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Updated: Sep 10, 2025
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Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System
Published on: May 1, 2019
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Interdomain Interactions Modulate Refolding Kinetics and Aggregation in a Monoclonal Antibody
Philipp Trolese1,2, Andrea Pierangelini1, Benedetta Fongaro1
1Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova 35131, Italy.
Journal of the American Society for Mass Spectrometry
|August 26, 2025
Summary
Antibody CH3 domain stability is crucial for preventing aggregation in therapeutic proteins like bevacizumab. Understanding domain-specific unfolding and refolding guides the design of more stable antibody therapeutics.
Area of Science:
- Biochemistry
- Protein Science
- Therapeutic Antibody Development
Background:
- Antibody stability is critical for therapeutic efficacy and shelf-life.
- Understanding protein unfolding and aggregation mechanisms is essential for biopharmaceutical development.
Purpose of the Study:
- To investigate the unfolding and refolding behavior of bevacizumab under denaturing conditions.
- To identify domain-specific contributions to antibody stability and aggregation.
- To provide insights for designing more stable therapeutic antibodies.
Main Methods:
- Dynamic Light Scattering (DLS) for aggregation detection.
- Circular Dichroism (CD) for structural analysis.
- Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) for unfolding kinetics.
Main Results:
- Distinct unfolding kinetics observed across antibody domains: CH2 and VH unfolded rapidly, CH3 unfolded slowly.
- Aggregation was detected after CH3 destabilization, indicating its critical role in preventing aggregation.
- Aggregation-prone regions identified in Fc and Fab portions, with VH CDR H1 showing aberrant protection post-refolding.
Conclusions:
- CH3 domain stability is paramount in preventing bevacizumab aggregation.
- Cooperative refolding of CH2-CH3 domains and CH3-CH3 interfaces is essential.
- Both constant and variable domains contribute to the complex, interdependent nature of monoclonal antibody aggregation.
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