasmid containing the human growth hormone gene. For detailed explanation, see Ref. [61].
Figure 1.7 The fibrinolytic system. The proenzyme plasminogen is activated to the active enzyme plasmin by tissue-type or urokinase-type plasminogen activator. Plasmin degrades fibrin into soluble fibrin degradation products. Inhibition of the fibrinolytic system may occur through plasminogen activators, by plasminogen activator inhibitors, or more directly via plasmin, mainly by α2-antiplasmin.
Chapter 2: Evolution of Antibody Therapeutics
Figure 2.1 Chronological, technological, and conceptual evolution of antibody therapeutics from 1891 to 2016. Rather than presenting the evolution linearly and chronologically only (
Y
-axis, from top to bottom), a second dimension (
X
-axis) has been added to better highlight the technological evolutions (gray boxes), which led to new concepts and therapeutic principles (pink boxes) and a large panel of therapeutic uses, which are categorized into neutralizing Abs (blue boxes), cytolytic Abs (red-brown boxes), antagonist Abs (green boxes), and bispecific Abs (dark gray box). The
Y
-axis starts in 1891, year of the first clinical use of serotherapy. The trajectory of the thin arrows indicates how today's applications originate from older therapeutic concepts, having gradually benefited from new technological inputs. The thick arrows indicate the importance of the deployment of the applications.
Chapter 3: Human Antibody Structure and Function
Figure 3.1 Schematic representation of a prototypic antibody IgG, which contains four polypeptide chains, two copies of each with two different chains, heavy (H) and light (L). The heavy chain has four domains: one variable (V
H
) followed by three constant domains (C
H1
, C
H2
, and C
H3
). The heavy chain also contains the hinge region. The light chain has one variable (V
L
) and one constant domain (C
L
). The N-terminal variable domains of both chains have hypervariable regions, also known as CDRs, interspersed with framework regions. Disulfide bonds connect the four chains. N-linked glycosylation site is found at the C
H2
domain.
Figure 3.2 Five major isotypes of antibodies. All subtypes contain light and heavy chains and each with one variable domain (V
H
) and different number of constant domains (C
H1
–C
H4
). Generally, two heavy (H) and two light (L) chains form a monomer Ig module as in IgG, IgD, and IgE. IgM is a pentamer with four constant domains, as in IgE, and an additional polypeptide, the J chain, connecting two monomer Ig units. The J chain is also found in IgA linking two units to form the IgA dimer. Carbohydrate moieties attached to N-glycosylation site of the penultimate constant domains are not shown in this figure.
Figure 3.3 (a) Cartoon ribbon diagram of an intact human antibody IgG1. The variable domains of heavy (V
H
) and light (V
L
) chains at the N-terminal are shown blue and purple, respectively. The constant domains of light (C
L1
) and heavy (C
H1
, C
H2
, and C
H3
) chains at the C-terminal are shown in orange. IgG contains the two Fab units that are linked to Fc. The carbohydrates (in green sticks) are found between the C
H2
domains of the IgG. The flexibility of IgG is achieved through the hinge regions that connect the Fab to the Fc and the elbow regions along the peptides linking V
H
to C
H1
and V
L
to C
L1
, as shown. (b) Structural comparison of variable and constant domains of IgG. Both domains share similar topology comprising of a pair of β sheets in which the β strands are labeled with A–G. The variable domain has CDR loops, whereas those loops in the constant domains are shorter and invariable. The CDR1 connects the β strands B and C, while CDR3 connects the β strands F and G. The variable domain has two additional β strands, C′ and C′′, that supports CDR2. The conserved intradomain disulfide bond is shown in green.
Figure 3.4 (a) Crystal structure of a Fab depicted in ribbons. The variable domains of heavy (V
H
) and light (V
L
) chains at the N-terminal are shown blue and purple, respectively, while the constant domains are in orange. (b) Close-up view showing the antigen-binding site as formed by the six CDR loops. The CDR H3 is located centrally and forms the primary antigen-binding loop of all CDRs. Note that CDR H3 makes significant interactions with other CDRs.
Figure 3.5 Ribbon diagrams of different IgG Fc fragments. X-ray crystal structures of human IgG Fc fragment (PDB 1H3X), monomeric Fc (PDB 4J12), monomeric and unglycosylated CH2 domain (PDB 3DJ9), monomeric CH3 (PDB 4B53), and CH3 dimer (PDB 5HSF).
Figure 3.6 Antibody diversity generated by genetic recombination, junctional mechanisms, and heavy/light chain pairing. Theoretical diversity could be estimated as of up to 10
12
different antibodies based on the number of germline gene segments contributing to functional antibodies and junctional diversity.
Figure 3.7 Five of the six CDR loops (L1, L2, L3, H1, and H2) with standard conformations, called canonical structures. The CDR H3 is highly variable in length, sequence, and structure, and therefore does not have any canonical structures. (a–e) The most abundant canonical structures for the five CDRs are shown. (f) For CDR H3, it is generally divided into three conformations – base, torso, and head (tip or crown region). The base region has always a kinked conformation or sometimes an extended conformation, and the torso region is either budged or non-bulged. The head region generally forms a classical β-turn motif or different conformations based on the torso region as well as its interactions with antigen and/or other CDRs.
