Orbitrap Based Mass Spectrometric Characterization of Antibody Drug Conjugates Engineered through Antibody Glycans

Methods: ADCs were made from SiteClickTM enzyme-based N-glycan labeling of tratuzumab with DIBO-MMAE toxin. Intact, F(ab’)2, scFc fragments and tryptic peptides of wild type antibody, azide-tagged intermediate, and ADCs were analyzed on Thermo ScientificTM Q ExactiveTM Plus and Thermo ScientificTM Orbitrap FusionTM mass spectrometers by LC-MS. Intact and fragment data were processed by Thermo Scientific Protein DeconvolutionTM 4.0 software. Peptide data was processed by Thermo ScientificTM PepFinderTM 2.0 software. Top down sequencing was performed by Prosight Lite software from Northeastern University.