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

12.6.3 Biophysical Functional Assays

12.6.4 Isothermal Calorimetry

12.6.5 Microscale Thermophoresis

12.7 Case Study for the Use of Biophysical Methods in the Elucidation of Structure Section of the License Application

12.8 Case Study for the use of Biophysical Methods in the Comparability Section of the License Application

12.9 Case Study for the use of Biophysical Methods in the Pharmaceutical Development Section of the License Application

12.10 Biophysical Methods for Evaluating Protein—Surface/Device Interaction

12.11 Implication for Biosimilars

12.12 Concluding Remarks

Acknowledgments

References

Index

Supplemental Images