Figure 3.8 Co-crystal structures of fully human antibody fragments (Fabs) with viral envelope glycoproteins: (a) HIV-1 gp120-Fab X5 (PDB 2B4C), (b) SARS CoV RBD-Fab 396 (PDB 2DD8), (c) HeVG-Fab m102.3 (PDB XXX), and (d) MERS CoV RBD-Fab m336 (PDB 4XAK). The antibodies against different infectious diseases were generated by the selection of human antibody fragments from
in vitro
libraries. Structural analysis revealed neutralizing epitopes on the viral envelope glycoproteins or its receptor binding domains (RBDs) recognized by CDRs of those antibodies (paratopes) as indicated by rectangular boxes. (e) Close-up views of amino acid residues that make up the interacting paratope and epitope surfaces.
Figure 3.9 (a) View of human IgG with a representative oligosaccharide structure attached to the N-glycosylation site at Asn297. (b) Commonly observed oligosaccharides on recombinant IgGs produced by standard cell lines.
Figure 3.10 Comparison of the complex crystal structures of (a) IgG Fc–FcγRI (PDB 4ZNE), (b) IgG Fc–FcγRIII (PDB 1T83), (c) IgE Fc–FcϵRI (PDB 1F6A), and (d) IgG Fc–FcαR1 (PDB 1OW0). The extracellular domains of the Fc receptors are shown in green, whereas one heavy chain of each Fc region is shown in yellow and the other in red. Carbohydrates are shown as gray sticks.
Chapter 4: Antibodies from Other Species
Figure 4.1 Phylogenetic tree of species of immunological interest. Appearance of unique features, isotypes, and binding structures are indicated in blue. Appearance of lymphoid organs and molecular mechanisms involved in antibody diversity are indicated in the bars on the right. AID stands for the activation-induced cytidine deaminase and GALT for gut-associated lymphoid tissue.
Figure 4.2 Comparison of different species' antibody structures. Common IgG antibody structure found in most species. The heavy chain only antibodies, or HCAbs, are found in camels, which only possess C
H
2 and C
H
3. The heavy chain only antibodies, IgNAR, are found in sharks, have 3–5 C
H
(C
H
1–5) regions. The stalk and knob structure found in ultralong cow antibodies. Cartoon (a) of the antibody and ribbon (b) diagrams of the variable region of each antibody type are shown. The CDR H3 regions are highlighted in each ribbon diagram. The PDB codes of the structures are indicated below each graphic.
Chapter 5: Human Antibody Discovery Platforms
Figure 5.1 Total and human monoclonal antibodies approved by the United States Food and Drug Administration (US FDA) by year. Clear bar: total number of antibodies approved in a given year; Solid bar: number of human antibodies approved in a given year; Line with solid circles: accumulated total of human antibodies approved over time.
Chapter 6: Beyond Antibodies: Engineered Protein Scaffolds for Therapeutic Development
Figure 6.1 Non-antibody scaffolds with β-sheet secondary structure. Engineered variable domains are highlighted in red. (a) Monobody/Adnectin/FN3 domain (PDB: 1FNF); (b) Fynomer/SH3 Domain (PDB ID: 1M27); (c) Anticalin/Lipocalin (PDB: 2HZQ); (d) Nanobody/VHH Domain (PDB: 4KRL).
Figure 6.2 Non-antibody scaffolds with α-helix secondary structure. Engineered variable domains are highlighted in red. (a) DARPin/Ankyrin repeats (PDB ID: 3HG0), (b) Affibody (PDB: 1LP1).
Figure 6.3 Non-antibody scaffolds with mixed secondary structure. Engineered variable domains are highlighted in red. Disulfide linkages are highlighted in yellow and represented as sticks. The GP2 protein in F is isolated because it does not contain disulfide linkages. (a) Avimer/A-domain binders (PDB ID: 1AJJ), (b) EETI-II knottin (PDB ID: 2IT7), (c) MCoTI-II cyclotide (PDB ID: 4GUX), (d) Kringle domain (PDB ID: 1I5K), (e) Kunitz domain (PDB ID: 1AAP), (f) GP2 (PDB ID: 2WNM).
Figure 6.4 Drugs derived from non-antibody scaffolds in clinical development. Each slice corresponds to a clinical target. Individual colors delineate phase of development. White circles represent a single drug in a certain phase of clinical development. AMD: Age-related macular degeneration, HAE: Hereditary angioedema. “Other” includes cachexia, pain, anemia, IBD, plaque psoriasis, and acute respiratory distress syndrome.
Chapter 7: Protein Engineering: Methods and Applications
Figure 7.1 Cross section of an FcRn-expressing endothelial cell showing (a) IgG recycling and (b) IgG transcytosis. After pinocytosis, IgG molecules enter acidic endosomes, bind to FcRn, and are rescued from a lysosomal degradation pathway. IgG-containing vesicles then cycle back or migrate to the cell surface where the IgG molecules are released.
Figure 7.2 Model of an Fc fragment derived from a human IgG1, based upon the X-ray structure corresponding to protein database ID number 1HZH [214]. Colored residues correspond to the various positions where single or multiple substitutions were shown to improve the affinity of the (a) human IgG/FcRn, (b) human IgG/CD16A, (c) human IgG/C1q, and (d) human IgG/CD32A interaction. Carbohydrates are shown as sticks.
