Characterizing Biotherapeutics E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Provides detailed and up-to-date coverage of analytical approaches for biologic drug modalities
The development of innovative biologic therapies has revolutionized medicine, but their complexity demands equally advanced analytical approaches. Characterizing Biotherapeutics: Analytical Methods for Diverse Modalities introduces the tools and techniques used to analyze these groundbreaking therapies. Designed to help readers characterize and troubleshoot increasingly diverse and sophisticated therapeutic molecules, this in-depth guide addresses different biologic drugs, their unique analytical challenges, and the methods that enable their analysis.
Organized into two comprehensive sections, Characterizing Biotherapeutics first delves into the fundamentals of analytical platforms, providing a robust foundation in techniques such as mass spectrometry and biophysical assays. The second section applies these methods to real-world scenarios, focusing on drug discovery, clinical evaluation, and commercial considerations in drug development. Authors Jennie R. Lill and Wendy Sandoval provide clear guidance tailored to the evolving demands of therapeutic innovations such as structural characterization, high-throughput biophysical assays, and RNA-based therapeutics.
Equipping researchers with the knowledge to navigate the challenges posed by increasingly complex biologics, Characterizing Biotherapeutics: Analytical Methods for Diverse Modalities:
- Discusses new and emerging biological molecule diversity and both traditional analytical methods
- Provides characterization strategies for large molecule (protein/antibody) based therapeutics
- Summarizes cutting-edge technologies for novel modalities and antibody protein therapeutics
- Incorporates the latest advancements in mass spectrometry, functional assays, and biophysical methods
- Addresses specialized analytical issues for membrane proteins and other challenging targets
- Presents methodologies for assessing safety and therapeutic potential
Characterizing Biotherapeutics: Analytical Methods for Diverse Modalities is an essential reference for analytical scientists, biologists, and mass spectrometrists involved in biomolecule analysis. It is also a valuable resource for graduate students taking advanced courses in biotechnology, drug development, and biotherapeutic analysis, as well as professionals in biotechnology and pharmaceutical industries working to advance biotherapeutic research and development.
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Table of Contents
Cover
Table of Contents
Title Page
Copyright
About the Editors
List of Contributors
Preface
1 Introduction
Exploring a Diversity of Biotherapeutic Approaches
Delivering Diverse Biotherapeutics to Expand Patient Treatment
Unlocking Innovation: The Role of Biophysical Techniques in Drug Discovery and Development
Increasing Throughput: The Role of High‐Quality Analytical Assays in Characterizing Biotherapeutics
The Next Frontier: Additional Bioanalytical Tools and Targets on the Horizon
Artificial Intelligence and Machine Learning: A Transformative Shift for Computational Tools
A Bioanalytical Call to Action!
References
2 Analytical Characterization of Bispecific Antibodies and Bispecific Molecules
Introduction
Bispecific Antibodies and Alternative Scaffolds with Tethered Domains
Identifying the Best Parental Antibody Pairs to Combine in a BsAb
Alternatives to Bispecific Antibodies: Antibody Mixtures
Characterization of the Bispecific Molecule
Characterization by Mass Spectrometry Methods
Conclusions
Abbreviations
References
3 Analytical Challenges in Analyzing IgM Biotherapeutics
Introduction
Characterization of Biotherapeutic mAbs
IgM Sequence and Structure
IgM Assembly Factors
IgM Glycosylation Patterns
IgM Disulfide Bond Networks
Conclusions
Acknowledgments
References
4 Mass Spectrometric Characterization of Therapeutic Recombinant Proteins
Introduction
Instrumentation
Software for the Analysis of Intact Molecular Weight Measurements
Emerging Mass Spectrometric Approaches for Characterizing Molecular Heterogeneity in Biotherapeutics
Tandem Mass Spectrometric Characterization of Biomolecules
Data Acquisition Methods
Quantitative Mass Spectrometric Methods
Computational Analysis of Tandem Mass Spectra
Conclusions and Perspectives
Abbreviations
References
5 Therapeutic Proteins and Hyphenated Methods for Characterization
Introduction
Methods
Considerations
Quality Control Feasibility
Conclusions
References
6 Methods for Characterization and Optimization of Peptide Therapeutics: Bioanalysis and Biotransformation
Introduction
Acknowledgment
References
Note
7 Structural Characterization of Biotherapeutics and New Modalities
Introduction
Antigens, Epitopes, and Paratopes
Recombinant Antigen Generation
N‐Linked Glycosylation
Manipulating N‐Linked Glycans on Antigens
Antibody Generation for Crystallography
Crystallization of Antibody/Antigen Complexes
Consideration for New Modalities and New Targets
Conclusion
Abbreviations
References
8 Modern Methods for Characterizing the Higher‐Order Structure (HOS) of Biotherapeutics
Protein Higher‐Order Structure (HOS)
Importance of HOS Characterization in Biotherapeutic Development
Hydrogen/Deuterium Exchange Mass Spectrometry (H/DX‐MS)
Hydroxyl Radical Protein Footprinting Mass Spectrometry (HRPF‐MS)
Covalent Labeling Mass Spectrometry (CL‐MS)
X‐Ray Crystallography
Cryogenic Electron Microscopy (Cryo‐EM)
Multiscale
in‐silico
Modeling of Biotherapeutics
Conclusion
References
9 Target Binding Assays for Biotherapeutics
Introduction
Assays to Determine Affinity of Biotherapeutics
Conclusions
References
10 Functional Assays for Screening Protein Biotherapeutics
Introduction
Development of a Screening Cascade
Biochemical Assays for Cellular Receptor‐Binding or Extracellular Targets
Cellular Assays for a Cell Signaling Readout
Fundamentals of Assay Development, Validation, and Implementation
Concluding Remarks
References
11 Immunogenicity Profiling of Biotherapeutics (Wiley Book – Bioanalytical Characterization of New Therapeutic Modalities)
Introduction
The Anti‐drug Antibody Response
Clinical Monitoring of ADAs
ADA Assay Development Challenges and Considerations for New Modalities
Integrating Immunogenicity Assessments, Harmonization, and Incorporation of Clinically Relevant Information into Drug Product Labeling
