Comparative glycosylation analysis of etanercept and its biosimilars using TMT labeling and ZIC-HILIC HPLC chromatography

Etanercept is a biopharmaceutical fusion protein composed of tumor necrosis factor receptor 2 (TNFR2) and the Fc region of human immunoglobulin G (IgG1), used to treat autoimmune diseases. Due to its heterogeneous glycosylation at multiple sites, a detailed glycoprotein analysis is essential for characterizing this biopharmaceutical. In this study, we present a workflow that integrates the enrichment and fractionation of TMT-labeled glycopeptides with ZIC-HILIC HPLC chromatography. This method was applied for an in-depth analysis of site-specific N- and O-glycopeptides from Etanercept, comparing the original product with two biosimilars. We identified 87 N-glycopeptides and 67 O-glycopeptides from three N-glycosylation sites and 15 O-glycosylation sites, respectively. For the first time, we report 12 O-glycopeptides from two novel O-glycosylation sites. The N149 site in the TNFR2 region accounted for over 70% of afucosylated complex-type glycans, while the N171 site predominantly featured fucosylated complex-type glycans. In the IgG region, the main glycan types were asialo fucosylated complex and high mannose. At all O-glycosylation sites, over 70% of the glycopeptides were of the core1 type. Glycosylation patterns among the three pharmaceuticals were similar in macro heterogeneity but differed in micro heterogeneity. Our method demonstrates that TMT labeling combined with ZIC-HILIC HPLC fractionation provides precise quantitation and higher throughput for site-specific N- and O-glycopeptide analysis in therapeutic fusion proteins, enabling the identification of novel glycosylation sites and characterization of low abundant glycosylation types.