Figure 7.3 Cross section of a cell showing the fate of (a) a conventional antibody binding to a membrane-bound antigen and (b) binding to a soluble antigen. In both situations, the antibody–antigen complex is degraded via proteolysis in the lysosome. A pH-dependent antigen binding antibody will dissociate from its antigen, either membrane-bound (c) or soluble (d), in the acidic endosome and is recycled back to the plasma via FcRn, enabling it to bind to another antigen.
Chapter 9: Antibody–drug Conjugates (ADCs)
Figure 9.1 ADC mode of action (for DNA-targeting warhead).
Figure 9.2 ADC components.
Figure 9.3 Auristatin-based warheads and payloads.
Figure 9.4 Kadcyla (where the mAb is trastuzumab).
Figure 9.5 Mylotarg.
Figure 9.6 Mafodotin (MMAF payload).
Figure 9.7 Maytansine-based payloads.
Figure 9.8 Govitecan payload.
Figure 9.9 Pyrrolobenzodiazepine-based warheads.
Figure 9.10 SYD985.
Figure 9.11 Release of duocarmycin payload from SYD985.
Figure 9.12 Tubulysin-based warheads and payloads.
Chapter 10: Pharmacokinetics of Therapeutic Proteins
Figure 10.1 Schematics for two separate case examples showing the effect of ADA on exposure by between-subject comparison. (a) Between-subject comparison of exposure time course. (b) Within-subject comparison of exposure at two different time points.
Figure 10.2 Binding of TGN1412 with the co-stimulatory receptor CD28.
Figure 10.3 Full TMDD model comprising drug distribution and drug–receptor interaction.
Chapter 11: Safety Considerations for Biologics
Figure 11.1 Key activities during early discovery of biologics.
Chapter 12: Immunogenicity of Biologics
Figure 12.1 Human monocyte-derived dendritic cells (white arrows) produced by
in vitro
culture of monocytes with IL-4, GM-CSF, and TNFα.
Figure 12.2 Peptide binding to HLA class II molecules. (a) Top view of peptide (stick model) sitting in the binding groove of MHC class II (surface model). The surface is colored to indicate the depth of cavities, with dark blue indicating the deepest pockets. (b) Lateral view of peptide orientation in the binding groove. Residues with side chains oriented downward make contact with MHC class II, whereas side chains oriented upward potentially contact the TCR [63]. Figure prepared using Swiss-PdbViewer (Guex, N. and Peitsch, M.C. (1997) SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 18, 2714–2723.) using PDB structure file 1FV1.
Figure 12.3 Potential interacting CMC variables that may influence immunogenicity.
Chapter 17: Protein Therapeutics in Respiratory Medicine
Figure 17.1 Stepwise approach to increasing asthma medications. Modified from GINA guidelines. SABA, short acting β-2 agonist; ICS, inhaled corticosteroid; LABA, long-acting β-2 agonist; LTRA, leukotriene receptor antagonist.
Figure 17.2 Pathways leading to Th1 (neutrophilic) and Th2 (eosinophilic) inflammation. Naïve Th0 cells differentiate following interaction with antigen -APCs and cytokine influence. The Th1 and Th2 pathways inhibit each other via IFN-γ and IL-4, respectively. APC, antigen presenting cell; Th, T helper; IL, interleukin; IFN-γ, interferon gamma.
Figure 17.3 Proposed parallel mechanisms of eosinophilic inflammation due to interactions of antigens and irritants at the air/epithelial layer interface. Antigens and irritants can induce eosinophilic inflammation via the IgE-mediated hypersensitivity pathway and also by IL-25- and IL-33-mediated activation of ILC-2 cells. IgE, immunoglobulin E; PGD2, prostaglandin D2; IL, interleukin; Th2, T-helper type 2; ILC2, innate lymphoid cell type 2; LTE4, leukotriene E4; CRTH2, chemokine receptor homologous molecule expressed on Th2 lymphocytes.
Figure 17.4 Cumulative number of exacerbations in each treatment group – DREAM study, 2012 [25]. Significantly reduced exacerbation rate at all doses of mepolizumab.
Figure 17.5 (a) IL-4Rα Type 1 and Type 2 receptors. Type 1 comprises the IL-4Rα and common cytokine receptor γ-chain (γc). Type 2 receptor is made from IL-4Rα subunit and IL-13Rα1. IL-4 binds to Type 1 and Type 2 receptors, while IL-13 binds to only the Type 2 receptor. (b) Dupilumab binds to the IL-4Rα subunit of both Type 1 and Type 2 receptors, stopping the binding of IL-4 and IL-13. IL, interleukin.
Chapter 20: Biosimilars
Figure 20.1 Number of mAbs clinical studies in the United States.
Figure 20.2 Major patents expiry for top-selling mAbs.
Figure 20.3 Herceptin price change in India.
Figure 20.4 Dossier required for biosimilar approval.
Figure 20.5 How similar is “highly similar”?
List of Tables
Chapter 2: Evolution of Antibody Therapeutics
Table 2.1 Monoclonal antibody therapeutics approved or in review in the European Union or the United States
Table 2.2 Fc fusion protein therapeutics approved in the European Union or the United States
Chapter 3: Human Antibody Structure and Function
Table 3.1 Different numbering schemes for antibody variable domain sequence annotation
Table 3.2 Properties and function of five different antibody isotypes
Table 3.3 Definitions of different antibody fragments and domains.
