Investigation of O-glycosylation heterogeneity of recombinant coagulation factor IX using LC-MS/MS

Recombinant coagulation factor IX (rFIX) has extraordinarily multiple post-translational modifications including N-glycosylation and O-glycosylation which have a drastic effect on biological functions and in vivo recovery. Unlike N-glycosylation extensively characterized, there are a few studies on O-glycosylation due to its intrinsic complexity. In-depth O-glycosylation analysis is necessary to better understand and assess pharmacological activity of rFIX. Results: We determined unusual O-glycosylations including O-fucosylation and O-glucosylation which were located at Serine 53 and 61, respectively in EGF domain. Other O-glycosylations bearing core 1 glycan moiety were found on activation peptide. Conclusion: This is the first comprehensive study to characterize O-glycosylation of rFIX using MS-based glycomic and glycoproteomic approaches. Site-specific profiling will be a powerful platform to determine bioequivalence of biosimilars.