RESUMEN
Essentials Glycosylation heterogeneity of recombinant proteins affects pharmacokinetics and immunogenicity. N-glycomics/glycoproteomics of plasma-derived Factor VIII and 6 recombinant FVIIIs were compared. Depending on cell line, significant differences to plasma-derived FVIII were observed. Recombinant FVIIIs expressed distinct and immunologically relevant epitopes. SUMMARY: Background/Objective Human factor VIII (FVIII) is a plasma glycoprotein, defects of which result in hemophilia A. Current substitution therapy uses FVIII products purified from human plasma or from various cell lines (recombinant FVIII) with different levels of B-domain deletion. Glycosylation is a post-translational protein modification in FVIII that has a substantial influence on its physical, functional and antigenic properties. Variation in glycosylation is likely to be the reason that FVIII products differ in their pharmacokinetics, pharmacodynamics and immunogenicity. However, the literature on FVIII glycosylation is inconsistent, preventing assembly into a coherent model. Seeking to better understand the glycosylation mechanisms underlying FVIII biology, we studied the N-glycosylation of human plasma-derived (pd)FVIII and six rFVIII products expressed in CHO, BHK or HEK cell lines. Methods FVIII samples were subjected to head-to-head detailed glycomic and glycoproteomic characterization using a combination of MALDI-MS and MS/MS, GC-MS and UPLC-UV-MSE technologies. Results/Conclusion The results of our study detail the N-glycan repertoire of pdFVIII to an unprecedented level, and for the first time, provide evidence of N-glycolylneuraminic acid (NeuGc) found on pdFVIII. Although site-specific glycosylation of rFVIII proved consistent with pdFVIII regardless of the expression system, the entire N-glycan content of each sample appeared significantly different. Although the proportion of biologically important epitopes common to all samples (i.e. sialylation and high-mannose) varied between samples, some recombinant products expressed distinct and immunologically relevant epitopes, such as LacdiNAc (LDN), fucosylated LacdiNAc (FucLDN), NeuGc, LewisX/Y and Galα1,3 Gal epitopes. rFVIII expressed in HEK cells showed the greatest glycomic differences to human pdFVIII.
RESUMEN
BACKGROUND: von Willebrand factor (VWF) is a key component for maintenance of normal hemostasis. Its glycan moieties, accounting for about 20% of its molecular weight, have been shown to affect many of its properties. Previous studies reported correlations between VWF secretion, half-life and the nature or presence of its N-glycans, and more importantly between VWF plasma level and the type of N-linked ABH antigens. Despite the presence of 10 predicted O-glycosylation sites, the O-glycome remains poorly characterized, impairing the complete elucidation of its influence on VWF functions. So far only a single glycan structure, a disialyl core 1 glycan, has been identified. OBJECTIVES: To define an exhaustive profile of the VWF O-glycan structures to help the understanding of their role in VWF regulation and properties. METHODS: Plasma-derived VWF O-linked sugars were isolated and analyzed using state-of-the-art mass spectrometry methodologies. RESULTS AND CONCLUSIONS: We provide here a detailed analysis of the human plasma-derived VWF O-glycome. Eighteen O-glycan structures including both core 1 and core 2 structures are now demonstrated to be present on VWF. Amongst the newly determined structures are unusual tetra-sialylated core 1 O-glycans and ABH antigen-containing core 2 O-glycans. In conjunction with current models explaining VWF activity, knowledge of the complete O-glycome will facilitate research aimed at providing a better understanding of the influence of glycosylation on VWF functions.