Isomerization and Oxidation in the Complementarity-Determining Regions of a Monoclonal Antibody: A Study of the Modification-Structure-Function Correlations by Hydrogen-Deuterium Exchange Mass Spectrometry

Anal Chem. 2016 Feb 16;88(4):2041-50. doi: 10.1021/acs.analchem.5b02800. Epub 2016 Jan 29.

Abstract

Chemical modifications can potentially change monoclonal antibody's (mAb) local or global conformation and therefore impact their efficacy as therapeutic drugs. Modifications in the complementarity-determining regions (CDRs) are especially important because they can impair the binding affinity of an antibody for its target and therefore drug potency as a result. In order to understand the impact on mAb attributes induced by specific chemical modifications within the CDR, hydrogen-deuterium exchange mass spectrometry (HDX MS) was used to interrogate the conformational impact of Asp isomerization and Met oxidation in the CDRs of a model monoclonal antibody (mAb1). Our results indicate that despite their proximity to each other, Asp54 isomerization and Met56 oxidation in CDR2 in the heavy chain of mAb1 result in opposing conformational impacts on the local and nearby regions, leading directly to different alterations on antibody-antigen binding affinity. This study revealed direct evidence of local and global conformational changes caused by two of the most common degradation pathways in the CDRs of a mAb and identified correlations between chemical modification, structure, and function of the therapeutic monoclonal antibody.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / metabolism
  • Antibody Affinity
  • Antigen-Antibody Reactions
  • Aspartic Acid / chemistry
  • CHO Cells
  • Complementarity Determining Regions / chemistry
  • Complementarity Determining Regions / metabolism
  • Cricetinae
  • Cricetulus
  • Deuterium / chemistry
  • Deuterium Exchange Measurement*
  • Enzyme-Linked Immunosorbent Assay
  • Hydrogen / chemistry
  • Isomerism
  • Kinetics
  • Mass Spectrometry*
  • Methionine / chemistry
  • Oxidation-Reduction
  • Protein Structure, Tertiary
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification

Substances

  • Antibodies, Monoclonal
  • Complementarity Determining Regions
  • Recombinant Proteins
  • Aspartic Acid
  • Hydrogen
  • Methionine
  • Deuterium