HILIC glycan analysisAnalysis of glycosylation is a topic of growing importance in a number of fields such as antibody and biopharmaceutical characterization and human glycoproteome characterization for disease biomarkers. Variations in glycosylation have been implicated in a number of conditions including autoimmune disease, cancer and infectious disease as well as the correct functioning of the immune system, more details of which can be found in the review article by Zauner et al entitled, Glycoproteomic Analysis of Antibodies, (link to article).

In addition, with increasing interest of using monoclonal antibodies and other glycoproteins as biopharmaceuticals, then the characterization of glycans is critical to ensure the biotherapeutic is safe, stable and effective in vivo. As can be seen in both of these fields, the characterization of glycans is a critical element and UHPLC column technology is evolving to allow such glycan characterization with improved accuracy, resolution and speed.

Typically, the characterization of N-linked glycans has taken the approach of separating enzymatically released and fluorescently-labeled glycans by hydrophilic interaction chromatography (HILIC) with subsequent fluorescent detection. However, HILIC columns separate 2-ab-glycans based on size, but lack any selectivity towards oligosaccharides with different sialic acid content.

The Poster note, An Ultra High Resolution Column and Mass Spectrometer for Isomeric Separation and the Structural Identification of Labeled N-linked Glycans, (downloadable pdf) describes the use of a new mixed mode HPLC column (Thermo Scientific™ GlycanPac™ AXR-1 column) for the separation of both neutral and charged glycans using an UHPLC instrument (Thermo Scientific Dionex™ UltiMate™ 3000 system). The column chemistry combines both weak anion exchange (WAX) for retention and selectivity of negatively charged glycans and reversed phase (RP) which facilitates the separation of glycans of the same charge according to their isomeric structure, polarity and size. The separated glycans were then detected using either a fluorescence or mass spectrometry (Thermo Scientific Orbitrap Fusion™ Tribrid™ Mass Spectrometer) detector.

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The results demonstrated that the use of this new mixed-mode column resolved twice the number of peaks and enabled the identification of four times more glycan structures compared to existing commercial HILIC columns for 2AB-labeled N-Linked glycans released from bovine fetuin.

The image accompanying this post is of the Caspofungin antifungal drug molecule.

Additional Resources

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Are you performing glycan analysis and if so which column chemistries are you using and with what results? I’d like to hear your experiences and comments.

Timothy Cross is a regional marketing manager within EMEA for HPLC in the Chromatography and Mass Spectrometry Division at Thermo Fisher Scientific Inc. As a former scientist and following his Professional Diploma and Postgraduate Professional Diploma in Marketing from the Chartered Institute of Marketing (UK), Timothy has worked in a variety of roles in the life science industry including R&D scientist, product manager, marketing manager and demand generation manager. This background enables Timothy to pursue his passion of truly understanding the scientist’s application needs and requirements and delivering the appropriate workflows, support and total solutions to drive and enhance these applications. Timothy received his B.Sc. in Biological Sciences and M.Sc. and Ph.D. in Immunology from the University of Birmingham (UK).