Xie, Yongjing and Mota, Letícia Martins and Bergin, Adam and O'Flaherty, Roisin and Jones, Aled and Morgan, Bethan and Butler, Michael (2021) High-throughput and high-sensitivity N-Glycan profiling: A platform for biopharmaceutical development and disease biomarker discovery. Analytical Biochemistry, 623. p. 114205. ISSN 0003-2697

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Abstract

Protein glycosylation contributes to critical biological function of glycoproteins. Glycan analysis is essential for the production of biopharmaceuticals as well as for the identification of disease biomarkers. However, glycans are highly heterogeneous, which has considerably hampered the progress of glycomics. Here, we present an improved 96-well plate format platform for streamlined glycan profiling that takes advantage of rapid glycoprotein denaturation, deglycosylation, fluorescent derivatization, and on-matrix glycan clean-up. This approach offers high sensitivity with consistent identification and quantification of diverse N-glycans across multiple samples on a high-throughput scale. We demonstrate its capability for N-glycan profiling of glycoproteins from various sources, including two recombinant monoclonal antibodies produced from Chinese Hamster Ovary cells, EG2-hFc and rituximab, polyclonal antibodies purified from human serum, and total glycoproteins from human serum. Combined with the complementary information obtained by sequential digestion from exoglycosidase arrays, this approach allows the detection and identification of multiple N-glycans in these complex biological samples. The reagents, workflow, and Hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-FLD), are simple enough to be implemented into a straightforward user-friendly setup. This improved technology provides a powerful tool in support of rapid advancement of glycan analysis for biopharmaceutical development and biomarker discovery for clinical disease diagnosis.

Item Type:	Article
Additional Information:	Cite as: Xie, Y., Mota, L.M., Bergin, A., O'Flaherty, R., Jones, A., Morgan, B. & Butler, M. 2021, "High-throughput and high-sensitivity N-Glycan profiling: A platform for biopharmaceutical development and disease biomarker discovery", Analytical biochemistry, vol. 623, pp. 114205-114205.
Keywords:	Glycosylation; Glycomics; Glycan profiling; Antibody; Human serum; Biomarker discovery; HILIC-FLD Exoglycosidase
Academic Unit:	Faculty of Science and Engineering > Chemistry Faculty of Science and Engineering > Research Institutes > Human Health Institute
Item ID:	17442
Identification Number:	https://doi.org/10.1016/j.ab.2021.114205
Depositing User:	Roisin O'Flaherty
Date Deposited:	17 Aug 2023 14:18
Journal or Publication Title:	Analytical Biochemistry
Publisher:	Elsevier Masson
Refereed:	Yes
URI:	Publisher
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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