Therapeutic Fc-Fusion Proteins -- Contents -- Preface -- List of Contributors -- 1 Introduction: Antibody Structure and Function -- 1.1 Introduction to Antibodies -- 1.2 General Domain and Structure of IgG -- 1.2.1 Structural Aspects Important for Fc Fusion(s) -- 1.2.1.1 Fc Protein-Protein Interactions -- 1.2.1.2 Fc Glycosylation -- 1.2.1.3 Hinge and Interchain Disulfide Bonds -- 1.3 The Neonatal Fc Receptor -- 1.3.1 FcRn Function and Expression -- 1.3.2 Species Difference in FcRn -- 1.3.3 Engineering to Modulate Pharmacokinetics -- 1.3.3.1 Fc Engineering -- 1.3.3.2 Other Engineering Efforts to Modify PK of an IgG or Fc Fusion -- 1.4 Introduction to FcgR- and Complement-Mediated Effector Functions -- 1.4.1 Cell Lysis and Phagocytosis Mediation -- 1.4.2 FcgR-Mediated Effector Functions -- 1.4.2.1 FcgR Biology -- 1.4.2.2 Expression Profiles -- 1.4.2.3 Therapeutic Relevancy -- 1.4.3 Complement -- 1.4.3.1 C1q Biology -- 1.4.3.2 Therapeutic Relevancy -- 1.4.4 Modifying Effector Functions -- 1.4.4.1 FcgR-Dependent Effector Function -- 1.4.4.2 Engineering -- 1.4.4.3 Glycoengineering -- 1.4.4.4 Reducing and Silencing Effector Function -- 1.5 Current Trends in Antibody Engineering -- 1.5.1 Bispecific -- 1.5.2 Drug Conjugates -- References -- Part One: Methods of Production for Fc-Fusion Proteins -- 2 Fc-Fusion Protein Expression Technology -- 2.1 Introduction -- 2.2 Expression Systems Used for Fc-Fusion Proteins -- 2.2.1 Expression Using Mammalian Cell Lines -- 2.2.1.1 Host Cells -- 2.2.1.2 Codon Optimization -- 2.2.1.3 Vectors -- 2.2.1.4 Stable versus Transient Expression -- 2.2.1.5 Viral Transduction and Transfection Methods -- 2.2.2 Expression Using Prokaryotic Cells -- 2.2.2.1 Vectors -- 2.2.3 Expression Using Baculovirus/Insect Cells -- 2.2.3.1 Host Cells -- 2.2.3.2 Vectors -- 2.2.3.3 Additional Considerations -- 2.3 Summary -- References.

3 Cell Culture-Based Production -- 3.1 Introduction -- 3.2 Basic Aspects of Industrial Cell Culture -- 3.2.1 The Central Role of the Production Cell Line -- 3.2.2 Production Systems -- 3.2.3 Production Mode: Fed-Batch or Perfusion? -- 3.2.4 Scale-Up -- 3.2.5 Raw Materials and Process Control -- 3.2.6 How to Develop or Optimize a Culture Production Process for Fc-Fusion Molecules -- 3.3 Speci.c Process Considerations for Fc-Fusion Molecules -- 3.3.1 Product Quality Challenges -- 3.3.2 Process Strategies and Process Parameters -- 3.3.2.1 Temperature and Misfolding -- 3.3.2.2 Other Process Parameters -- 3.3.2.3 Glycosylation -- 3.4 Case Studies -- 3.4.1 LTBr-Fc (Baminercept) -- 3.4.2 rFVIIIFc -- 3.5 Conclusions -- References -- 4 Downstream Processing of Fc-Fusion Proteins -- 4.1 Introduction and Overview of Fc-Fusion Proteins -- 4.2 Biochemistry of Fc-Fusion Proteins -- 4.3 Purification of Fc-Fusion Proteins from Mammalian Cells -- 4.3.1 Platform Approaches for Downstream Purification -- 4.3.2 Comparison of Protein A Chromatography, Viral Inactivation, and Polishing Steps -- 4.4 Purification of Fc-Fusion Protein from Microbial Systems -- 4.5 Future Innovations in Fc-Fusion Protein Downstream Processing -- 4.6 Conclusions -- References -- 5 Formulation, Drug Product, and Delivery: Considerations for Fc-Fusion Proteins -- 5.1 Challenges of Molecule Design and Protein Formulation -- 5.2 The Promise of Fc-Fusion Proteins -- 5.3 Current Landscape of Commercial Antibody-Related Products -- 5.4 Fc Conjugates Compared to mAb Counterparts -- 5.5 Factors in Selecting Liquid versus Lyophilized Formulations -- 5.6 Advantages and Disadvantages of a Lyophilized Product -- 5.7 The General Lyophilization Formulation Strategy for Fc-Fusion Proteins -- 5.7.1 pH and Buffer -- 5.7.2 Stabilizing Agents (Cryoprotectant and Lyoprotectant) -- 5.8 Bulking Agent.

