Therapeutic Antibodies: Methods and Protocols

✍ Scribed by Gunnar Houen

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 347
• Series: Methods in Molecular Biology
• Edition: 1
• Category: Library

✦ Synopsis

This detailed book covers methods for studying, producing, and analyzing therapeutic antibodies, measuring their concentration, developing neutralizing antibodies for them, and for predicting and monitoring their therapeutic efficacy and clinical effects. These biologics are the fastest growing pharmaceutical drug group and have had tremendous clinical and scientific impact in cancer, autoimmune diseases, infectious diseases, and other immune-related diseases, making the content of this volume essential. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible methods, and tips on troubleshooting and avoiding known pitfalls. 

Authoritative and practical, Therapeutic Antibodies: Methods and Protocols serves as an ideal guide for researchers working with the production of, research on, and development of therapeutic antibodies as well as for clinicians using therapeutic antibodies in daily work with patients.

✦ Table of Contents

Dedication
Preface
Contents
Contributors
Chapter 1: Therapeutic Antibodies: An Overview
1 Introduction
2 Immunoglobulin (Ig) Therapy
2.1 Polyclonal Human Ig Therapy
2.2 Animal Ig Preparations
2.3 Monoclonal Antibody (MAb) Therapy
3 Therapeutic Antibodies
3.1 Humanized and Human Antibodies
3.2 Therapeutic Antibody Conjugates
3.3 Designed and Engineered Antibodies
4 Discussion
5 Notes
References
Chapter 2: Treatment of Autoimmune Diseases with Therapeutic Antibodies: Lessons Learned from PID Patients Allow for Stratific...
Abbreviations
1 Introduction
2 Cytokine Inhibitors
3 Anti-Integrin Antibodies
4 Complement Cascade Inhibition
5 Lymphocyte Depletion
6 Discussion
References
Chapter 3: B-Cell Analysis for Monitoring Patients Undergoing B-Cell Depletion for the Treatment of Autoimmune Diseases
Abbreviations
1 Introduction
2 Pre-analytical Phase
2.1 Preparation of Fluorescence-Labeled Monoclonal Antibody Mix
2.1.1 Materials and Equipment (See Note 1)
2.1.2 Methods
2.2 Instrument Settings of the Flow Cytometer
2.2.1 Materials
2.2.2 Methods
2.3 Collection of Blood
2.3.1 Materials
2.3.2 Methods
3 Analytical Phase
3.1 Single-Platform B-Cell Quantification
3.1.1 Materials and Equipment (See Note 1)
3.1.2 Methods
3.2 B-Cell Phenotyping
3.2.1 Materials and Equipment (See Note 1)
3.2.2 Methods
4 Post-analytical Phase
4.1 Quality Control
4.2 Interpretation by the Laboratory Specialist
5 Notes
6 Discussion and Conclusion
References
Chapter 4: Assessment of Therapeutic Antibody Developability by Combinations of In Vitro and In Silico Methods
1 Introduction
2 Requirements for Therapeutic Antibody Development
2.1 Expression and Purification
2.2 Sample Preparation and Transport
2.3 Storage and Administration
3 Biophysical and Biochemical Determinants of Developability
3.1 Chemical Stability
3.2 Conformational Stability and its Link to Aggregation
3.3 Solubility
3.4 Colloidal Stability and its Link to Self-Association, Aggregation, and Viscosity
3.5 Specificity
3.6 Immunogenicity
3.7 Biochemical and Biophysical Properties are Closely Interlinked
4 Experimental Measurements of Developability Potential
4.1 Biophysical Characterization Assays