Table 3.4 Germline V
H
–V
L
pairing occurrences as observed in 766 human antibodies as derived from the Protein Data Bank
Table 3.5 Canonical structures for CDRs H1, H2, L1, L2, and L3 are classified into different CDR clusters as implemented in PyIgClassify.
Table 3.6 Databases and tools for analyzing antibody sequence and structural data
Chapter 4: Antibodies from Other Species
Table 4.1 Different immunoglobulin isotypes found in each species
Table 4.2 Presence and use of light chains in various species
Chapter 5: Human Antibody Discovery Platforms
Table 5.1 Human antibodies approved for marketing in major markets as of January 2016.
a
Table 5.2 Source of variable sequences for approved and Phase III clinical candidate mAbs.
a
Table 5.3 Use of genes in formation of serum Igs
Table 5.4 Examples of antibodies in preclinical and clinical trials that have been sourced from human B cells
Table 5.5 Examples of human antibody libraries and associated display technologies
Table 5.6 Companies with transgenic animal platforms producing human antibodies upon immunization
Chapter 6: Beyond Antibodies: Engineered Protein Scaffolds for Therapeutic Development
Table 6.1 Characteristic qualities of prominent non-antibody scaffolds
Chapter 8: Bispecifics
Table 8.1 Description of DT and PE variants used for immunotoxin generation
Table 8.3 Ongoing immunotoxin clinical trials
Table 8.4 Recently completed immunotoxin clinical trials
Table 8.2 Examples of Bispecifics
Table 8.5 Immunocytokines in the clinic
Chapter 9: Antibody–drug Conjugates (ADCs)
Table 9.1 Tabulated summary of vedotin ADCs in Phase II clinical trials
Table 9.2 Summary of PBD ADC clinical trial
Chapter 11: Safety Considerations for Biologics
Table 11.1 Comparison between small molecules and biologics
Table 11.2 Key considerations for an early safety prediction evaluation
Table 11.3 Examples demonstrating variations in species specificity
Table 11.4 Benefits and considerations of alternate approaches to nonclinical safety assessment.
Chapter 12: Immunogenicity of Biologics
Table 12.1 TLR ligands identified from natural, synthetic, and protein therapeutic sources
Table 12.2 Values provided are the total number of peptide contacts (either side chain or main chain) from six crystal structures including both human and murine peptide/MHC II/TCR complexes (1u3h, 1j8h, 1fyt, 1ymn, 1zgl, 1d9k) [74]
Table 12.3 Identified immunogenicity risks for approved therapeutic proteins and antibodies
Chapter 14: Stability, Formulation, and Delivery of Biopharmaceuticals
Table 14.1 Example of a stability program for sterile drug products intended to be stored in a refrigerator (5 °C)
Table 14.2 Overview of analytical methods possibly to study drug product stability targeting different instabilities
Chapter 16: Antibody-Based Therapeutics in Oncology
Table 16.1 Solid Tumor Cell-Surface Targeting Antibodies in Clinical Development
Table 16.2 Check-point blockade antibodies in clinical development
Table 16.3 Costimulatory antibodies in clinical development
Table 16.4 Bispecific antibodies in clinical development or approved for the treatment of cancer
Chapter 17: Protein Therapeutics in Respiratory Medicine
Table 17.1 Biomarkers of eosinophilic inflammation used in RCTs with monoclonal antibodies to preselect patients in adult asthma
Table 17.2 Anti-IL-5 programs
Table 17.3 Anti-IL-5 efficacy and safety
Table 17.4 Effects of different cytokine blockers on clinical measures and biomarkers
Chapter 18: Antibodies for the Prevention, Treatment, and Preemption of Infectious Diseases
Table 18.1 mAbs in development
Chapter 19: Rescue Therapies
Table 19.1 Antidotes/reversal agents acting through high-affinity binding
Table 19.2 Antidotes/reversal agents acting as degrading enzymes
Table 19.3 Molecular weights and estimated pharmacokinetic properties of antibodies and fragments
Table 19.4 Examples for antitoxins/antisera
Chapter 20: Biosimilars
Table 20.1 Characteristics of small molecule drugs compared to biologics
Table 20.2 Definitions of biosimilar products worldwide
Table 20.3 Top 10 best-selling drugs of 2012
Table 20.4 EMA approved biosimilar products.
Table 20.5 Guidance comparison between EMA, FDA, and WHO
Chapter 21: Future Horizons and New Target Class Opportunities
Table 21.1 Key parameters for functional intracellular delivery of therapeutic proteins
Methods and Principles in Medicinal Chemistry
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Protein Therapeutics
Volume 1
Edited by Tristan Vaughan, Jane Osbourn, and Bahija Jallal
Protein Therapeutics
Volume 2
Series Editors
Prof. Dr. Raimund Mannhold
Rosenweg 7
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Prof. Dr. Gerd Folkers
Collegium Helveticum
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MedImmune Ltd.
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Dr. Jane Osbourn
MedImmune Ltd.