The Clinical Impact of an ADA
Assays Employed to Assess Immunogenicity Risk
In Silico
Approaches for Immunogenicity Risk Assessment
T‐Cell and Other Cellular Assays
MHC II Immunopeptidome
Data Visualization and Curation
Engineering Best Practices Post‐immunogenicity Hotspot Data Elucidation
Conclusion
Abbreviations
References
Note
12 High‐Throughput Biophysical Assays for Developability Assessment
Introduction
High‐Throughput Intact Mass Spectrometry
Aggregation
Size Variants
Hydrophobicity
Charge Variants
Viscosity
Conformational Stability
Chemical Stability Assessment
Leveraging AI/ML for Molecule Assessment
Future of Developability Assessments
References
Note
13 Establishing Analytical Methods for RNA‐Based Therapeutics Characterization
Introduction to RNA‐Based Therapeutics
Applications of RNA‐Based Drugs
Quality Attributes and Techniques for the Characterization of mRNA
Conclusion
References
14 Analytical Methods Shaping Cell Therapy Characterization
Introduction
Fundamentals of T‐Cell Immunology in Cell Therapy
Measuring Immune Receptor Expression
Functional Cell‐Based Assays for
In Vitro
Characterization
Cell Phenotyping
Immune Monitoring: BCR and TCR Sequencing
Conclusions
Acknowledgment
References
15 Characterizing Nucleic Acid‐, Gene‐, and Cell‐Based Therapeutics with Sequencing Technologies
Introduction
DNA Sequencing
Nucleic‐Acid‐Based Therapeutics
Gene‐Based Therapies
Cell‐Based Therapies
Immune Repertoire Sequencing Facilitates Therapeutic Advances
Conclusions and Future Perspectives
Abbreviations
References
16 Mass Spectrometry‐Based Methods to Investigate Protein–Protein Interactions in Cells and Their Application in Drug Discovery
Introduction
Affinity Purification Mass Spectrometry (AP‐MS)
Cross‐Linking Mass Spectrometry (XL‐MS)
Proximity Labeling Mass Spectrometry (PL‐MS)
Concluding Remarks and Other Interactomic Technologies
Abbreviations
References
17 Receptomics and Extracellular Interactomics for Biotherapeutic Characterization
Introduction
Receptomic and Extracellular Interactomics Technology Overview
Natural Products and Peptides
Antibodies and Related Proteins
Lipid Nanoparticles (LNPs), Extracellular Vesicles (EVs), Virus‐Like Particles (VLPs) and Viruses
Cell Therapies
Summary
Abbreviations
References
18 Membrane Proteins: Challenging Biotherapeutic Targets
Membrane Proteins: Challenging Biotherapeutic Targets Families of Membrane Proteins
Membrane Protein Solubilization Methods
MS Analysis of Membrane Proteins
Conclusion
Acknowledgments
References
19 Chemoproteomics Approaches for Diverse Modalities
Introduction
Chemoproteomics Approaches
Probe‐Based (Direct) Target Deconvolution
Target Deconvolution Without Chemical Probes
Proximity‐Labeling Approaches
Mechanism‐Centric (Indirect) Proteomics Methods
Covalent Drugs
Protein Degraders
Antibody–Drug Conjugates and Oligonucleotides
Concluding Remarks
Acknowledgments
Abbreviations
References
20 Drug Delivery Modalities and Mechanisms
Introduction
Delivery of Proteins
Delivering Genes to the Immune System: Enabling Cell‐Specific Targeting
Tissue‐Specific Delivery: Targeting the Brain Parenchyma
An Analytical Perspective to Drug‐Delivery Technology Development
Conclusions/Future Perspectives
Abbreviations
References
Index
End User License Agreement
List of Tables
Chapter 2
Table 2.1 Techniques to purify and characterize bispecific antibodies.
Chapter 3
Table 3.1 Comparison of IgG1 mAbs with various IgM assemblies to highlight d...
Chapter 5
Table 5.1 Native ESI‐UV/MS identification results of CrossMAb size variants ...
Table 5.2 Difference capabilities between ACE‐MS and AC‐MS workflows.
Chapter 6
Table 6.1 Recommended controls for
in vitro
permeability assays.
Table 6.2 Pharmacokinetic and pharmacokinetic–pharmacodynamic characteristic...
Chapter 8
Table 8.1 Complementarity of structural methods for characterizing protein h...
Table 8.2 Experimental components of HRPF‐MS.
Chapter 9
Table 9.1 Common technologies to determine affinity of biotherapeutics to th...
Chapter 10
Table 10.1 Controls for assay normalization.
Chapter 11
Table 11.1 Strategies to de‐risk the immunogenic potential of biotherapeutic...
Chapter 14
Table 14.1 How are MHC class I tetramers generated?
Table 14.2
In vitro
characterization assays to measure cell function.
Chapter 18
Table 18.1 List of FDA‐approved mAbs for membrane proteins.
Chapter 20
Table 20.1 Various clinically approved PEGylated protein therapeutics.
Table 20.2 Various clinically approved lipidated peptide therapeutics.
Table 20.3 Various clinically approved products of PLGA‐based LAD systems fo...
Table 20.4 Preclinical studies with successful
in situ
delivery of CAR‐T the...
Table 20.5 Strategies for DC‐targeted LNPs.
Table 20.6 Clinical trials with peptides and proteins being administered IN ...
Table 20.7 Preclinical studies demonstrated efficacy or distribution to the ...
Table 20.8 Biological compositions in humans for different parenteral sites ...
List of Illustrations
Chapter 2
Figure 2.1 Bispecific antibody modes of action. (a) Retarget cytotoxic T‐ or...
Figure 2.2 The bispecific antibody light‐ and heavy‐chain pairing problems. ...
Figure 2.3 Examples of bispecific antibodies are classified by their mode of...
Figure 2.4 Production technologies of bispecific antibody formats and their ...
Chapter 3
Figure 3.1 Schematic diagram of IgM monomer with domains, disulfide bonds, a...
Figure 3.2 Sequence alignment of the heavy chain constant regions of human i...
Figure 3.3 Schematic models of IgM pentameric assemblies formed endogenously...
Figure 3.4 Relative abundances of glycan types and features determined from ...
Figure 3.5 Site‐specific glycan composition of recombinant IgM pentamers and...
Figure 3.6 MS‐based identification of the grass carp IgM tailpiece cysteine ...
Chapter 4
Figure 4.1
MALDI ionization
. The uncharged analyte is mixed with an uncharge...
Figure 4.2
Electrospray ionization
. Schematic of the path of the analyte fro...
Figure 4.3
Quadrupole time‐of‐flight (Q‐TOF) mass spectrometer
...
Figure 4.4
Native MS analysis of an RGY‐antibody hexamer
. Native mass ...
Figure 4.5
Conventional drift‐tube ion mobility spectrometry (DT‐IMS)
...
Figure 4.6
High‐performance liquid chromatography (HPLC)
. Analytes are...
Figure 4.7
Capillary electrophoretic system for mass spectrometry
. The analy...
