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

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 ...