Milstein Building, Granta Park
Cambridge CB21 6GH
United Kingdom
Dr. Bahija Jallal
MedImmune LLC.
1 Medimmune Way
Gaithersburg, MD 20878
USA
Cover credit: Background picture (infusion bags) - Photodisc; antibody - fotolia/molekuul.be
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Preface
The number of marketed protein therapeutics [1–3] has increased enormously since the introduction of the first recombinant protein, human insulin, into the clinic several decades ago. Protein therapeutics play a very significant role in many various fields of medicine, and their use continues to steadily broaden. There are several key advantages of protein therapeutics over small-molecule drugs that contribute to this [1]:
Proteins often exhibit highly specific and complex functions that cannot be mimicked by simple chemical compounds.
The larger binding interface of a protein therapeutic enables them to be engineered with high affinity for their target.
Due to their high level of specificity, there is often less potential for protein therapeutics to interfere with normal biological processes and cause off-target effects.
Recombinant technology allows the production of proteins that provide a novel function or activity.
Because the body naturally produces many of the proteins that are used as therapeutics, these agents are often well tolerated and are less likely to elicit immune responses.
For diseases in which the product of a gene is absent or defective, protein therapeutics can provide an effective replacement treatment.
The clinical development and approval time for protein therapeutics may be faster than for small-molecule drugs [4].
Recombinant proteins can also be used in combination with both other large molecule, or indeed small-molecule, drugs to provide additive or synergistic benefit.
Many successful uses of protein therapeutics are documented in this volume. However some challenges still remain for the discovery and development of protein therapeutics: (i) The current route of administration is typically parenteral. Development of oral biologics remains largely an aspiration at this time. (ii) Although the production of recombinant proteins is becoming increasingly efficient and cost–effective, it remains relatively expensive compared to that of small molecules. (iii) The body may mount an immune response against the therapeutic protein. In some cases, this immune response can neutralize the protein and reduce the efficacy of the potential drug.
Taken together, the early success of recombinant insulin production in the 1970s created an atmosphere of enthusiasm and hope, which was followed by an era of disappointment when the vaccine attempts, nonhumanized monoclonal antibodies, and cancer trials in the 1980s were largely unsuccessful. Despite these setbacks, significant progress has been made. With the large number of protein therapeutics both in current clinical use and in clinical trials for a range of disorders, one can confidently predict that protein therapeutics will have a further expanding role in future medicine and may – together with cell and gene therapy – dominate over classical therapeutic approaches based on small-molecule drugs.
Accordingly, this is an appropriate time to review our current knowledge and future perspectives of protein therapeutics as realized in this volume by experts in the field both from industry and academia. It is organized in six sections, first of which introduces the past and present development of protein therapeutics in the chapters on “Early Recombinant Protein Therapeutics” and “Evolution of Antibody Therapeutics.” The second section is dedicated to antibodies as the ultimate scaffold for protein therapeutics and is covered in two chapters on “Human Antibody Structure and Function” as well as “Antibodies from Other Species.” Discovery and engineering of protein therapeutics are described in the next section comprising detailed chapters on “Human Antibody Discovery Platforms,” “Beyond Antibodies: Engineered Protein Scaffolds for Therapeutic Development,” “Protein Engineering: Methods and Applications,” “Bispecifics,” and on “Antibody–Drug conjugates.” Physiological and manufacturing considerations are given in the follow-up section including overviews on “Pharmacokinetics,” “Safety Considerations,” “Immunogenicity,” “Expression Systems for Manufacture,” and a chapter on “Stability, Formulation, and Delivery.” The section on Clinical Applications discusses in detail “Protein Therapeutics in Autoimmune and Inflammatory Diseases, Oncology, Respiratory, and Infectious Diseases.” Chapters on “Rescue Therapies” and “Biosimilars” supplement this section. Future horizons and new target class opportunities are the topics of the final section.
The series editors thank Tristan Vaughan, Jane Osbourn, and Bahija Jallal for organizing this volume and for identifying and working with such excellent authors. Last but not least we thank Frank Weinreich and Waltraud Wüst from Wiley-VCH for their valuable contributions to this project and to the entire book series.
May 2017
Raimund Mannhold, Düsseldorf Gerd Folkers, Zürich Helmut Buschmann, Aachen
References
1 Leader, B., Baca, Q.J., and Golan, D.E. (2008) Protein therapeutics: a summary and pharmacological classification.
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2 Tsomaia, N. (2015) Peptide therapeutics: targeting the undruggable space.
Eur. J. Med. Chem.
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3 Carter, P.J. (2011) Introduction to current and future protein therapeutics: a protein engineering perspective.
Exp. Cell Res.
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4 Reichert, J.M. (2003) Trends in development and approval times for new therapeutics in the United States.
Nat. Rev. Drug Discov.
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, 695–702.
A Personal Foreword
To a diabetic patient, it is hard to imagine a world without biosynthetic insulin. For a new parent of a premature baby, Synagis provides potentially life-saving prevention of respiratory syncytial virus. And for someone suffering the devastating effects of rheumatoid arthritis, Humira, the world's first fully human antibody, introduced in 2002, is so effective that it has become the world's best-selling medicine. Today, biologics such as these account for nearly half of all new drug approvals across the globe and nearly 25% of overall sales. In fact, in 2015, 6 biologics were among the top 10 best-selling drugs worldwide. With more than 500 biopharmaceuticals on the market, biologics represent the fastest growing sector of this industry, targeting illnesses such as cancer, asthma, cardiovascular disease, infectious diseases, multiple sclerosis, hepatitis, inflammatory disease, and so many others.
