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

Abstract

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.

Figure 1.

Schematic workflow for site-specific glycosylation analysis of hyperglycosylated proteins. This workflow centers on glycopeptide-level characterization, supported by released glycan profiling and intact protein mass analysis. Released glycans were independently derivatized via amidation and permethylation to cross-validate glycan identification and enhance confidence in the glycan database construction, thereby facilitating glycopeptide assignment. At the glycopeptide level, nonspecific enzymatic digestion was used to produce short and uniform glycopeptides, enabling comprehensive coverage of densely glycosylated regions. A one-tube dual derivatization strategy, involving dimethylation followed by amidation, was applied to stabilize sialic acids and improve the detection of sialylated glycopeptides. Additionally, site occupancy analysis and intact protein mass profiling were conducted to provide an overview of the glycosylation levels.

Figure 2.

MALDI-TOF MS spectra of sialic acid derivatized N-glycans released from (a) darbepoetin alfa and (b) EPO-XL, respectively. [Legend: H, hexose; N, N-acetylhexosamine; F, fucose; S, N-acetylneuraminic acid (sialic acid, Neu5Ac); O-Ac, O-acetylation; α−2,3, α2,3-linked sialic acid. The number following the condensed code represents the quantity of theses residues. The structures in SFGN are putative glycan structures based on MS/MS analysis and biosynthetic rules.]

Figure 3.

Comparative analysis of N- and O-glycopeptides without derivatization (left) and with derivatization (right). [Legend: H, hexose; N, N-acetylhexosamine; F, fucose; S, N-acetylneuraminic acid (sialic acid, Neu5Ac). The number following the condensed code represents the quantity of theses residues.]

Figure 4.

Identification of a novel O-glycosylation site at Ser120 in the EPO-XL protein via (a) sceHCD and (b) EThcD MS/MS analysis of the derivatized glycopeptide AIS(HexNAcHexNeuAc)PPDAA. Glycan fragments are shown in red, peptide fragments are in blue, and those containing the glycan motif are in bold. Italicized labels in the figure indicate amino acid residues or oxonium ions modified by derivatization, including dimethylation of the peptide N-terminus, amidation of the peptide C-terminus, and amidation of sialic acid. The peak assignments are provided in Tables S2 and S3 in the Supporting Information.

Figure 5.

Site-specific N- and O-glycosylation analysis of derivatized darbepoetin alfa and EPO-XL. Glycan abundance was calculated by the sum of glycopeptides with same glycans in the same site, and relative abundance was normalized at each site. [Legend: H, hexose; N, N-acetylhexosamine; F, deoxyhexose; S, N-acetylneuraminic acid; G, N-glycolylneuraminic acid. The number following the condensed code indicates the quantity of the corresponding residue.]