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Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System
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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.

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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.

Keywords:
aggregationhydrogen−deuterium exchange mass spectrometrymonoclonal antibodiesrefoldingunfolding

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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.