Etanercept is a recombinant Fc fusion protein widely used to treat rheumatic diseases. This protein is highly glycosylated and contains numerous O- and N-glycosylation sites. Since glycosylation is recognized as an important critical quality attribute (CQA) that can affect immunogenicity, solubility, and stability of Fc fusion proteins, it should be thoroughly characterized. In this work, hydrophilic interaction chromatography (HILIC) was combined with high-resolution mass spectrometry (HRMS) by using a quadrupole time-of flight mass spectrometer to assess glycosylation of etanercept at the middle-up level of analysis (fragments of ca. 25-30 kDa). In addition, a combination of different enzymatic digestion procedures (i.e., glycosidase, sialidase, and protease) was systematically employed to facilitate spectra deconvolution. With the developed procedure, the main post-translational modifications (PTMs) of etanercept were assessed, and a global overview of the subunit-specific distribution of the glycosylation pattern was obtained at a middle-up level of analysis.

Center of Immunology Pierre Fabre 5 Avenue Napoléon 3 BP 60497 Saint Julien en Genevois France

Department of Analytical Chemistry Faculty of Pharmacy in Hradec Králové Charles University Hradec Králové Czech Republic

Department of Chemistry Gustavus Adolphus College St Peter Minnesota 56082 United States

Section of Pharmaceutical Sciences School of Pharmacy Geneva Lausanne University of Geneva Centre Médical Universitaire Rue Michel Servet 1 1211 Geneva Switzerland

Waters Corporation 34 Maple Street Milford Massachusetts 01757 3696 United States

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Comparison of human IgG glycopeptides separation using mixed-mode hydrophilic interaction/ion-exchange liquid chromatography and reversed-phase mode

Glycan-specific precipitation of glycopeptides in high organic content sample solvents used in HILIC