Site-specific enrichment of highly sialylated N-glycans in an erythropoietin-hybrid Fc fusion protein.

Erythropoietin-hybrid Fc fusion protein (EPO-hyFc), comprising an EPO domain fused to a hybrid IgD-IgG4 Fc, is a next-generation erythropoiesis-stimulating agent with an approximately two-fold longer serum half-life than darbepoetin alfa, a clinically established long-acting EPO formulation. Although sialylation at Asn-38 and Asn-83 is known to modulate serum stability and half-life, the site-specific N-glycosylation of EPO-hyFc has not been characterized. Here, we performed comprehensive N-glycan profiling using LC-MS/MS glycomics combined with nano-LC-MS/MS glycoproteomic analysis of Glu-C-digested peptides. In total, 23 N-glycans (15 sialylated and 8 neutral) were identified. Site-normalized quantification revealed that Asn-24 was mainly occupied by mono- and di-sialylated glycans (64.9%), whereas Asn-38 (76.9%) and Asn-83 (87.7%) were enriched in tri- and tetra-sialylated structures. The Fc site (Asn-261) contained mainly non-sialylated glycans (94.4%). The average number of sialic acids per N-glycan was 2.2, and the sialic acid-capping ratio was 90.9%, indicating extensive terminal sialylation across the EPO sites. These results provide the first site-specific characterization of N-glycosylation in EPO-hyFc and offer structural and quantitative insights for optimizing its stability, pharmacokinetics, and therapeutic efficacy.