Comparative O-Glycosylation Analysis of Etanercept Originator and Biosimilars Using Orthogonal LC-MS Workflows

Etanercept is a highly glycosylated Fc-fusion protein whose dense, heterogeneous O-glycosylation is challenging to characterize yet relevant to biosimilarity assessment. We compared O-glycosylation in five originator (Enbrel) batches, three biosimilars (Erelzi, Benepali and Nepexto) and the EDQM chemical reference standard using orthogonal LCMS workflows on an Orbitrap Excedion Pro BioPharma mass spectrometer. Native intact mass analysis after N-glycan and sialic acid removal, and middle-up analysis after IdeS digestion, characterized O-glycan macroheterogeneity together with C-terminal lysine and N-terminal leucine-loss variants. Site-specific mapping combined the O-glycoproteases OgpA and IMPa with HCD-triggered EThcD fragmentation. The same 13 O-glycosites were identified in every sample, carrying predominantly sialylated core 1 O-glycans (72%-80% sialylated) with low levels of Tn antigen. In contrast, Benepali and Nepexto showed lower O-glycan occupancy (approximately 17.3 versus 18.9-19.1 O-glycans per molecule) and broader glycoform distributions, and Benepali showed markedly elevated leucine loss (35% of TNF domains). Subunit analysis resolved nearisobaric leucine-loss and di-lysine variants that were ambiguous at the intact level, while EThcD localized O-glycans within densely glycosylated peptides. This multi-level workflow provides a generalizable approach for site-specific O-glycosylation analysis and comparability assessment of O-glycosylated biotherapeutics.