Biophysical Methods for Biotherapeutics -- Contents -- Preface -- About the Editor -- List of Contributors -- Section 1 Early Discovery Stages and Biotherapeutic Candidate Selection -- 1 Biophysical Methods Applied in Early Discovery of a Biotherapeutic: Case Study of an Egfr-Igf1r Bispecific Adnectin -- 1.1 Introduction -- 1.2 Target Identification -- 1.3 Target Generation -- 1.3.1 Multiple Constructs Strategy -- 1.4 Hit Evaluation -- 1.4.1 Qualitative and Rapid Self-Association Check -- 1.4.2 Qualitative and Rapid Thermal Stability Check -- 1.4.3 Confirmation of Binding -- 1.5 Lead Selection -- 1.5.1 Self-Association -- 1.5.2 Thermal Stability -- 1.5.3 Binding Affinity, Kinetics, and Epitope -- 1.6 Lead Optimization -- 1.7 Lead Formatting -- 1.7.1 Solubility -- 1.7.2 Thermal Unfolding Behavior -- 1.8 Final Development Candidate Selection -- 1.9 Concluding Remarks -- Acknowledgment -- References -- 2 X-ray Crystallography for Biotherapeutics -- 2.1 Introduction to X-ray Crystallography -- 2.1.1 Early X-Ray Crystallography for Biologics -- 2.2 Modern X-ray Crystallography -- 2.2.1 Construct Design and Protein Production -- 2.2.2 Macromolecular Crystallization -- 2.3 X-ray Data Collection -- 2.3.1 Crystal Mounting -- 2.3.2 Collecting a Data Set -- 2.3.3 Data Reduction -- 2.4 Solving the Structure of the Crystal -- 2.4.1 Molecular Replacement -- 2.4.2 Heavy Atom Techniques -- 2.4.3 Confirming the Validity of a Solution -- 2.4.4 Building and Refining the Structure -- 2.5 Understanding the Target Through Structure -- 2.5.1 The Model -- 2.5.2 The Protein Databank and Related Resources -- 2.5.3 Information Provided by X-Ray Crystallography -- 2.6 Applications of X-ray Crystallography to Biotherapeutics -- 2.6.1 Antibody-Based Biotherapeutics -- 2.6.2 Antibody Design -- 2.6.3 Protein Receptor Interactions.

2.7 Future Applications of Crystal Structures in Biotherapeutics -- 2.7.1 Protein Engineering -- 2.8 Conclusion -- Acknowledgments -- References -- 3 Solubility and Early Assessment of Stability for Protein Therapeutics -- 3.1 Introduction -- 3.2 Measuring Protein Solubility -- 3.2.1 Direct Measurement of Solubility: Concentration to Precipitation -- 3.2.2 Indirect Assessment of Solubility: The Second Virial Coefficient (B22) and Self-Interaction Chromatography -- 3.3 Assessment of Protein Stability -- 3.3.1 Thermal Stability -- 3.3.2 Aggregation -- 3.3.3 Chemical Modifications -- 3.4 Computational Predictions -- 3.4.1 Identifying Aggregation Promoting Regions -- 3.4.2 Interaction Hot Spots -- 3.5 Enhance the Solubility of Biotherapeutics -- 3.5.1 Site-Directed Mutagenesis -- 3.5.2 Pegylation -- 3.5.3 Glycosylation -- 3.5.4 Formulation Optimization -- 3.6 Development of Rapid Methods to Identify Soluble and Stable Biotherapeutics -- 3.7 Concluding Remarks -- References -- Section 2 First-in-Human and Up To Proof-of-Concept Clinical Trials -- 4 Biophysical and Structural Characterization Needed Prior to Proof of Concept -- 4.1 Introduction -- 4.2 Biophysical Methods for Elucidation of Protein Structure and Physiochemical Properties -- 4.2.1 Protein Primary Structure -- 4.2.2 Protein Secondary and Tertiary Structures -- 4.2.3 Quaternary Structure -- 4.2.4 Posttranslational Modifications -- 4.3 Biophysical and Structural Characterization Data -- 4.4 Case Study-Characterization of Higher Order Structure of a Fusion Protein with Biophysical Methods -- 4.5 Biophysical and Structural Characterization Data in Analytical Comparability Assessments -- 4.5.1 Case Study-Product Formulation Change -- 4.5.2 Case Study-Cell Line and Process Change -- 4.6 Summary and Future Perspectives -- Acknowledgments -- References.

5 Nucleation, Aggregation, and Conformational Distortion -- 5.1 Introduction -- 5.2 Nonnative Aggregation Involves Multiple Competing Processes -- 5.2.1 Aggregation Rates, Pre-Equilibration, and Rate-Determining Step(s) -- 5.2.2 Nucleation versus Growth in the Context of Stability of Biotherapeutics -- 5.3 Importance of Conformational Changes in Forming/Nucleating Aggregates -- 5.3.1 Measuring Global and Local Unfolding/Conformational Changes -- 5.3.2 Measuring Nonnative Structures in Aggregates and Detecting Nuclei -- 5.4 Conformational Changes During Aggregate Growth -- 5.5 Surface-Mediated Unfolding and Assembly -- 5.5.1 Additional Challenges Presented by Interface-Mediated Aggregation -- 5.5.2 Potential Roles of Surfactants -- 5.6 Summary -- References -- 6 Utilization of Chemical Labeling and Mass Spectrometry for the Biophysical Characterization of Biopharmaceuticals -- 6.1 Mass Spectrometry of Biopharmaceuticals -- 6.2 Introduction to Hydrogen/Deuterium Exchange -- 6.3 Applications of Hydrogen/Deuterium Exchange and Mass Spectrometry to Proteins -- 6.4 Introduction to Covalent Labeling Techniques -- 6.5 Overview and Applications of Hydroxyl Radical Footprinting to Mass Spectrometry -- 6.6 Overview and Applications of Chemical Cross-Linking to Mass Spectrometry -- 6.7 Overview and Applications of Specific Amino Acid Labeling to Mass Spectrometry -- 6.8 Conclusions -- References -- 7 Application of Biophysical And High-Throughput Methods in the Preformulation of Therapeutic Proteins-Facts and Fictions -- 7.1 Introduction -- 7.2 Considerations for a Successful Protein Drug Product -- 7.2.1 Formulation Composition -- 7.2.2 Testing under Different Stress Conditions -- 7.2.3 Primary Packaging/Container Closure -- 7.3 Protein Preformulation Strategies -- 7.3.1 Developability Assessment and Molecule Candidate Selection.

