Native Hydrophobic Interaction Chromatography Hyphenated to Mass Spectrometry for Characterization of Monoclonal Antibody Minor Variants.

Conventionally, hydrophobic interaction chromatography (HIC) uses mobile phases with high salt concentration that are not compatible with mass spectrometry (MS). Here we describe development of an HIC method coupled with MS detection (HIC-MS) utilizing an aqueous mobile phase with a low concentration of a volatile salt for characterizing recombinant monoclonal antibody (mAb) post-translational modifications (PTMs). The ability of HIC to separate the oxidation and free thiol variants of the mAbs enables their isolation and rapid characterization of these attributes under native conditions, an important step toward understanding the role they play.