4.1.1 Assays Probing Conformational Stability
4.1.2 Assays Probing Colloidal Stability
4.1.3 Assays Probing Aggregation and Aggregation Propensity
4.1.4 Assays Probing Solubility
4.1.5 Assays Probing Viscosity
4.2 Developability Screening Assays
4.2.1 High-Throughput, Low-Material Stability Measurements
4.2.2 Assays Probing for Nonspecific Interactions
4.2.3 Assays Probing Self-Association and Aggregation Propensity
4.3 Discussion on Developability Screening Assays
4.4 Microfluidics for Developability Assessment
5 In Silico Predictions of Developability
5.1 Machine Learning-Based Algorithms
5.2 Structure-Based and Sequence-Based Computational Methods
5.3 Structure-Based Predictions Using Structural Models
5.4 Empirical Algorithms for the Identification of Developability Liabilities
5.5 The CamSol Method of Predicting Solubility and Developability Potential
6 Conclusions: Incorporation of Silico Tools in Antibody Discovery Pipelines
References
Chapter 5: The Therapeutic Antibody Profiler for Computational Developability Assessment
1 Introduction
2 Materials
2.1 Web Requirements
2.2 Reimplementation
3 Methods
4 Notes
References
Chapter 6: Short Read-Length Next Generation DNA Sequencing of Antibody CDR Combinations from Phage Selection Outputs
1 Introduction
2 Materials
2.1 Phage Harvest and ssDNA Template Preparation
2.2 CDR Strip Generation and Amplification
2.3 Next Generation DNA Sequencing
2.4 CDR Strip Bioinformatics
3 Methods
3.1 Phage Harvest
3.2 ssDNA Isolation
3.3 CDR Strip Synthesis
3.3.1 CDR Strip ccc-dsDNA Synthesis
3.3.2 CDR Strip PCR Amplification
3.4 Next Generation Sequencing
3.5 Parsing CDR Sequences from NGS Data
3.6 Reconstruction of Desired Antibody Sequences
4 Notes
References
Chapter 7: Large-Scale Transient Production in ExpiCHO-S with Enhanced N-Galactosylation-Sialylation and PEI-Based Transfection
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Plasmid DNAs
2.3 Transfection
2.4 Titer Estimation and Quality Control for Antibody and Fc Fusions
3 Methods
3.1 Routine Cell Culture
3.2 Plasmid DNA Preparation
3.3 Transfection of ExpiCHO-S Cells with Enhanced N-Linked Galactosylation and Sialylation (Fig. 1)
3.4 Transfection of ExpiCHO-S Cells with PEImax (Fig. 2)
3.5 Titer Estimation and Quality Control for Antibody and Fc Fusions
4 Notes
References
Chapter 8: Production of Therapeutic Single-Chain Variable Fragments (ScFv) in Pichia pastoris
1 Introduction
2 Materials
2.1 Cloning and P. pastoris Transformation
2.2 Protein Expression
2.3 Purification
3 Methods
3.1 Cloning
3.2 Transformation of P. pastoris with the Expression Vector
3.3 Selection of Most Productive Clones
3.4 Study of Protein Expression
3.5 Protein Expression
3.5.1 Lab Scale Expression
3.5.2 Scale-up in a 5L-Bioreactor Expression
3.6 Protein Purification
4 Notes
References
Chapter 9: Purification of Therapeutic Antibodies by Protein A Affinity Chromatography
1 Introduction
1.1 Steps for Optimization of Protein A Affinity Chromatography Process
1.2 Steps Toward Establishing Protein A Affinity Chromatography Platform
2 Materials
2.1 Equipment and Consumables
2.2 Chemicals
2.3 Chromatography Column
2.4 Buffer Preparation for Chromatography
3 Methods
3.1 Sample Preparation
3.2 Purification Process for Protein A Affinity Chromatography
3.3 Aggregate Analysis
