Reversed-phase liquid chromatography–mass spectrometry of site-specific chemical modifications in intact immunoglobulin molecules and their fragments

The employment of a diphenyl column for the separation of intact monoclonal antibodies (mAbs) and their fragments by reversed-phase HPLC is discussed as a novel approach for the characterization of chemical modifications in a site-specific manner. Chromatographic separation of the intact mAb07 on the diphenyl support resulted in the separation of the cysteinylated from the non-cysteinylated mAb. A detected mass increase of 119 Da by mass spectrometric sequence analysis confirmed the cysteinylation. Furthermore, the diphenyl column resolved site-specific oxidation of five different methionine residues in the heavy chain (HC) of mAb03. Oxidized mAb03 HC eluted as five distinct peaks with shorter retention times than the corresponding peak representing unoxidized HC. Analysis of these peaks by in-line mass spectrometric analysis confirmed the site-specific oxidation of five different methionine residues. In another application, the diphenyl column was able to resolve free sulfhydryl groups containing Fc and Fab fragments, which were generated by limited proteolysis with endoproteinase Lys-C. The free sulfhydryl groups were responsible for a mass shift of approximately 2 Da. Their identity was further confirmed by N-ethylmaleimide labeling, which caused a shift in their chromatographic retention and led to a mass increase of 250 Da. This is the first report about chromatographic resolution on a reversed-phase column that results in site-specific separation of chemical modifications in intact mAb and mAb fragments.