Figure 4.8
Top‐down and bottom‐up mass spectrometry‐based analysis of an ant
...
Figure 4.9
Peptide fragment MS/MS ion series
. If the charge is retained on t...
Chapter 5
Figure 5.1 Bispecific antibody proteoforms and product variants analyzed by ...
Figure 5.2 Comparison of fast‐SEC with UV detection (a) versus native ESI‐UV...
Figure 5.3 Affinity separation using ACE and AC is illustrated by FcγRIIIa a...
Chapter 6
Figure 6.1 The structures of cyclosporine A, ocreotide, and monomethyl auris...
Figure 6.2 Stability of Octreotide in buffers containing different bile salt...
Figure 6.3 PAMPA permeability correlation with different lipophilicity descr...
Figure 6.4 Permeability versus (a) EPSA, (b) Δlog
K
W
IAM
, (c) Δlog
P
oct–tol
...
Figure 6.5 The identification of significant ADME liabilities during drug di...
Figure 6.6 Schematic of a bidirectional Transwell permeability assay with th...
Figure 6.7 Cells expressing HaloTag are pulsed with chloroalkane‐tagged mole...
Figure 6.8 1. HeLa cells are transiently transfected with a NanoLuc‐HaloTag ...
Figure 6.9 Tissues and matrices considered for
in vitro
metabolic stability ...
Figure 6.10 The mechanism of deamidation and isomerization begins with a nuc...
Chapter 7
Figure 7.1 Perjeta (red/yellow) and Herceptin (blue/green) bound to the EGF ...
Figure 7.2 A stalk epitope on hemagglutinin (HA) that neutralizes most strai...
Figure 7.3 Comparison of antibody epitopes on VEGF. Surface representation s...
Figure 7.4 2D [
1
H–
15
N] HSQC spectrum of uniformly
15
N‐
13
C‐labeled and 50% de...
Figure 7.5 Epitope mapping by NMR. (a) Superposition of 2D
1
H‐
15
N HSQC spect...
Chapter 8
Figure 8.1 Schematic representation of protein structural levels, illustrati...
Figure 8.2 A general workflow of hydrogen/deuterium exchange mass spectromet...
Figure 8.3 The whole workflow of HRPF‐MS. Hydroxyl radicals can be generated...
Figure 8.4 Schematic workflow for DEPC‐based covalent labeling mass spectrom...
Figure 8.5 X‐ray crystallographic structure determination workflow.
Figure 8.6 The binding epitope of VEGF to the humanized anti‐VEGF antibody i...
Figure 8.7 General SPA Workflow: 1 – Characterized and highly purified prote...
Figure 8.8 A panel of Fabs against the SARS‐CoV‐2 spike domain was character...
Figure 8.9 The primary levels of resolution in biomolecular simulations (qua...
Chapter 9
Figure 9.1 A biotherapeutic protein binding to its target on the surface of ...
Figure 9.2 Examples of binding affinity of therapeutics to their targets det...
Figure 9.3 Simulated sensorgrams of experiments performed using (a) multi‐cy...
Figure 9.4 Representative SPR sensorgram of Obinutuzumab binding to CD20 exp...
Figure 9.5 Schematic of (a) saturation and (b) competition cell‐based equili...
Figure 9.6 Principles of kinetic exclusion assay to determine binding affini...
Figure 9.7 Pre‐equilibrium cell‐based assays. (a) An example of a real‐time ...
Chapter 10
Figure 10.1 Screening cascade to find the lead antibody candidates. This gen...
Figure 10.2 Example of screening cascade tier 2 and tier 3 assays for a bisp...
Figure 10.3 Example of screening cascade tier 2 and tier 3 assays for a prot...
Figure 10.4 Application and analysis of assay controls to assess assay unifo...
Chapter 11
Figure 11.1 Overview of the key steps in the generation of a T‐cell‐dependen...
Figure 11.2 PBMCs from healthy donors were stimulated with testing molecules...
Figure 11.3
In vitro
BrdU‐based T‐cell proliferation assay for immunogenicit...
Figure 11.4 The DC:CD4+ T‐cell restimulation assay. (a) Experimental setup o...
Figure 11.5 Peptide binding properties of MHC II molecules. Left panel shows...
Figure 11.6 The isolation and differentiation of monocytes from PBMCs into i...
Chapter 12
Figure 12.1 Workflow for incorporation of high‐throughput biophysical assays...
Figure 12.2 Application of RF‐MS for the identification of mispaired bispeci...
Figure 12.3 Overview of biophysical assays to monitor aggregation in biother...
Figure 12.4 High‐throughput MZE separation of (a) mAb A and (b) mAb C before...
Figure 12.5 Improved correlation between k
D
and viscosity with the addition ...
Figure 12.6 The MMS weighted spectral difference (WSD) across the measured c...
Figure 12.7 An overview of an end‐to‐end MAM set up that includes liquid han...
Figure 12.8 Schematic overview of the antibody discovery, engineering and ma...
Chapter 13
Figure 13.1 Applications of RNA‐based drugs including therapeutic protein pr...
Figure 13.2 Structure and function of messenger RNA.
Figure 13.3
In vitro
transcription (IVT) mRNA manufacturing process.
Figure 13.4 List of quality attributes and associated techniques for mRNA.
Figure 13.5 RNAse T1 Oligonucleotide mapping LC‐MS/MS profile of the SARS‐Co...
Figure 13.6 Importance of using orthogonal methods to assess mRNA. Similar r...
Figure 13.7 Overview of sequencing applications for mRNA vaccine QC using Il...
Figure 13.8 Analytical HPLC‐based methods for measurement of mRNA poly(A) ta...
Chapter 14
Figure 14.1 T‐cell activation by antigen presenting cells. The image outline...
Figure 14.2 T‐cell differentiation subsets and associated cell surface marke...
Chapter 15
Figure 15.1 Sequencing technologies. (a) Sanger sequencing uses a mix of reg...
Figure 15.2 Sequencing methods for nucleic acid‐based therapeutic developmen...
Figure 15.3 CRISPR edits with Cas9. A guide RNA (gRNA) is directed against a...
Figure 15.4 Immune repertoire sequencing. (a) Schematic of VDJ recombination...
Chapter 16
Figure 16.1 Affinity purification methods for AP‐MS. (a) Common immunoprecip...
Figure 16.2 Cross‐linking methods for XL‐MS. (a) Cross‐linking reagents, gen...
Figure 16.3 Proximity labeling methods for PL‐MS. (a) Proximity‐labeling rea...
Figure 16.4 Other interactomic methods. (a) In Virotrap, a bait protein of i...
Chapter 17
Figure 17.1 Schematic of biotherapeutics.