5.9 Surfactant -- 5.10 The Impact of Residual Moisture -- 5.11 Practical Considerations for Low-Protein-Concentration Lyophilized Products -- 5.12 Drug Delivery Considerations -- 5.13 Device Considerations -- 5.14 Assessing Feasibility of a Multidose Formulation -- 5.15 Overage Considerations -- 5.16 Summary -- References -- 6 Quality by Design Applied to a Fc-Fusion Protein: A Case Study -- 6.1 Introduction -- 6.1.1 Atacicept: A Novel Immunomodulator with B Cell Targeting Properties -- 6.1.2 Molecular Characteristics -- 6.1.3 Quality by Design Concept -- 6.2 Critical Quality Attributes -- 6.3 Critical Process Parameters -- 6.4 Process Characterization -- 6.5 Global Multistep Design Space -- 6.6 Robustness Studies -- 6.7 Adaptive Strategy -- 6.8 Engineering Design Space -- 6.8.1 Principle of the Engineering Design Space -- 6.8.2 The Shear Stress as One Element of the Engineering Design Space -- 6.9 Control Strategy -- 6.9.1 Process Controls -- 6.9.2 Testing Controls -- 6.9.3 Process Monitoring -- 6.9.4 Material Control -- 6.10 Continuous Process Verification -- 6.11 Expanded Change Protocol and Continual Improvement -- 6.12 Business Case -- References -- 7 Analytical Methods Used to Characterize Fc-Fusion Proteins -- 7.1 Background -- 7.2 Product Characterization -- 7.2.1 Physiochemical Analysis -- 7.2.1.1 Measurement of Strength by Absorbance at 280nm -- 7.2.1.2 Determination of Identity and Evaluation of Charge Variants -- 7.2.1.3 Measurement of Purity and Integrity -- 7.2.1.4 Mass Analysis and Confirmation of Primary Structure -- 7.2.1.5 Oligosaccharide Analysis -- 7.2.1.6 Purity (Product-Related Variants) -- 7.2.2 Measurement of Potency -- 7.2.3 Process-Related Impurities and Contaminants -- 7.2.3.1 Host Cell Protein -- 7.2.3.2 Residual DNA -- 7.2.3.3 Residual Protein A -- 7.2.3.4 Tests for Contaminants.

