Characterization of glycosylation sites for a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein by liquid chromatography–mass spectrometry peptide mapping

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

“…There are also some O-linked glycan sites occupied with complex O-linked glycans. 24 For this reason, it is very difficult to get detail information about glycosylation heterogeneity at an intact level. 29 These oligosaccharides are a complex mixture of different glycans, making them challenging for characterization.…”

“…23 In a study of CTLA4-Ig fusion protein, 3 N-linked sites and one O-linked sites were reported. 24 N-glycosylation at N76, N108, and N207 was identified, but detailed information about the glycosylation was not provided. The specific N-glycosylation heterogeneity at each individual site and the O-linked glycosylation sites (and site heterogeneity) of the fusion protein are still ambiguous.…”

“…The specific N-glycosylation heterogeneity at each individual site and the O-linked glycosylation sites (and site heterogeneity) of the fusion protein are still ambiguous. 24 For definite conclusions to be drawn about the processing and biological function of CTLA4-Ig fusion protein glycosylation, a complete characterization of glycosylation is required. This is also necessary to validate the similarity of a biosimilar to the reference product.…”

Rapid Fc glycosylation analysis of Fc fusions with IdeS and liquid chromatography mass spectrometry

“…Additionally, O-linked glycosylation at Ser129 and Ser139 has also been identified through peptide mapping. 15 For IdeS digestion, 4 uL 25 mg/mL abatacept reconstituted from lyophilized powder was directly diluted in 96 uL 150 mM sodium chloride, 20 mM sodium phosphate pH 6.6 and incubated with 1 uL IdeS (Bulldog Bio, Portsmouth, NH) at 37°C for 30 min. Because the reported O-linked glycosylation and sialylation might complicate the assignment and quantitation of N-linked glycan structures, we treated 50 ug and 10 ug of IdeS digested abatacept with 1 uL PNGase F (New England BioLabs) and 1 uL neuraminidase (New England BioLabs), respectively, at 37°C for 30 min.…”

Mass spectrometry for glycosylation analysis of biopharmaceuticals

“…LC-ESI-IT-MS and MS/MS have been applied to assign the site of a novel O-linked modification in a mAb and the N-and O-glycosylation sites in an Ig fusion protein, respectively, by using CID [49,53]. Though LC-ESI-IT-MS is widely used for glycopeptide analysis in biopharmaceuticals, LC-ESI-(Q)TOF-MS is gaining interest due to its higher resolution and mass accuracy [10,51,54,55].…”