Figure 17.2 Biochemical fractionation and affinity purification both start w...
Figure 17.3 Tagging and format requirements differentiates the different app...
Chapter 18
Figure 18.1 Examples of Fab binding to different membrane protein types: (a)...
Figure 18.2 Illustration of different approaches used to solubilize membrane...
Figure 18.3 (a) Structures of detergents with the different HLB and packing ...
Figure 18.4 Synthetic high‐density lipoprotein (sHDL) nanodiscs made using 2...
Figure 18.5 Workflow for in‐cell labeling of mTNFα. mTNFα expressed in HEK29...
Figure 18.6 Mitochondria isolated from mouse heart tissue were treated with ...
Chapter 19
Figure 19.1 | Emerging therapeutic modalities and pharmacological concepts i...
Figure 19.2 Schematic overview of chemoproteomics principles to characterize...
Figure 19.3 Chemoproteomics method portfolio to characterize activity and se...
Figure 19.4 Distinguishing on‐target, off‐target and downstream effects of p...
Figure 19.5 Types of targeted protein degradation technologies. (a) Differen...
Chapter 20
Figure 20.1 (a) Common parenteral routes of administration for proteins, pep...
Figure 20.2 Various examples of clinically approved nonmodifying polymeric L...
Figure 20.3 Chemical structures of poly(lactic acid) (PLA), poly(lactic‐co‐g...
Figure 20.4 Therapeutic release mechanisms of PLGA by (a) diffusion through ...
Figure 20.5 Chemical structures of poly(ethylene‐vinyl acetate), poly(uretha...
Figure 20.6 Examples of reservoir‐based devices.
Figure 20.7 The PDS ocular implant, ancillary devices, and position
in situ
....
Figure 20.8 Zero‐ and first‐order drug release kinetics as a function of tim...
Figure 20.9
In vitro
drug release of ranibizumab from the Port Delivery Syst...
Figure 20.10 (a) Targeted LNPs can be leveraged for immune‐cell‐specific del...
Figure 20.11 General workflow for approved CAR‐T therapies. Blood is taken f...
Figure 20.12 Schematic representation of select strategies used for delivery...
Figure 20.13 Analytical toolbox with traditional and drug‐delivery‐specific ...
Figure 20.14 Considerations for characterization of a controlled‐release dru...
Guide
Cover
Table of Contents
Title Page
Copyright
About the Editors
List of Contributors
Preface
Begin Reading
Index
End User License Agreement
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Characterizing Biotherapeutics
Analytical Methods for Diverse Modalities
Edited by
Jennie R. LillProteomic and Genomic TechnologiesGenentech IncSouth San Francisco, CA
Wendy SandovalBotanicaSebastopol, CA, United States
Copyright © 2025 by John Wiley & Sons, Inc. All rights reserved, including rights for text and data mining and training of artificial technologies or similar technologies.
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About the Editors
Jennie R. Lill has been a leader in the biophysical space, enabling Drug Discovery and Development for over 30 years. She currently leads various teams in deciphering new drug targets and characterizing a broad range of therapeutic modalities. Jennie has been at Genentech for the past 21 years, leading their Genomic and Proteomic initiatives. Jennie has two sons, Joe and Charlie, who inspire her to do her best every day!
Wendy Sandoval worked for over 30 years in Research at Genentech where she led the Translational Mass Spectrometry team and focused on native mass spectrometric methods to characterize complex biotherapeutics. She has coauthored over 100 manuscripts and 2 books. She currently owns and runs Botanica (https://botanicanurseryandgardens.com/) in Sebastopol, CA, a public botanical garden, and takes her meetings under a massive Dawn Redwood canopy. Her mother, Ame, and daughters, Nicolina and Olivia, assist in managing the 7.5 acre oasis.
List of Contributors
Corey E. Bakalarski
Proteomic and Genomic Technologies
Genentech Inc.
South San Francisco, CA
USA
Mercedesz Balasz
Biochemical and Cellular Pharmacology
Genentech
South San Francisco
CA, USA
Marcus Bantscheff
Target Discovery, GSK
Heidelberg
Germany
and
Research and Early Development
Roche Pharma
Basel
Switzerland
Hayley M. Bennett
Department of Proteomic and Genomic Technologies
Genentech Research and Early Development
South San Francisco, CA
USA
Maureen Beresini
Department of Biochemical and Cellular Pharmacology
Genentech Inc.
South San Francisco, CA
USA
Tess Branon
Proteomic & Genomic Technologies Department
Genentech Inc.
South San Francisco, CA
USA
Ashley Byrne
Department of Proteomic and Genomic Technologies
Genentech Research and Early Development
South San Francisco, CA
USA
Julien Camperi
Cell Therapy Engineering and Development
Genentech
South San Francisco, CA
USA
Shengya Cao
Development
Inceptive
Palo Alto, CA
USA
Naincy R. Chandan
Discovery Technology
Nurix Therapeutics, Inc.
San Francisco, CA
USA
Jieming Chen
gRED Computational Sciences
Genentech
South San Francisco, CA
USA
Sivan Cohen
Bioanalytical Sciences
Genentech, Inc.
South San Francisco, CA
USA
Phillip Chu
Genentech, Inc.
South San Francisco, CA
USA
Michael Dillon
Department of Antibody Engineering
Genentech Research and Early Development
South San Francisco, CA
USA
H. Christian Eberl
Target Discovery, GSK
Heidelberg
Germany
Alberto Estevez
Thomson Instrument Company
Carlsbad, CA
USA
Amin Famili
Department of Synthetic Molecule Analytical Chemistry
Genentech
South San Francisco, CA
USA
Abel Ferrel
Independent Researcher
San Francisco, CA
USA
Emily Freund
Department of Molecular Biology
Genentech
South San Francisco, CA
USA
Manasi Gaikwad
Center for Mass Spectrometry and Optical Spectroscopy (CeMOS)
Mannheim University of Applied Sciences
Mannheim
Germany
Carolina Galan
Department of Molecular Biology
Genentech
South San Francisco, CA
USA
Amy Gilbert
Adaptive Biotechnologies
Seattle, WA
USA
Alissa D. Guarnaccia
Department of Proteomic and Genomic Technologies
Genentech
South San Francisco, CA
USA
and
Proteomic and Genomic Technologies
Genentech Inc.
South San Francisco, CA
USA
Axel Guilbaud
Protein Analytical Chemistry
Genentech
South San Francisco, CA
USA
Albert J.R. Heck
Biomolecular Mass Spectrometry and Proteomics
Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences
University of Utrecht
Utrecht
The Netherlands
and
Netherlands Proteomics Center
Utrecht
The Netherlands
Suk‐Joon Hyung
Genentech, Inc.