As a student of physiology and biochemistry in Paris, I was struck by the possibility of modifying proteins to increase their potential to treat illness. My postdoctoral work in molecular biology and oncology at the Max-Planck Institute for Biochemistry in Munich allowed me to focus on the analysis of epidermal growth factor receptor (EGF-R and HER2) signaling and to investigate the antitumor properties of the novel secreted tumor-associated antigen 90K.
My later work building the translational sciences function at Sugen further heightened my interest. But the day I met a patient with renal cell carcinoma who was successfully being treated with Sutent, a multitargeted receptor tyrosine kinase (RTK) inhibitor, made me realize that my true calling was to work in the biopharmaceutical industry to discover new drugs to help patients. Today, as the head of MedImmune, the global biologics early research and development unit of AstraZeneca, I am even more excited by the possibilities of using protein therapeutics to not just treat or prevent disease but to provide a durable cure for so many illnesses affecting patients across the globe.
This book dissects the field of protein therapeutics, from its early struggles to its promising future, and provides a thorough look into this dynamic industry. It touches on exciting developments around immuno-therapies for oncology – advancements I never thought were possible in my lifetime. It delves into the considerable energy going into the development of antibody drug conjugates and their potential for new therapies. Other topics include human and nonhuman antibodies; technological advances in protein therapeutics, including human antibody discovery platforms, nonantibody scaffolds and antibody mimetics; protein engineering and physiological and manufacturing considerations around pharmacokinetics, immunogenicity, safety, and manufacturing; and clinical applications for protein therapeutics.
Protein Therapeutics concludes with a view into innovation of the future, including the potential to target protein therapeutics across the blood–brain barrier for the treatment of diseases ranging from brain tumors to Alzheimer's disease and how protein therapeutics could be delivered intracellularly to gain a better understanding of protein interactions and, for example, modify the RAS/MAPK pathway that could be potentially transformative for the treatment of leukemia and other cancers.
With contributions from recognized academic and industry experts, including Professor Pierre De Meyts, the leading academician in the field of insulin research and one of the fathers of protein therapeutics, and Herren Wu, MedImmune's chief technology officer who offers just a glimpse at potential future innovations, Protein Therapeutics will be a valuable addition to a field that is profoundly changing the way we treat disease.
Gaithersburg, MD
December 2016
Bahija Jallal
Acknowledgments
We would like to express our gratitude to Karen Stanger for her proactive stewardship over early stages of this project and to Fay Larman who helped us see it through to completion. We are, of course, most indebted to all the authors for taking time out from their daily activities to make so many excellent and insightful contributions to this handbook, as well as to all the reviewers for providing valuable critique and comment. Finally, we would like to especially thank all of you, the scientists, who have contributed so much to this most exciting, dynamic, and motivational field. There is nothing more rewarding than having the opportunity to meet a patient who is benefitting from a protein therapeutic that you have had the good fortune to have helped discover and develop.
Part IIntroduction to Protein Therapeutics: Past and Present
Chapter 1Early Recombinant Protein Therapeutics
Pierre De Meyts1,2,3
1Department of Cell Signalling, de Duve Institute, Catholic University of Louvain, Avenue Hippocrate 75, 1200, Brussels, Belgium
2De Meyts R&D Consulting, Avenue Reine Astrid 42, 1950, Kraainem, Belgium
3Global Research External Affairs, Novo Nordisk A/S, 2760, Måløv, Denmark
1.1 Introduction
The successful purification of pancreatic insulin by Frederick Banting, Charles Best, and James Collip in the laboratory of John McLeod at the University of Toronto in the summer of 1921 [1–3], as reviewed in the magistral book of Bliss [4], ushered in the era of protein therapeutics. Banting and McLeod received the Nobel Prize in Physiology or Medicine in 1923. The discovery of insulin was truly a miracle for patients with Type 1 diabetes, for whom the only alternative to a quick death from ketoacidosis was the slow death by starvation on the low-calorie diet prescribed by Allen of the Rockefeller Institute [5–7]. Insulin went into immediate industrial production (from bovine or porcine pancreata) from the Connaught laboratories of the University of Toronto and, under license from the University of Toronto by Eli Lilly and Co. in the United States, by the Danish companies Nordisk Insulin Laboratorium and Novo (who merged in 1989 as Novo Nordisk), and by the German company Hoechst (now Sanofi), all of which remain the major players in the insulin business today.
Insulin also turned out to be a blessing for scientists interested in protein structure. It was the first protein to be sequenced [8, 9], earning Fred Sanger his first Nobel Prize in 1958. It was the first protein to be assembled by total peptide synthesis [10–14]. It was the first peptide hormone to have its minute blood levels measured by radioimmunoassay [15], earning Rosalyn Yalow the Nobel Prize in Physiology or Medicine in 1977. The structure of insulin was solved by X-ray crystallography in 1969 by Nobel Laureate Dorothy Hodgkin and her team in Oxford [16], providing a rationale for detailed structure–activity relationships studies [17] and, later, for protein engineering of insulin and insulin analogs.
