EPO-hyFc N-Glycan Profiling Reveals Site-Specific Sialylation Patterns Influencing Stability

Therapeutic peptides, while potent, often suffer from short plasma half-lives, limiting their clinical utility. Erythropoietin (EPO) is a crucial hormone stimulating red blood cell production, and its recombinant forms are vital for treating anemia. However, even long-acting EPO formulations like darbepoetin alfa require frequent dosing. EPO-hyFc, a fusion protein combining EPO with a hybrid IgD-IgG4 Fc, aims to extend serum half-life. Sialylation, the addition of sialic acid residues to N-glycans, is a known modulator of protein stability and pharmacokinetics, particularly at Asn-38 and Asn-83 in EPO. Understanding the site-specific N-glycosylation of EPO-hyFc is critical for optimizing its therapeutic properties.

Researchers performed a comprehensive N-glycan characterization of EPO-hyFc, a novel erythropoiesis-stimulating agent. The study utilized LC-MS/MS glycomics to identify overall N-glycan profiles and nano-LC-MS/MS glycoproteomic analysis of Glu-C-digested peptides for site-specific quantification. This dual-approach allowed for detailed mapping of glycosylation patterns across different N-glycosylation sites within the EPO-hyFc molecule. The primary objective was to characterize the distribution and type of sialylated N-glycans at each specific glycosylation site.

The analysis identified a total of 23 distinct N-glycans, comprising 15 sialylated and 8 neutral structures on EPO-hyFc. Site-normalized quantification revealed significant differences in sialylation patterns across the protein. Asn-24 was predominantly occupied by mono- and di-sialylated glycans, accounting for 64.9% of its N-glycans. In contrast, Asn-38 showed a high enrichment of tri- and tetra-sialylated structures, representing 76.9% of its glycans. Asn-83 exhibited an even higher enrichment, with 87.7% of its glycans being tri- and tetra-sialylated. The Fc site (Asn-261) displayed a starkly different profile, containing mainly non-sialylated glycans at 94.4%. The overall average number of sialic acids per N-glycan across the EPO sites was 2.2, and the sialic acid-capping ratio was 90.9%, indicating extensive terminal sialylation.

Asn-38 and Asn-83, known to modulate serum stability, were highly enriched in tri- and tetra-sialylated structures, with 76.9% and 87.7% respectively.