South San Francisco, CA
USA
Saeed Izadi
Pharmaceutical Development
Genentech Inc.
South San Francisco, CA
USA
Eric Janezic
Biochemical and Cellular Pharmacology
Genentech Inc.
South San Francisco, CA
USA
Christine C. Jao
Department of Structural Biology
Genentech Inc.
South San Francisco, CA
USA
Hiruni S. Jayasekera
Department of Chemistry and Biochemistry
The University of Arizona
Tucson, AZ
USA
Robert S. Jones
Drug Metabolism and Pharmokinetics
Genentech, Inc.
South San Francisco, CA
USA
S. Cyrus Khojasteh
Genentech, Inc.
South San Francisco, CA
USA
Sascha Knecht
Target Discovery, GSK
Heidelberg
Germany
Felix Kuhne
Analytical Characterization, Pharma Technical Development
Roche Diagnostics GmbH
Penzberg
Germany
M. Violet Lee
Bioanalytical Sciences
Genentech
South San Francisco, CA
USA
Paola Di Lello
Department of Structural Biology
Genentech, Inc.
South San Francisco, CA
USA
Dennis H. Leung
Genentech, Inc.
South San Francisco, CA
USA
Amritha Lewis
Adaptive Biotechnologies
Seattle, WA
USA
Yinyin Li
Biochemical and Cellular Pharmacology
Genentech
South San Francisco, CA
USA
Jennie R. Lill
Department of Proteomic and Genomic Technologies
Genentech
South San Francisco, CA
USA
and
Proteomic and Genomic Technologies
Genentech Inc.
South San Francisco, CA
USA
and
Proteomic and Genomic Technologies
Genentech
South San Francisco, CA
USA
Rachel P. Liu
Protein Analytical Chemistry
Genentech Inc.
South San Francisco, CA
USA
T. Noelle Lombana
Department of Antibody Engineering
Genentech Research and Early Development
South San Francisco, CA
USA
and
ReCode Therapeutics
Menlo Park, CA
USA
Kelly Loyet
Department of Biochemical and Cellular Pharmacology
Genentech Inc.
South San Francisco, CA
USA
Patrick Lupardus
Department of Structural Biology
Genentech, Inc.
South San Francisco, CA
USA
Bin Ma
Genentech, Inc.
South San Francisco, CA
USA
Michael T. Marty
Department of Chemistry and Biochemistry
The University of Arizona
Tucson, AZ
USA
Rebecca Melmon
Adaptive Biotechnologies
Seattle, WA
USA
Madyson Migliozzi
Department of Pharmaceutical Development
Genentech
South San Francisco, CA
USA
Zora Modrusan
Department of Proteomic and Genomic Technologies
Genentech Research and Early Development
South San Francisco, CA
USA
Farhana Afrin Mohona
Department of Chemistry and Biochemistry
The University of Arizona
Tucson, AZ
USA
Huy Nguyen
Genentech, Inc.
South San Francisco, CA
USA
Emma Pelegri‐O'Day
Amgen Research, Amgen Inc.
South San Francisco, CA
USA
Qui T. Phung
Proteomic and Genomic Technologies
Genentech
South San Francisco, CA
USA
Emile Plise
Genentech, Inc.
South San Francisco, CA
USA
Markus A. Queisser
Modality Platform Technologies, GSK
Medicines Research Centre
Stevenage
UK
Roshan M. Regy
Pharmaceutical Development
Genentech Inc.
South San Francisco, CA
USA
Amber D. Rolland
Biomolecular Mass Spectrometry and Proteomics
Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences
University of Utrecht
Utrecht
The Netherlands
and
Netherlands Proteomics Center
Utrecht
The Netherlands
Ola M. Saad
Genentech, Inc.
South San Francisco, CA
USA
and
Bioanalytical Sciences
Genentech
South San Francisco, CA
USA
Sudeshna Sadhu
Adaptive Biotechnologies
Seattle, WA
USA
Wendy Sandoval
Department of Proteomic and Genomic Technologies
Genentech
South San Francisco, CA
USA
and
Proteomic and Genomic Technologies
Genentech Inc.
South San Francisco, CA
USA
Rahamthulla Shaik
Adaptive Biotechnologies
Seattle, WA
USA
Whitney Shatz‐Binder
Department of Pharmaceutical Development
Genentech
South San Francisco, CA
USA
Christoph Spiess
Department of Antibody Engineering
Genentech Research and Early Development
South San Francisco, CA
USA
Alavattam Sreedhara
Department of Pharmaceutical Development
Genentech
South San Francisco, CA
USA
Nithya Srinivasan
Amgen Research
Amgen Inc.
Thousand Oaks, CA
USA
William Stephenson
Department of Proteomic and Genomic Technologies
Genentech Research and Early Development
South San Francisco, CA
USA
Yu Tong Tam
Department of Pharmaceutical Development
Genentech
South San Francisco, CA
USA
Devin B. Tesar
Department of Pharmaceutical Development
Genentech
South San Francisco, CA
USA
John C. Tran
Genentech, Inc.
South San Francisco, CA
USA
Egemen Tutuncuoglu
Adaptive Biotechnologies
Seattle, WA
USA
Lingfei Wang
Protein Analytical Chemistry
Genentech Inc.
South San Francisco, CA
USA
Xiangdan Wang
Bioanalytical Sciences
Genentech Inc.
South San Francisco, CA
USA
Aaron T. Wecksler
Protein Analytical Chemistry
Genentech Inc.
South San Francisco, CA
USA
and
Department of Protein Analytical Chemistry
Genentech
South San Francisco, CA
USA
Sara Wichner
Department of Protein Chemistry
Genentech
South San Francisco, CA
USA
Zhaojun Yin
Bioanalytical Sciences
Genentech, Inc.
South San Francisco, CA
USA
Qinying Yu
Genentech, Inc.
South San Francisco, CA
USA
Xing Zhang
Genentech, Inc.