7.3 Characterization of the Reference Standard -- 7.4 Typical Product Release and Stability Assays -- 7.5 Analytical Method Qualification and Validation -- References -- Part Two: Case Studies of Therapeutic Fc-Fusion Proteins -- 8 Introduction to Therapeutic Fc-Fusion Proteins -- 8.1 Therapeutic Fc-Fusion Proteins -- 8.2 Background -- 8.3 Fc-Fusion Constructs Have Increased In Vivo Stability -- 8.4 Immunoglobulin-Mediated Effector Function -- 8.5 Considerations in Fc-Fusion Protein Design -- 8.6 Fc-Fusion Proteins Approved for Use in the United States -- 8.6.1 Alefacept -- 8.6.2 Etanercept -- 8.6.3 Abatacept and Belatacept -- 8.6.4 Aflibercept -- 8.6.5 rFVIIIFc and rFIXFc -- 8.6.6 Rilonacept -- 8.6.7 Romiplostim -- 8.6.8 Trebananib -- 8.7 Concluding Remarks -- References -- 9 Alefacept -- 9.1 Introduction -- 9.2 Chronic Plaque Psoriasis -- 9.3 Conventional Treatments for Psoriasis -- 9.4 Preclinical Development -- 9.4.1 CD2/LFA-3 -- 9.4.2 Fusion Protein Alefacept (LFA3TIP) -- 9.5 Preclinical Primate Studies -- 9.6 Phase 1 and 2 Human Clinical Studies -- 9.7 Phase 3 Studies -- 9.7.1 Study Design -- 9.7.1.1 Eligibility -- 9.7.1.2 Dosing and Blood Work -- 9.7.1.3 Endpoints -- 9.7.1.4 Statistical Analysis -- 9.7.1.5 Intravenous Studies 711 and 724 -- 9.7.1.6 Intramuscular Studies 712 and 717 -- 9.7.2 Efficacy -- 9.7.2.1 Patient Population -- 9.7.2.2 CD4 Monitoring -- 9.7.2.3 PASI and PGA Results -- 9.7.2.4 Quality of Life -- 9.7.2.5 Remittance -- 9.7.3 Multiple Courses of Treatment -- 9.8 Clinical Pharmacology -- 9.9 Clinical Safety -- 9.9.1 Adverse Events -- 9.9.2 Infection -- 9.9.3 Cancer -- 9.9.4 Laboratory Tests -- 9.10 Amevive Discontinued -- References -- 10 Etanercept -- 10.1 Introduction -- 10.1.1 TNF Structure and Function -- 10.1.2 TNF Receptor Types -- 10.1.3 TNF Receptor Signaling -- 10.1.4 Role of TNF in Chronic Inflammatory Disease.

10.1.5 Rheumatoid Arthritis -- 10.1.6 Juvenile Idiopathic Arthritis -- 10.1.7 Psoriatic Arthritis -- 10.1.8 Ankylosing Spondylitis -- 10.1.9 Crohn's Disease -- 10.1.10 Ulcerative Colitis -- 10.1.11 Psoriasis -- 10.2 Design, Construction, and Characterization of TNFR-Fc-Fusion Protein -- 10.2.1 State of Therapeutic Antibodies and Rationale for a Receptor-Fc-Fusion Protein -- 10.3 Etanercept Preclinical Development -- 10.3.1 Binding and TNF Inhibitory Activity -- 10.3.2 Inhibition of TNF Activity -- 10.3.3 Preclinical Efficacy -- 10.3.4 Pharmacokinetics and Pharmacodynamics -- 10.3.5 Toxicology -- 10.4 Etanercept Key Clinical Trials -- 10.4.1 Rheumatoid Arthritis -- 10.4.2 Polyarticular Juvenile Idiopathic Arthritis -- 10.4.3 Psoriatic Arthritis -- 10.4.4 Ankylosing Spondylitis -- 10.4.5 Plaque Psoriasis -- 10.4.6 Other Potential Indications -- 10.5 Competitive Landscape -- 10.6 Conclusions -- References -- 11 Abatacept and Belatacept -- 11.1 Introduction -- 11.2 Design, Construction, and Characterization of Abatacept -- 11.3 Immunosuppressive Properties of Abatacept -- 11.4 Rational Design and Characterization of Belatacept -- 11.5 Belatacept Activity in Primate Renal Transplant Studies -- 11.6 Abatacept Clinical Development -- 11.7 Belatacept Clinical Development -- 11.8 Concluding Remarks -- References -- 12 Aflibercept -- 12.1 Introduction -- 12.2 Clinical Indications -- 12.2.1 Age-Related Macular Degeneration -- 12.2.2 Macular Edema with CRVO -- 12.2.3 Metastatic Colorectal Cancer -- 12.3 Characterization of Aflibercept -- 12.4 Preclinical Studies with Aflibercept -- 12.5 Clinical Studies with Aflibercept -- 12.5.1 Aflibercept and AMD -- 12.5.2 Aflibercept and Cancer -- 12.5.2.1 Single-Agent Phase 1 Studies -- 12.5.2.2 Combination Phase 1 Studies -- 12.5.2.3 Single-Agent Phase 2 Studies -- 12.5.2.4 Combination Phase 2 and 3 Studies -- 12.6 Summary.

References.