In-depth site-specific O-Glycosylation analysis of therapeutic Fc-fusion protein by electron-transfer/higher-energy collisional dissociation mass spectrometry

Unexpected O-glycosylations, including O-xylosylations and mucin-type O-glycosylations, have been reported in recent glycosylation analyses of Fc-fusion proteins produced in mammalian cell expression systems. This observation suggests that therapeutic proteins with novel structures can undergo unintended O-glycosylations, having implications regarding their efficacy and safety. Therefore, the implementation of O-glycosylation analysis during product developmental is essential. However, detail site-specific O-glycosylation analysis is difficult because no consensus sequence for mucin-type O-glycosylations is known, and O-glycopeptides often contain multiple or continuous glycosylation sites. Recently, a new mass spectrometric fragmentation method called electron-transfer/higher-energy collisional dissociation (EThcD) has been used for site-specific glycosylation analysis. In this study, we conducted site-specific O-glycosylation analysis of commercially available GLP1-Fc fusion protein with (G4S)3 linker peptide using liquid chromatography/mass spectrometry (LC/MS) with EThcD and a glycoproteomic database search. We successfully identified unexpected O-xylosylations at Ser residues in the (G4S)3 linker peptide, mucin-type O-glycosylations at Thr and Ser residues in the GLP-1 peptide, and Ser residues in the (G4S)3 linker peptide. This study is the first to report these unexpected O-xylosylations and mucin-type O-glycosylations in this therapeutic fusion protein. Mammalian-cell production of therapeutic fusion proteins that contain novel structures may require exhaustive O-glycosylation analysis to ensure their quality, efficacy, and safety.