Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry

• DOI: 10.1016/j.ab.2011.04.030
• Publication Date: 2011-04-30T18:36:50Z
• Authors: Guillaume Chevreux; Nolwenn Tilly; Nicolas Bihoreau
• Keywords: 0301 basic medicine; Spectrometry, Mass, Electrospray Ionization; Glycosylation; Antibodies, Monoclonal; Recombinant Proteins; 3. Good health; Cysteine Endopeptidases; 03 medical and health sciences; Bacterial Proteins; Disulfides; Immunoglobulin Heavy Chains; Protein Processing, Post-Translational; Chromatography, High Pressure Liquid

We describe a fast and informative method to investigate the posttranslational modifications of monoclonal antibodies (MAbs). The MAb is first digested by a specific enzyme that cleaves heavy chains under the hinge domain. After reduction of disulfide bridges, three polypeptide chains of approximately 25 kDa are released and analyzed by liquid chromatography-mass spectrometry (LC-MS). By bisecting the heavy chains prior to MS analysis, this method provides a better MS resolution and facilitates the study of the N-linked glycans as well as of other modifications (loss of C-terminal lysine, pyroglutamination, and oxidation). The sample preparation and analysis can be performed within few hours.