Comparison of the Analysis of Fluorophore Labeled N-Glycans Using Hilic-Uplc and Multicapillary Gel Electrophoresis

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Comparison of the Analysis of Fluorophore Labeled N-Glycans Using Hilic-Uplc and Multicapillary Gel Electrophoresis

Csenge Filep 1 , Beáta Borza 1 , Márton Szigeti 1 , Zuzana Demianova 2 , András Guttman 1,2

1 Horvath Csaba Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary, 2 Sciex, Brea, United States

As glycomics research is getting more and more important for the biopharmaceutical industry, there is an increasing need for rapid and reproducible analytical methods to characterize and monitor N-glycosylation of therapeutic antibodies. [1] It is well known that the glycosylation pattern plays a key role in the biological activities of glycobiotherapeutics. Approaches to comprehensively analyze complex carbohydrates comprise numerous bioanalytical methods including mass spectrometry, liquid chromatography, capillary electrophoresis, NMR and exoglycosidase array based sequencing. [2] In this poster the most frequently used high performance liquid phase separation methods for carbohydrate analysis - ultrahigh pressure hydrophilic interaction liquid chromatography (HILIC-UPLC) and capillary electrophoresis (CE) - will be compared (Figure 1), using a multicapillary electrophoresis system. The latter has the great advantage of allowing the analysis of up to 12 samples in parallel, thus supporting high throughput. The effectiveness of the two methods will be compared by separating different fluorophore labeled partitioned carbohydrate libraries (e.g. high-mannose, afucosyl biantennary and fucosyl biantennary) and several high profile glycosylated therapeutic proteins (e.g., Humira and Enbrel).

Figure 1. Analysis of fluorophore-labeled N-glycans of Enbrel by HILIC UPLC (upper panel) and multicapillary gel electrophoresis (lower panel).

Á. Szekrényes et al., “Multi-site N-Glycan mapping study 2: UHPLC,” Electrophoresis, vol. 39, no. 7, pp. 998–1005, Apr. 2018.

A. Guttman, A., Rathore, A.S., Krull, “Bioanalytical Tools for the Characterization of Biologics and Biosimilars,” LCGC North Am., vol. 30, no. 5, 2012.

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