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

Affiliation

Faculty of Materials Science and Engineering, Department of Chemistry, University of Miskolc, 3515 Hungary. Electronic address: [Email]

Abstract

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.

Keywords

Capillary electrophoresis,Cetuximab glycosylation,Charge variants,Peptide mapping,

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