Unveiling the Accurate Site-Specific N- and O-Glycosylation of Hyperglycosylated Erythropoietin Drugs by an Integrated Approach

Hyperglycosylated proteins with a high sialic acid content show great promise in the development of long-acting biotherapeutics. However, their structural complexity and heterogeneity pose significant challenges to traditional analytical methods, which often fail to provide comprehensive glycan information across all glycosylation sites, leading to ambiguities in characterization. Despite the fact that long-acting hyperglycosylated erythropoietin (hyperEPO) has been available on the market for over two decades, its site-specific glycosylation profile remains ambiguous due to closely spaced glycosylation sites and large glycans that contain labile sialic acids substituents. Here, using hyperEPO as an example, we developed an integrated workflow that incorporates an experimentally cross-validated glycan database, optimized nonspecific digestion, and a streamlined glycopeptide derivatization method to enhance sialylated peptide detection, enabling comprehensive site-specific characterization of both N- and O-glycosylation. We applied this strategy to compare the glycosylation profiles of the commercial hyperEPO drug darbepoetin alfa and a novel high-potency analogue, EPO-XL. We found that both proteins exhibited high site occupancy, large tetra-sialylated glycans, and similar types of sialic acid linkages but differed markedly in site-specific glycoform distributions. Notably, EPO-XL contained extensive LacNAc structures at all five N-glycosylation sites, and a previously unreported O-glycosylation site at S120 was identified alongside the canonical S126 site. Collectively, this study presents the first site-specific N- and O- glycosylation profiles of hyperEPO proteins, offering valuable guidance for the quality control and rational design of these therapeutics. The state-of-the-art analytical strategy introduced here holds great potential to advance the site-specific glycosylation characterization of proteins with complex glycosylation.