For about 60 years after the discovery of insulin, major therapeutic advances were in formulation to improve pharmacokinetic and pharmacodynamic (PK/PD) properties and provide longer acting insulins, and in purification (reviewed in Ref. [18]). The introduction of the first insulin injection pen in 1985 (Novopen) made insulin treatment somewhat more bearable for diabetic patients.
In 1982, insulin once again was at the forefront of a therapeutic revolution by becoming the first DNA recombinant protein therapeutics on the market (human insulin, Humulin by Eli Lilly), kickstarting the biotechnology era. By 2008, 130 protein therapeutics (of which 95 were produced by genetic engineering) had been approved by the US Food and Drug Administration (FDA) [19], and in 2016, 206 [20]. These comprise hormones, interferons, interleukins, hematopoietic growth factors, tumor necrosis factors, blood clotting factors, thrombolytic drugs, enzymes, monoclonal antibodies, and vaccines.
In this introductory chapter, I will attempt to retrace the pioneering early steps in the development of recombinant protein therapeutics.
1.2 The Birth of Genetic Engineering
The term “molecular biology” is said to have been coined in 1938 by Warren Weaver, head of the Division of Natural Sciences at the Rockefeller Foundation. While the field developed since the 1930s, it got a major impetus in 1953 with the discovery of the double-helical structure of DNA by Watson and Crick [21, 22], who deduced from it the mechanism of genetic self-duplication. For this achievement, Watson and Crick were awarded the Nobel Prize in Physiology or Medicine in 1962.
While studying the structure, function, and replication of the genes of simian virus 40 (SV40), Paul Berg of Stanford University had the idea in the early 1970s that viral DNA could be used to transduce inserted nonviral genes into mammalian cells. He and his colleagues succeeded in developing a general way to join two DNAs together in vitro, a process called DNA recombination. This involved synthesizing cohesive ends onto the end of DNA segments and covalently joining them in vitro using DNA ligase. They succeeded in inserting both lambda phage genes and the galactose operon of Escherichia coli into circular SV40 DNA [23]. Recombinant DNA technology was born. Paul Berg received the Nobel Prize in Chemistry in 1980 for this work, shared with Walter Gilbert and Fred Sanger (his second Nobel Prize) for their work on sequencing DNA methods. Interestingly, this landmark paper does not report successful expression of the recombinant DNA into mammalian cells (in part due to biohazard concerns), but Berg and colleagues later developed suitable vectors for this purpose (Figure 1.1) [24].
Figure 1.1 Paul Berg's construction of hybrid genome.
(From his speech: The Nobel Prize in Chemistry 1980. Nobelprize.org. Nobel Media AB 2014. Web. 12 Jan 2017. http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1980/ [24].)
Berg's very basic goal was to insert bacterial, viral, and simple eukaryotic genes into mammalian cells, in order to understand better how human genes are organized and function. What triggered the rise and explosive growth of the biotechnological industry was a different postulate: how to transduce mammalian genes encoding proteins of therapeutic importance into bacteria or simple eukaryotes (and eventually mammalian cells). The key experiments toward this end were done by a team of scientists that included Stanlry N. Cohen from Stanford University, as well as Herbert Boyer and Howard Goodman from the University of California, San Francisco (UCSF), who were to become major players in the nascent biotechnology industry [25, 26].
They transferred and expressed frog ribosomal DNA into bacterial cells using a constructed plasmid vector, pSC101. This plasmid contained a single site for the restriction enzyme EcoRI and a gene for tetracycline resistance. EcoRI was used to cut the frog DNA into small fragments, which were combined with the plasmid that had also been cleaved with EcoRI. The aligned sticky ends of the DNA fragments were joined using DNA ligase. The plasmids were then transferred into E. coli and plated onto a growth medium containing tetracycline. The cells that incorporated the plasmid carrying the tetracycline resistance gene grew and formed colonies of bacteria, some of which carried the frog ribosomal RNA gene. The colonies were shown to express frog ribosomal DNA.
This led to the very first biotechnology patent granted to Cohen and Boyer in 1980, to the Wolf Prize in Medicine in 1981 and the National Medal of Science in 1988 to Cohen, and to the Lasker Award in 1980 and National Medal of Science in 1990 to Boyer.
Berg Boyer and Cohen with other leading molecular biologists raised concern in 1974 about the potential biohazards of recombinant DNA [27], and organized the influential Asilomar Conference in February 1975, which resulted in stringent National Institutes of Health (NIH) guidelines for recombinant DNA work in June 1976; however, these were later relaxed when the technology became more familiar.
Herbert Boyer made a bold move in April 1976 by founding the biotechnology company Genentech in South San Francisco with a young venture capitalist Robert A. Swanson. Genentech to this day (acquired by Roche, Switzerland, in 2009) remains one of the most successful biotech companies. Since 1976, more than 2000 such companies have been founded, and major pharmaceutical companies have subsequently strived to become more biotechnologically oriented.
