Epitope mapping of an interleukin receptor for three therapeutic antibodies by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)

By
  • Eric Largy
  • Caroline Cajot
  • Arnaud Delobel
Date
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Monoclonal antibodies represent a rapidly expanding class of biotherapeutics, valued for their high selectivity towards specific targets. However, their physicochemical characterisation remains challenging due to their structural complexity, variability and dynamic behaviour. Understanding the molecular interaction between mAbs and their antigens is therefore essential, particularly to elucidate their mechanism of action and functional differences. Epitope mapping plays a key role in this process by identifying the precise binding regions on the antigen. In this context, Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) offers a solution-based approach to investigate protein structure and interaction dynamics under near-physiological conditions.

This application note presents a case study of epitope mapping for three monoclonal antibodies targeting an interleukin receptor, and the full technical content is available for download.

HDX-MS CAPABILITIES DEMONSTRATED

  • HDX-MS enables epitope mapping at the scale of a few amino acids, even for complex protein systems
  • Experiments are performed in solution under near-physiological conditions, preserving protein dynamics
  • High sequence coverage achieved (99.6%) with strong peptide redundancy (6.44 peptides per amino acid)
  • Excellent reproducibility with very low retention time deviation (average 0.009 min across 399 peptides)
  • Ability to compare bound vs. unbound states to identify statistically significant protection regions and define epitope

HOW THIS SUPPORTS YOUR PROGRAMME?

HDX-MS provides detailed and reliable insights into antigen–antibody interactions, supporting the development and characterisation of therapeutic antibodies.

  • Enables precise identification of antibody binding regions on the antigen
  • Supports comparison of multiple mAb candidates targeting the same antigen
  • Provides insight into binding modes and differences in interaction strength
  • Facilitates understanding of structure–function relationships
  • Delivers statistically robust data through replicated experiments and quantitative analysis

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