Orthogonal Middle-up Approaches for Characterization of the Glycan Heterogeneity of Etanercept by Hydrophilic Interaction Chromatography Coupled to High-Resolution Mass Spectrometry
Language English Country United States Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- Bacterial Proteins chemistry MeSH
- Chromatography methods MeSH
- Etanercept chemistry MeSH
- Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase chemistry MeSH
- Glycosylation MeSH
- Mass Spectrometry methods MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Neuraminidase chemistry MeSH
- Peptide Hydrolases chemistry MeSH
- Streptococcus pyogenes enzymology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Bacterial Proteins MeSH
- Etanercept MeSH
- Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase MeSH
- Neuraminidase MeSH
- Peptide Hydrolases MeSH
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 Chemistry Gustavus Adolphus College St Peter Minnesota 56082 United States
Waters Corporation 34 Maple Street Milford Massachusetts 01757 3696 United States
References provided by Crossref.org
Glycan-specific precipitation of glycopeptides in high organic content sample solvents used in HILIC