3.4 Quantification of HCP
3.5 Picogreen Assay for Quantification of DNA
4 Notes
References
Chapter 10: Ion Exchange Chromatographic Methods for Purification of Therapeutic Antibodies
1 Introduction
2 Materials
2.1 Sample
2.2 Chromatography System, Consumables and Reagents
2.3 Concentration Analysis
2.4 Aggregate Analysis
2.5 Charge Variant Analysis
3 Methods
3.1 Cation Exchange Chromatography
3.2 Charge Variant Analysis
3.3 Aggregate Analysis
3.4 Pooling of Fractions
4 Notes
References
Chapter 11: Structure-Indicated LC-MS/MS Bioanalysis of Therapeutic Antibodies
1 Introduction
2 Materials
2.1 Sample Processing for Fab-Selective Proteolysis of Monoclonal Antibodies
2.2 Preparation of Trypsin-Immobilized Nanoparticles
2.3 LC-MS/MS Analysis
3 Methods
3.1 Preparation of Control Serum or Plasma
3.2 Preparation of Trypsin-Immobilized Nanoparticles (See Note 3)
3.3 Confirmation of Signature Peptides for Therapeutic Antibodies
3.4 Verification of Selected Signature Peptides in Biological Matrix
3.5 LC and MS Parameters
3.5.1 Parameters for LC
3.5.2 Autosampler Setting for Decreasing the Carry-over
3.5.3 ESI Interface Parameters (Fig. 6)
3.5.4 MS Parameters
3.6 Multiple Sample Assay
4 Notes
References
Chapter 12: Use of PASEF for Accelerated Protein Sequence Confirmation and De Novo Sequencing with High Data Quality
1 Introduction
2 Materials
2.1 Materials for Proteolytic Digests and Proteins Used
2.1.1 Materials for Trypsin Digests
2.1.2 Materials for Elastase Digests
2.1.3 Materials for Chymotrypsin Digests
2.2 Instrumentation, Software and Labware
3 Methods
3.1 Sample Digestion and Preparation for LC
3.1.1 The Sample
3.1.2 Cysteine Reduction and Denaturation
3.1.3 Cysteine Alkylation
3.1.4 Protease Digestion
Trypsin Digestion
Elastase Digestion
Chymotrypsin Digestion
3.1.5 Determination of the Protein Concentration
3.2 Reversed Phase Chromatography
3.2.1 Materials
3.2.2 Method
3.3 Mass Spectrometry
3.4 Parameters: Data Processing and Analysis for Sequence Confirmation
3.5 Parameters: Data Processing and De Novo Sequence Analysis
3.6 Results from Sequence Confirmation of Nivolumab and Dulaglutide
3.6.1 Nivolumab
3.6.2 Dulaglutide
3.7 Results for De Novo Antibody Sequence Determination
4 Notes
References
Chapter 13: Nano-Microscopy of Therapeutic Antibody Aggregates in Solution
1 Introduction
2 Materials
2.1 Reagents, Analytical Instruments, and Software
3 Methods
3.1 Preparing Antibody Samples
3.2 Setting Antibody Samples into the SE-ADM System
3.3 Observation of Antibody Aggregates Using the SE-ADM System
3.4 Particle Size Analysis from SE-ADM Images Using the ImageJ Software
3.5 Fractal Dimension Calculation Using the Box-Counting Method
4 Notes
Appendix
References
Chapter 14: Measuring Self-Association of Antibody Lead Candidates with Dynamic Light Scattering
1 Introduction
1.1 mAb Self-Association
1.2 Importance of Viscosity in the Development of mAbs
1.3 Methods to Measure mAb Self-Association
1.4 Screening Self-Association Under Formulation and Process Conditions
2 Materials
3 Methods
3.1 Detailed Experimental Procedure of kD Measurements
3.2 Detailed Experimental Procedure of the pH Screen
3.3 Case Studies Highlighting the Application of the pH Screen
3.4 Conclusion
4 Notes
References
