Characterization of IgG Glycosylation Using Intact Protein Analysis and Peptide Mapping

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Himanshu S. Gadgil , Da Ren, Paul Rainville, Reb J. Russell II and Jeff R. Mazzeo [Waters]
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IgG; Glycosylated IgG
BioSuite C18 PA-A 3 µm Peptide Analysis Column 2.1 mm 250 mm BioSuite 250 HR SEC 5 µm Column 7.8 mm 300 mm Oasis® HLB 5 µm Extraction Column - steel 2.1 mm 20 mm
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Glycosylation is a co-translational and a post-translational modification found in proteins. N-linked glycosylation is found in the Fc region of Immunoglobulins. The covalently bound oligosaccharides vary in composition and branching within different classes of immnoglobulins. Glycosylation is vital for bioactivity and pharmacokinetics of biotherapeutic proteins. We characterized glycosylation in monoclonal IgG by “top down” and “bottom up” approaches using intact protein mass analysis and peptide mapping. The intact protein analysis was carried out using SEC-MS and RP-MS. Global mass analysis of glycosylated, partially glycosylated and deglycosylated IgG revealed that the sugar moiety has mass of approximately 1468 Daltons corresponding to the asialo-biantennary N-linked oligosaccharide with core fucose. Signature ion scan for mass 204 in the peptide map of IgG revealed 3 distinct glycol-peptide peaks. Based on the mass of these resolved peaks we were able to confirm that they were composed of different glycoforms, confirming the heterogeneity observed at the intact protein analysis. The MS/MS analysis of the G0 glycopeptide was also obtained and the composition of the oligosaccharides confirned by daughter ion assignments.

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