N- and O-Glycosylation Analysis of Etanercept Using Liquid Chromatography and Quadrupole Time-of-Flight Mass Spectrometry Equipped with Electron-Transfer Dissociation Functionality

Etanercept is a highly glycosylated therapeutic Fc-fusion protein that contains multiple N- and O-glycosylation sites. An in-depth characterization of the glycosylation of etanercept was carried out using liquid chromatography/mass spectrometry (LC/MS) methods in a systematic approach in which we analyzed the N- and O-linked glycans and located the occupied O-glycosylation sites. Etanercept was first treated with peptide N-glycosidase F to release the N-glycans. The N-glycan pool was labeled with a 2-aminobenzamide (2-AB) fluorescence tag and separated using ultraperformance liquid chromatography–hydrophilic interaction liquid chromatography (UPLC-HILIC). Preliminary structures were assigned using Glycobase. These assignments, which included monosaccharide sequence and linkage information, were confirmed by exoglycosidase array digestions of aliquots of the N-glycan pool. The removal of the N-glycans from etanercept facilitated the selective characterization of O-glycopeptides and enabled the O-glycans to...