1.3 Recombinant Human Insulin
1.3.1 The Race to Clone the Human Insulin Gene and to Produce Human Recombinant Insulin
The race to produce the first recombinant protein therapeutics has been vividly described in the excellent book of Hall [28]. By 1976, Eli Lilly and Co. became concerned that the supply of animal pancreata would become insufficient at some point in the future to meet the needs of diabetic patients worldwide. This was supported by a report of the National Diabetes Advisory Board published 2 years later [29]. Lilly was aware of the promise of genetic engineering and had established an in-house group of scientists to evaluate its possibilities.
Forty years ago, in May 1976, Lilly convened one of its periodic academia–industry symposia (the 16th since 1922), focused this time on the theme of making insulin by genetic engineering. Leading molecular biologists and insulin researchers from all over the United States were invited: Donald F. Steiner (the discoverer of proinsulin [30]) and Shu Jin Chan from the University of Chicago, Grady Saunders and Peter Lomedico from University of Texas, William J. Rutter, Raymond Pictet, and Howard M. Goodman from UCSF, and William (Bill) Chick, Argiris Efstratiadis, and Walter Gilbert (who would later become a co-winner of the 1980 Nobel Prize in Chemistry) from Harvard University.
Bill Chick presented his results with establishing an insulin-producing cell line from an irradiated rat insulinoma, which became an obvious tool for attempting to isolate the rat insulin gene (rat and mice have two, in fact). The race was on [28].
The following year, Axel Ullrich and colleagues in the UCSF team led by Bill Rutter and Howard Goodman reported the construction of recombinant bacterial plasmids containing complementary DNAs (cDNAs) for the coding sequence or rat preproinsulin I and the A sequence or rat preproinsulin II [31]. In 1980, Graeme Bell in the Rutter–Goodman team determined the sequence of the human insulin gene [32]. The same year, Talmadge and colleagues in the Gilbert team showed that bacteria (E. coli) can process mature rat preproinsulin to proinsulin [33].
However, in the end the race to produce recombinant human insulin was won by a team that scooped all others by choosing a radically different approach from those trying to use cloned genes: they chose to use completely synthetic DNA.
Herbert Boyer missed the Lilly May 1976 meeting because he was busy founding Genentech in April, and forming an alliance with a team at the City of Hope National Medical Center in Duarte, California, in order to produce recombinant human insulin, and ultimately sell it [28].
The City of Hope team included Arthur (Art) D. Riggs, a geneticist and molecular biologist whose major interests were how genes are turned on and off, X-chromosome inactivation, and DNA methylation, basically founding the now popular field of epigenetics. Riggs had recruited in 1975 a highly talented Japanese organic chemist Keiichi Itakura (then in Ottawa), who was at the forefront of the difficult art of making synthetic DNA.
Riggs and Itakura had realized the potential power of combining chemical DNA synthesis technology and recombinant DNA technology, and Herbert Boyer had independently come to the same conclusion. The project got strong support from Rachmiel Levine, the Medical Director of the City of Hope, considered by many to be the father of modern diabetes research. Riggs and Itakura chose to first try the synthetic approach on a simpler peptide hormone, somatostatin, with only 14 amino acids. The project was rejected for funding by the NIH as unrealistic, but with Boyer's collaboration and Genentech's support, the effort to make somatostatin in E. coli was completed successfully in 1977 [34]. This represented the first synthesis of a functional polypeptide product from a gene of chemically synthesized origin. Then, in 1978, the City of Hope–Genentech team reported the successful chemical synthesis of separated genes for the A chain and B chain of human insulin [35], and in 1979 their successful expression in E. coli [36]. Synthetic genes for human insulin A and B chains were cloned separately in plasmid pBR322. The cloned synthetic genes were then fused to an E. coli β-galactosidase gene to provide efficient transcription and translation and a stable precursor protein. The insulin peptides were cleaved from β-galactosidase, detected by radioimmunoassay, and purified. Complete purification of the A chain and partial purification of the B chain were achieved. These products were mixed, reduced, and reoxidized. The presence of insulin was detected by radioimmunoassay.
This triumph launched the biotechnology industry. It also endeared Genentech with investors [28]. Some of the key players behind this historical milestone are shown in Figure 1.2. In this figure are David Goeddel, one of the first scientists hired by Genentech and who later became Genentech's research director, and Roberto Crea, who soon after the insulin project set up the DNA chemistry lab at Genentech.
Figure 1.2 From left to right: Keiichi Itakura, Art Riggs, David Goeddel, and Roberto Crea.
(Picture courtesy of City of Hope, used with permission of the scientists pictured.)
The process (Figure 1.3) was licensed by Genentech to Eli Lilly and Co. [38]. Crystals of the recombinant insulin are shown in Figure 1.4. In 1981, my group showed that the recombinant insulin had the same affinity for the insulin receptor as native insulin [39]. On October 29, 1982, recombinant human insulin was approved by the FDA and went on the market under the name Humulin. The two-chain recombination process was later replaced by the expression in E. coli of a gene for human proinsulin, which after expression and purification was then enzymatically converted to insulin [40]. More details about the precise processes can be found elsewhere [18, 41].
Figure 1.3 Production of insulin from separate genes encoding the A and B chains of human insulin.
(From Ref. [37], used with permission. © E.P. Kroeff.)
Figure 1.4 Zinc crystals of recombinant human insulin produced from the separate chains recombination process.
(From Ref. [38], used with permission © Raven Press 1981.)