7.3.2 High-Throughput Formulation Development -- 7.3.3 Surrogate Parameters from Biophysical Studies during Formulation Screening -- 7.4 Conclusions -- References -- 8 Bioanalytical Methods and Immunogenicity Assays -- 8.1 Introduction -- 8.1.1 Biotherapeutic Modalities -- 8.1.2 Application of Bioanalytical and Immunogenicity Assays in Discovery and Development of Biotherapeutics: Stage-Specific Requirements -- 8.2 Assays to Assess PK -- 8.2.1 PK Assay Development Considerations -- 8.2.2 PK Assay Validation Considerations -- 8.2.3 PK Assay Life Cycle -- 8.3 Biomarker Assays -- 8.4 Assays for Detection and Prediction of Anti-drug Antibodies -- 8.4.1 Anti-drug Antibody Screening Assays -- 8.4.2 Neutralizing Antibody Assays -- 8.4.3 Assays for Immunogenicity Prediction -- 8.5 New Trends: Biosimilars, Biobetters, Antibody-Drug Conjugates -- 8.6 Conclusions -- References -- 9 Structures and Dynamics of Proteins Probed by UV Resonance Raman Spectroscopy -- 9.1 Introduction -- 9.1.1 Background and Historical Perspective -- 9.1.2 Resonance Raman Scattering -- 9.1.3 Secondary Structure -- 9.1.4 Aromatic Amino Acids -- 9.1.5 Considerations for UVRR -- 9.2 Experimental -- 9.2.1 Excitation Source -- 9.2.2 Sample Cell -- 9.2.3 Detection System -- 9.2.4 Other Considerations -- 9.3 Applications of UVRR Spectroscopy to Membrane-Associated Peptides -- 9.3.1 Model Peptides for Soluble and Membrane Protein Folding -- 9.3.2 Antimicrobial Peptides (AMPs) -- 9.3.3 Toxins -- 9.3.4 Engineered AMPs for Enhanced Efficacy -- 9.3.5 Fibril-Forming Peptides -- 9.4 Protein Conformational Changes -- 9.5 Challenges and Beneffiits of UVRR Spectroscopy -- 9.6 Conclusion -- Acknowledgments -- References -- 10 Freezing- and Drying-Induced Micro- and Nano-heterogeneity in Biological Solutions -- 10.1 Introduction -- 10.2 Freezing-Induced Heterogeneity -- 10.3 Drying-Induced Heterogeneity.

10.4 Methods of Detection -- 10.4.1 Conventional Microscopy -- 10.4.2 Electron Microscopy -- 10.4.3 Thermal Analysis -- 10.4.4 Spectroscopy -- 10.5 Summary -- Acknowledgments -- References -- Section 3 Phase III and Commercial Development -- 11 Late-Stage Product Characterization: Applications in Formulation, Process, and Manufacturing Development -- 11.1 Introduction -- 11.2 Strategies in Using Biophysical Methods in Late-Stage Development -- 11.2.1 Progression from Early- to Late-Stage Development -- 11.2.2 Protein Instability and Process/Manufacturing Unit Operations -- 11.3 Analytical Methods Applications Considerations -- 11.3.1 Spectroscopic Methods -- 11.3.2 Aggregates and Subvisible Particulate Analysis -- 11.3.3 Emerging Applications -- 11.4 Concluding Remarks -- 11.4.1 Accelerated Stability Studies Considerations -- 11.4.2 Conclusions -- References -- 12 Biophysical Analyses Suitable For Chemistry, Manufacturing, and Control Sections of the Biologic License Application (Bla) -- 12.1 Introduction -- 12.2 The Biophysical Tool Box -- 12.3 Common Biophysical Methods for Assessing Folded Structure -- 12.3.1 Circular Dichroism Spectroscopy -- 12.3.2 Vibrational Spectroscopy -- 12.3.3 X-Ray Crystallography -- 12.3.4 Nuclear Magnetic Resonance Spectroscopy -- 12.3.5 Differential Scanning Calorimetry -- 12.3.6 Fluorescence Spectroscopy -- 12.3.7 Ultraviolet Spectroscopy -- 12.4 Common Biophysical Methods for Assessing Size Heterogeneity, Association State, Aggregation -- 12.4.1 Analytical Ultracentrifugation -- 12.4.2 Light Scattering -- 12.5 Methods for Assessing Subvisible Particulates -- 12.5.1 Light Obscuration -- 12.5.2 Imaging Techniques -- 12.6 Evolving Biophysical Technologies -- 12.6.1 Asymmetric Flow Field-Flow Fractionation for Assessing Protein Association State.

12.6.2 Mass Spectrometric-Based Methodologies for Assessing Higher Order Structure.