South San Francisco, CA
USA
Zhenru Zhou
Proteomic and Genomic Technologies
Genentech
South San Francisco, CA
USA
Preface
The focus of this book is to introduce and describe analytical approaches for biologic drug modalities. The intervention of diseases once deemed an unsurmountable task is now becoming a reality due to recent breakthroughs in our understanding of human biology, therapeutic intervention approaches, and drug delivery mechanisms. Such transformative medicines require sophisticated analytical tools to identify new pathways for intervention, profile the therapeutic moieties, discover biomarkers, and monitor pharmacokinetic and dynamic profiles after patient administration. Developing these analytical approaches and often the software or algorithms associated with them requires the heroic collaboration of diverse disciplines, working together to develop and refine these custom tools that can transform medicine. Biologics, or biotherapeutics, are medicines made up of biological material and are often large and complicated molecules, sometimes heterogeneous mixtures. Although sub‐microscopic, biologics are much larger than typical chemical drug molecules, presenting particular challenges for characterization, production, and stability. Biologic drug modalities encompass a wide swath of molecules, such as bispecific antibodies, IgM‐based antibodies, peptides, RNA, and cell and gene therapies, each with unique challenges that require carefully designed analytical strategies. In this book, we discuss a variety of biologic drugs, their unique analytical challenges, and the methods that enable their analysis. By delving into these advancements, we aim to shed light on how innovative bioanalytical tools are pivotal in transforming scientific discoveries into effective, safe, and accessible treatments for patients worldwide.
May 2025
Jennie R. Lill
Wendy Sandoval
1Introduction
Alissa D. Guarnaccia, Wendy Sandoval, and Jennie R. Lill
Department of Proteomic and Genomic Technologies, Genentech, South San Francisco, CA, USA
Exploring a Diversity of Biotherapeutic Approaches
The focus of this book is to introduce and describe analytical approaches for biologic drug modalities. Biologics, or biotherapeutics, are the types of medicines that are made up of biological material. Biologics are often large and complicated molecules and sometimes can be heterogeneous mixtures. Although still on the submicroscopic scale, biologics are much larger than typical chemical drug molecules, and the larger size of biologics presents a particular challenge for characterization, production, and stability. Biologic drug modalities encompass a wide swath of molecules; bispecific antibodies, IgM‐based antibodies, peptides, RNA, and cell and gene therapies all have unique challenges that require carefully designed analytical strategies to bring them from the bench to the patient. In this book, we discuss a variety of biologic drugs, their unique analytical challenges, and the methods that enable their analysis.
Antibody‐Based Therapies
Antibody‐based therapies, such as bispecific antibodies and IgM‐based therapies, are powerful, specific, and often durable treatments. Bispecific antibodies (BsAbs) bind to two different targets simultaneously, enabling profound specificity in therapeutic targeting. BsAbs are a therapeutic game‐changer in cancer, immune diseases, ocular diseases, and neurological disorders [1]. Analytically, BsAbs pose special challenges due to their multiple polypeptide chains and their dual‐target specificity. In Chapter 2, we explore how advances in mass spectrometry have significantly enhanced our ability to characterize BsAbs. Next, we move to the realm of even larger biological therapeutics and focus on IgM‐based therapies which involve complexes of six antibodies (Chapter 3). There are two key advantages to hexameric IgM‐based therapies. First, IgM complexes tend to have higher avidity compared to IgG antibodies, enabling better disease targeting. Second, IgM complexes can induce cell agglutination and complement activation, which is crucial for destroying diseased cells [2]. Analytically, IgM modalities are quite challenging due to their size, polymeric nature, extensive glycosylation, and intricate disulfide bond networks. But advances in MS instrumentation, data analysis software, and sample preparation techniques are enabling us to overcome such challenges and advance antibody‐based therapies as powerful medicines (Chapters 4 and 5).
Peptide‐Based Therapies
In Chapter 6, we cover peptide therapies, both linear and cyclic. Peptide modalities offer unique advantages for specific target engagement due to their flexible structures their stabile pharmacokinetic properties due to modifications with a wider range of amino acids, i.e. non‐natural amino acids [3, 4]. Advances in peptide technology have enabled peptide therapies for a variety of diseases in the clinic, but their development is not trivial. Peptide therapeutics need to have optimal solubility and lipophilicity and require various in vitro bioanalytical assays to refine the characteristics that are suitable for a therapeutic. In Chapter 6, the authors discuss formulation development, optimization, and examples of approved peptide therapeutics.
RNA‐Based Therapies
RNA‐based therapies are rapidly advancing [5] and can be engineered in remarkable ways. RNA in the forms of antisense oligonucleotides (ASOs) and siRNA can be used to degrade transcripts or stabilize transcripts. RNA in the form of lipid‐packaged mRNAs can be used as vaccines. A remarkable example of RNA‐based therapy advances is the technologies that enabled the COVID‐19 mRNA vaccines [6] and garnered the 2023 Nobel Prize in Physiology or Medicine [7, 8]. Currently, the transformative potential of mRNA vaccines is expanding beyond infectious diseases to treat cancer [9]. RNA‐based therapeutics come with their own unique set of technical, analytical, and delivery challenges. Analytical methods like UV spectroscopy, Reverse Transcriptase‐Polymerase Chain Reaction (RT‐PCR), capillary electrophoresis, High performance liquid chromatography (HPLC), and mass spectrometry are used to characterize RNA‐based therapeutics. As RNA technology evolves rapidly, so too are the analytical tools needed to ensure high‐quality production. We discuss this and more in Chapters 13 and 15.
Cells as Therapies
Cell‐based therapies have shown enormous potential as medicines [10] and are being implemented in oncology and autoimmunity. For example, T‐cell‐based therapies have transformed treatment for various malignancies and immune disorders, offering new hope for patients. Cell therapies are arguably the most complex type of therapy we discuss, and in Chapter 14, we describe the invaluable analytical methods that are used to characterize these modalities, from preclinical development to post‐treatment monitoring in patients.
Small Molecules as Therapies
Although this book focuses on biotherapeutics, we would be remiss to exclude small molecules. Small molecules, typically chemical compounds of <900 Da, are an essential modality in targeting disease. Recently, the area of small molecules in drug discovery and development has been expanding. Bifunctional molecules in particular are opening up new therapeutic opportunities because these enable specific targeting of a protein to a particular location including to the proteasome, to the lysosome, to an RNA, or to a particular cellular compartment [11, 12]. Currently, the most clinically advanced form of bifunctional molecules are protein degraders, such as Proteolysis‐targeting chimeras (PROTACs), that chemically tether a target protein to the proteasome to induce its degradation [13, 14]. Bifunctional molecules are enabling scientists to address challenging targets that may have previously been considered “undruggable.” In terms of biotherapeutics, combining small molecules with antibodies has been transformative for drug development. Engineered antibody–drug conjugates (ADCs) enable more targeted, less toxic drug delivery to specific tissues [15, 16]. Analytically, the complexity of ADCs requires diverse analytical approaches to investigate their synthesis, stability, and pharmacological profiles, which is covered in depth in Chapter 19. Altogether, many modalities on their own can be transformative, but combining different modalities can also lead to innovation in medicine.
