Orthogonal Middle-up Approaches for the Characterization of the Glycan Heterogeneity of Etanercept by Hydrophilic Interaction Chromatography Coupled to High Resolution Mass Spectrometry.

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Summary of "Orthogonal Middle-up Approaches for the Characterization of the Glycan Heterogeneity of Etanercept by Hydrophilic Interaction Chromatography Coupled to High Resolution Mass Spectrometry."

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 QTOF/MS instrument to assess glycosylation of etanercept at the middle-up level of analysis (fragments of circa 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.


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Name: Analytical chemistry
ISSN: 1520-6882


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