Analysis of cetuximab N-Glycosylation using multiple fractionation methods and capillary electrophoresis mass spectrometry

•Annotation of cetuximab N-glycan species by multiple exoglycosidase digestions.•Identification of site-specific cetuximab N-glycosylation by C4 fractionation in combination with CE-MS profiling.•Quantification of glycosylation site occupancy by heavy water incorporation.•CE-MS glycan analysis across cetuximab chargevariants. Site-specific glycosylation of Cetuximab was characterized in this study using multiple fractionation methods and capillary electrophoresis coupled to mass spectrometry (CE-MS) based glycomics. IdeS digested Cetuximab with subsequent reduction was fractionated using reversed-phase chromatography resulting in 3 fragments; Fd, Lc and Fc/2. Glycan release of the different fragments was performed in 18O enriched water providing the possible quantification of site occupancy. 2-AA labelled glycan structures were annotated by CE-MS profiling in combination with exoglycosidase sequencing, revealing potential structures with terminal α-galactose and N-glycolyl-neuraminic acid (NGNA) mainly originating from the Fd fragment. Glycosylation analysis was also performed on different charge variants of Cetuximab that were separated using pH gradient cation-exchange chromatography to investigate the impact of glycosylation on the net charge of the protein.