Delivering Diverse Biotherapeutics to Expand Patient Treatment
Alongside the development of biotherapeutic drug modalities, there has also been significant advancement in drug delivery strategies. Many therapeutic approaches once deemed failures are now being revived by optimized delivery systems. Oral delivery often does not work well for larger, complex modalities such as antibodies or cells, which instead require intravenous, subcutaneous, or intramuscular routes of delivery. Additionally, innovative delivery mechanisms such as nanoparticles [17] and ocular implants [18] are enabling certain therapies to have longer and localized activity that requires fewer dosages and can improve patient access and quality of life. In Chapter 20, we explore how innovations in delivery strategies are enhancing efficacy while minimizing side effects.
Unlocking Innovation: The Role of Biophysical Techniques in Drug Discovery and Development
To unlock the new modes of drug development, we must first understand the molecular details of how cells and proteins function. Various methods enable this, including biophysical methods that discern not only the shape of proteins but also how proteins interact with one another. In Chapters 7 and 8, we explore how structural techniques – such as nuclear magnetic resonance (NMR) spectroscopy, X‐ray crystallography, and cryo‐electron microscopy (Cryo‐EM) – provide high‐resolution insights that can inform and drive drug discovery. We also discuss how mass‐spectrometry‐based approaches (Chapter 16) and non‐mass‐spectrometry approaches (Chapter 17) enable molecular characterizations of cellular interactions and cell–cell communication processes.
Mass spectrometry is a workhorse technology in biotherapeutic development. Because of its importance – and applicability to so many therapeutic modalities – we delve deep into how mass spectrometry advances are powering biotherapeutic development (Chapters 4 and 5). In order to characterize complex biotherapeutic molecules, mass spectrometry often requires other methods in tandem, such as separation techniques and ionization techniques. Hyper‐specialized approaches are often needed to successfully analyze a particular protein class. For example, membrane proteins – with strong hydrophobicity and high levels of post‐translational modifications – pose unique challenges, and we dedicate Chapter 18 to discussing these specialized analytical methods. With high sensitivity and versatility in its application, mass spectrometry has proven invaluable in analyzing biotherapeutics and continues to advance in sensitivity and capability.
Increasing Throughput: The Role of High‐Quality Analytical Assays in Characterizing Biotherapeutics
In Vitro Assays
Getting the right assay is crucial for knowing if a potential therapeutic is safe and on‐target. In vitro assays that measure binding or function are critical for the targeted analysis of biotherapeutics. Tailored methods enable end‐to‐end validation throughout the drug development process, from initial validation of a target, to optimization of the best candidate molecule to pursue, to quality control later in the development process. These in vitro assays are simplified systems with minimal components (in vitro means in a test tube) that enable quantitative measurements. Tailored assays can be used to identify biophysical properties such as binding kinetics (Chapter 9), biological activity (Chapter 10), and a variety of other custom characteristics that inform therapeutic potential (Chapter 12). We discuss each of these in detail in the relevant chapters. Ultimately, we want to highlight that the iterative process of assay development, assay implementation, and assay validation is essential for advancing preclinical and clinical candidates.
In Silico Assays
Although many assays are physically performed, sometimes assays can be orchestrated computationally with simulations, or in silico. In silico assays can sometimes be a critical aid in the development of biotherapeutics, particularly when there is a large amount of data or complicated patterns involved. In Chapter 11, we discuss how adverse immune reactions related to biotherapeutic administration can be monitored in patients throughout the drug development process using an in silico approach. The ability of a substance to elicit an immune response is called immunogenicity, and when immunogenicity occurs toward a therapeutic, it can be detrimental – causing more harm than good. When a patient's immune system recognizes a biological drug as a foreign entity, the immune system generates antibodies against the drug, inducing an anti‐drug antibody (ADA) response. Adverse immune responses like ADA responses are becoming an increasing concern as more highly engineered biotherapeutics and delivery devices enter the clinic. In silico algorithms can be used to recognize immunogenic patterns and pinpoint particularly immunogenic regions in a protein sequence. Predicting immunogenicity is a key example of how in silico assays can enable therapeutic molecules to be optimized as safe and effective medicines.
Sequencing‐Based Assays
Sequencing‐based assays are often crucial in drug development. DNA sequencing technologies have revolutionized the development and characterization of therapeutics, and sequencing is also essential to analyze and inform the quality control of therapies that rely on DNA or RNA sequences. In Chapter 15, we discuss ASOs, small interfering RNAs (siRNAs), DNA and mRNA vaccines, and gene therapies. All of these require sequencing‐based assays to ensure the purity, integrity, and efficacy of these therapeutics. Sequencing enables the detection of synthesis‐related errors, off‐target effects, and structural variations. And sequencing is also important for understanding therapeutic potential of certain drugs for specific patients. Genomic sequencing of patients can help identify biomarkers that can inform patient response to a particular biotherapeutic. Thus, sequencing‐based assays are enabling more precise applications of medicine.
The Next Frontier: Additional Bioanalytical Tools and Targets on the Horizon
In the drug discovery and development industry, the mantra “drug the undruggable” has become a guiding principle. But what exactly is this “undruggable” space? New technical capabilities and biological insights have turned targets once thought of as undruggable – KRAS (named as it was found in Kirsten rat sarcoma virus) for example [19] – into breakthrough therapies. The idea of “undruggable” does not need to be a limitation in the way we think about exploring disease‐causing targets.
One approach to expand the view of therapeutic targets is by exploring the dark genome, encoding the dark proteome [20]. Dark genomic sequences encode potential regulatory molecules that do not conform to conventional rules of transcription or translation and have therefore largely been overlooked. Today, we have tools and understandings that enable us to probe these types of regions. For example, ribosome profiling (Ribo‐seq) enables unbiased translational profiling of all sequences engaged by a ribosome [21]. And mass spectrometry‐based proteomics enables us to detect proteins that are not part of the canonical reference genome. For mass spectrometry‐based methods, there are unique analytical challenges, such as a larger search space for peptide sequences and enzymatic digestion limitations. But technical advances and computational algorithms are overcoming these limitations [22]. Deeper biological insights, such as those obtained by interrogating the dark genome, could lead to future breakthrough therapies.
