In-depth O-glycosylation characterization and comparison of commercially available etanercept biosimilars by using Electron Activated Dissociation.

Glycosylation is one of the important post-translational modifications in protein drugs expressed by eukaryotic cells. It is closely related to the conformation, stability, and solubility of therapeutic proteins and holds vital biological significance. Etanercept contains 26 O-glycosylation sites with high glycosylation heterogeneity, which makes the analysis of O-glycosylation highly challenging. In this paper, using liquid chromatography-mass spectrometry (LC-MS), 11 O-glycosylation on the single chain of etanercept were first identified at the intact protein level, with Core1 and Core2 O-glycans. Second, 11 O-glycosylation sites were identified with the electron activation dissociation (EAD) fragmentation mode at the glycopeptide level, and their glycans were comprehensively characterized. The major O-glycan structures were Core1 and Core2, which were consistent with those observed at the intact protein level. Third, the etanercept innovator (Y1) and six biosimilars or follow-on products (Y2-Y7) from different manufacturers were also compared. Compared with the etanercept innovator, the results showed that etanercept biosimilars or follow-on products from different manufacturers exhibited pronounced differences in O-glycosylation modification due to variation in production processes. Based on a comprehensive assessment of consistency in deconvoluted spectra and the glycosylation ratios, etanercept biosimilar Y2 was the most similar to the innovator Y1. In-depth studies of etanercept's O-glycosylation can effectively be applied to the etanercept innovator's comparative analysis and biosimilars. Furthermore, the LC-MS method combined with the EAD fragmentation technique can offer detailed and precise data support for the comprehensive quality control of complex glycoprotein drugs and the optimization of production processes.