Chapter 15: Intact Mass Quantitation of Therapeutic Antibodies for Pharmacokinetic Studies Using Immuno-Purification
1 Introduction
2 Materials
2.1 Immunoaffinity (IA) Immobilization
2.2 IA Purification
2.3 LC-MS
3 Methods
3.1 IA Immobilization
3.2 IA Purification
3.3 LC-MS
3.4 Data Analysis and Quantitation
4 Notes
References
Chapter 16: Detection of Residual Host Cell Proteins in Biotherapeutic Drugs by Concatenated 2D LC-MS/MS
1 Introduction
2 Materials
2.1 Enzyme Digestion
2.2 First Dimensional Offline High-pH RPLC Fractionation and Concatenation
2.3 Second Dimensional Online Low-pH RP-UHPLC-MS/MS
3 Methods
3.1 Enzyme Digestion
3.2 First Dimensional Offline High-pH RPLC Fractionation and Concatenation
3.3 Second Dimensional Online Low-pH RP-UHPLC-MS/MS
3.4 Data Analysis
4 Notes
References
Chapter 17: Bioassays for the Evaluation of Target Neutralization and Complement-Dependent Cytotoxicity (CDC) of Therapeutic A...
1 Introduction
2 Materials
2.1 Evaluation of Target (sTNFα) Neutralization
2.2 Evaluation of CDC Induction
2.2.1 Transfection of HEK-293T Cells
2.2.2 CDC Assay
2.3 Equipment and Software
3 Methods
3.1 Assessment of Target (sTNFα) Neutralization
3.1.1 Standardization of the sTNFα Concentration for Induction of Cell Adhesion Molecules on Endothelial Cells
3.1.2 sTNFα Neutralization with Infliximab
3.2 Analysis of CDC Induction
3.2.1 Generation of mTNFα+ HEK-293T Target Cells
3.2.2 CDC Assay
4 Notes
References
Chapter 18: Methods for Functional Characterization of FcRn Interactions with Therapeutic Antibodies and Fc-Fusion Proteins
1 Introduction
2 Materials
2.1 Transcytosis Assay
2.2 ELISA
2.3 Lucifer Yellow Assay and Trans-Epithelial Electrical Resistance (TEER) Measurement
3 Methods
3.1 pH Gradient Transcytosis Assay
3.2 Neutral pH Transcytosis Assay
3.3 ELISA Method for Quantification of Transcytosis
4 Notes
References
Chapter 19: Method for Measurement of Antibody-Dependent Cellular Phagocytosis
1 Introduction
2 Materials
2.1 Macrophage Differentiation
2.2 Phagocytosis Assay
3 Methods
3.1 Monocyte Isolation and Macrophage Culture Procedure
3.2 ADCP Procedure
3.3 Flow Cytometry and Data Analysis
4 Notes
References
Chapter 20: Bioluminescent Bridging Immunoassay for Anti-Drug Antibody (ADA) Detection
1 Introduction
2 Materials
2.1 Reagents
2.2 Lab Equipment
3 Methods
3.1 Labeling Antibody with Biotin
3.2 Labeling Antibody with NanoLuc
3.2.1 Labeling Trastuzumab with HaloTag Ligand
3.2.2 Conjugating NanoLuc-HaloTag Protein to Antibody-HaloTag Ligand
3.3 NanoLuc Bridging Immunoassay in the Absence of Free Drug
3.4 Improving Drug Tolerance of NanoLuc Bridging Immunoassay
4 Notes
References
Chapter 21: A Surface Plasmon Resonance-Based Assay for Simultaneous Measurement of Serum Concentrations of Therapeutic Antibo...
1 Introduction
2 Materials
2.1 Instrumentation
2.2 Ligand Immobilization
2.3 Determination of the Serum Concentration of the Therapeutic Antibody and the Anti-drug Antibody
3 Methods
3.1 Ligand Immobilization on the Sensor Surface
3.1.1 Pre-concentration Assay
3.1.2 Ligands Immobilization
3.2 Determination of IFX and Anti-IFX Antibodies Concentration in Serum
3.2.1 Preparation of Samples Used for Calibration Curve Generation and Quality Controls
3.2.2 Startup Cycles
3.2.3 Sample Analysis
3.2.4 Concentration Calculation Based on Bioanalytical Assay
4 Notes
References
Index