A second example of promising therapeutic targets on the horizon is protein isoforms. Protein isoforms are the different forms of a particular protein that are the result of alternative splicing, germline mutations, or posttranslational modification. Many modern drug targets are a mixture of protein isoforms, such as Tau in neurodegeneration or various glycosylated moieties on cell surface proteins such as Her2. Sometimes, only one of the protein isoforms will be pathogenic, but teasing out these variations and how they impact disease is challenging. Interrogating protein isoforms is a worthwhile effort because targeting one protein isoform specifically can be the key to developing an effective medicine. Omics methods are promising analytical tools for elucidating protein isoforms. For example, long‐read next‐generation sequencing technologies (Chapter 15) can delineate alternative splicing mechanisms [23], and top‐down proteomics (Chapter 4) can determine the details of post‐translational modifications [24]. Additionally, emerging technologies such as single‐molecule sequencing enable the investigation of individual protein copies from a minimal number of cells [25]. Importantly, it is not only the development of new tools that will enable the next wave of therapeutic innovation but also the combinations of existing tools that will enhance our understanding of the molecular underpinnings of disease and inform the development of exquisitely targeted therapeutics.
Artificial Intelligence and Machine Learning: A Transformative Shift for Computational Tools
Alongside the therapeutic advancements we have discussed is a data revolution that promises to redefine personalized medicine. Computational systems are gaining new capabilities in analyzing, integrating, and handling data. Machine learning (ML) and artificial intelligence (AI) are enabling computer capabilities that are changing many aspects of human life, including healthcare and the drug development process. AI is being applied to protein structure [26, 27], to predicting protein interaction sites [28] and to interrogating drug–drug interactions in patients [29]. Notably, the 2024 Nobel Prize in Chemistry was awarded for the development of AlphaFold that has enabled amazing advances in protein structure prediction [30].
Data is also now being collected and analyzed at speeds and quantities never seen before. The prevalence of wearable monitoring devices and the significant increase in data collected from patients with specific diagnoses [31, 32] are enabling treatments to be tailored to the specific genetic, environmental, and lifestyle factors of individual patients. Algorithms are now being integrated into drug discovery with the promise of streamlining identification of drug candidates and optimal patient populations [33–35]. However, challenges such as insufficient training data, transparency in AI decision‐making, and the integration of heterogeneous data into decision‐making models must be carefully addressed. Despite these hurdles, new data technologies are here to stay, not as a replacement for human expertise, but as a powerful tool that aids researchers and healthcare professionals in developing superior medicines. Although the book does not have a specific chapter on this topic, many chapters touch upon AI and ML in drug discovery, and integrating AI and ML into data‐driven decision‐making will undoubtedly transform the ways we discover, analyze, and develop medicines.
A Bioanalytical Call to Action!
The promise of more personalized and effective treatments is becoming a reality, enabling us to imagine a future where healthcare is not only more advanced but also tailored specifically to the needs of each individual patient. One of the ways this can be achieved is with better analytical technologies and efficient integration of biological data. The bioanalytical mission is to provide the most efficient, accurate, and cost‐effective analytical tools to characterize therapeutics at every step of the way, from their inception to their arrival on the market. In this book, we take a tour of different kinds of bioanalytical methods that enable innovative therapeutics, and we discuss the advantages and limitations of each of them. Notably, the analytical tools often evolve in parallel with the therapy, and new modalities prompt the development of analytical innovations. Ultimately, the persevering bioanalytical efforts are rewarded by enabling and accelerating the development of new medicines that transform the lives of patients.
Biologic drug modalities are a major driver of revenue growth in the biopharmaceutical industry. In 2021, the most prominent biotechnology products based on sales and FDA approvals were biotherapeutics [36]. Consistently, in 2023, four of the six top‐selling biopharma products were peptide‐ or antibody‐based therapeutics (pembrolizumab, adalimumab, semaglutide, and dupilumab). Clearly, therapeutic options are evolving toward biologic therapies. However, these advances are not without their challenges. As drugs are becoming more complicated, they are also becoming more expensive and more difficult to manufacture [37]. Once the biologic drugs are made, sophisticated storage infrastructure is often required to keep these therapies stable before they are administered to patients. Using biotherapeutics, we have realized the potential to revolutionize patient care, and now patient accessibility is a crucial part of this revolution that needs to be carefully considered. Investments from pharmaceutical companies and innovative research technologies will help to develop groundbreaking biotherapeutic drugs and get them to the people who need them. Bioanalytical scientists bear a piece of this responsibility to aid in reducing the costs associated with delivering therapies to patients. One approach to achieve this is by developing efficient analytical methods to identify better drugs on a faster timescale. Our hope is that the information in this book helps equip the next generation of scientists to develop excellent and innovative therapies that transform the treatment of disease and bring better access to patients.
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2Analytical Characterization of Bispecific Antibodies and Bispecific Molecules
Michael Dillon1, T. Noelle Lombana1, 2, and Christoph Spiess1
1Department of Antibody Engineering, Genentech Research and Early Development, South San Francisco, CA, USA
2ReCode Therapeutics, Menlo Park, CA, USA
Introduction
Bispecific antibodies (BsAbs) and bispecific molecules have specificity for two distinct protein antigens compared to a monospecific antibody (MAb) which binds only a single antigen. The idea of using a BsAb as a therapeutic has been around for almost as long as its monospecific counterpart, however, during this time, over 100 MAbs were clinically approved compared to only 15 BsAbs. One of the main reasons why BsAbs have lagged behind is that they are far more complex to produce. Over the last decade, significant advances in the production and characterization of BsAbs contributed to their approval and commercialization, and over 500 BsAbs have been under clinical investigation covering a range of therapeutic indications. The majority of the BsAbs target the oncology disease area [1, 2], while immunology, ocular, and neurobiology indications are also being explored.
Bispecific Antibody Applications
There are many therapeutic reasons to choose a bispecific despite the increased complexity of production and characterization versus the more straightforward development of a monospecific. Adding an additional layer of specificity has the advantage of bestowing antibodies with new modes of action, increasing potency or efficacy, or even combatting biological redundancy [3, 4]. BsAbs function in several ways, namely by bringing two cells together (retargeting effector cells to tumor cells), binding molecules on the same cell (to block, activate, or induce proximity), neutralizing two soluble receptor ligands, or delivering antibodies to the brain (Figure 2.1). Additionally, BsAbs can be utilized to improve cancer diagnostics and imaging.
Figure 2.1 Bispecific antibody modes of action. (a) Retarget cytotoxic T‐ or NK‐cells to kill tumor cells, (b) inactivate signaling of two soluble ligands or receptors, (c) activate signaling by a ligand mimetic, or (d) delivery across the blood–brain barrier.
The classical application of BsAbs is the retargeting of effector cells to kill tumor cells, which is achieved by binding a receptor on each cell type. In their original application, these molecules retargeted natural killer (NK) cells and macrophages via CD16 to eradicate target cells [5], but now mainly focus on redirecting cytotoxic T‐cells [6]
