Detailed Characterization of Monoclonal Antibody Receptor Interaction Using Affinity Liquid Chromatography Hyphenated to Native Mass Spectrometry

Anal Chem. 2017 May 16;89(10):5404-5412. doi: 10.1021/acs.analchem.7b00211. Epub 2017 Apr 26.

Abstract

We report on the online coupling of FcRn affinity liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS) in native conditions to study the influence of modifications on the interaction of recombinant mAbs with the immobilized FcRn receptor domain. The analysis conditions were designed to fit the requirements of both affinity LC and ESI-MS. The mobile phase composition was optimized to maintain the proteins studied in native conditions and enable sharp pH changes in order to mimic properly IgGs Fc domain/FcRn receptor interaction. Mobile phase components needed to be sufficiently volatile to achieve native MS analysis. MS data demonstrated the conservation of the pseudonative form of IgGs and allowed identification of the separated variants. Native FcRn affinity LC-ESI-MS was performed on a therapeutic mAb undergoing various oxidation stress. Native MS detection was used to determine the sample oxidation level. Lower retention was observed for mAbs oxidized variants compared to their intact counterparts indicating decreased affinities for the receptor. This methodology proved to be suitable to identify and quantify post-translational modifications at native protein level in order to correlate their influence on the binding to the FcRn receptor. Native FcRn affinity LC-ESI-MS can tremendously reduce the time required to assess the biological relevance of the IgG microheterogeneities thus providing valuable information for biopharmaceutical research and development.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / chemistry
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / metabolism*
  • Chromatography, Affinity
  • Chromatography, High Pressure Liquid
  • Glycosylation
  • Hydrogen-Ion Concentration
  • Immunoglobulin Fc Fragments / chemistry
  • Immunoglobulin Fc Fragments / metabolism
  • Mice
  • Peptide Mapping
  • Peptides / analysis*
  • Peptides / isolation & purification
  • Protein Binding
  • Receptors, Fc / chemistry
  • Receptors, Fc / metabolism*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Spectrometry, Mass, Electrospray Ionization*

Substances

  • Antibodies, Monoclonal
  • Immunoglobulin Fc Fragments
  • Peptides
  • Receptors, Fc
  • Recombinant Proteins