Rapid N-glycan release from glycoproteins using immobilized PNGase F microcolumns

Szigeti, Márton and Bondar, Judit and Gjerde, Douglas and Keresztessy, Zsolt and Szekrényes, Ákos and Guttman, András (2016) Rapid N-glycan release from glycoproteins using immobilized PNGase F microcolumns. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. ISSN 1570-0232 (In Press)

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Abstract

N-glycosylation profiling of glycoprotein biotherapeutics is an essential step in each phase of product development in the biopharmaceutical industry. For example, during clone selection, hundreds of clonesshould be analyzed quickly from limited amounts of samples. On the other hand, identification of diseaserelated glycosylation alterations can serve as early indicators (glycobiomarkers) for various pathological conditions in the biomedical field. Therefore, there is a growing demand for rapid and easy to automate sample preparation methods for N-glycosylation analysis. In this paper, we report on the design and implementation of immobilized recombinant glutathione S-transferase (GST) tagged PNGase F enzymemicrocolumns for rapid and efficient removal of N-linked carbohydrates from glycoproteins. Digestionspeed and efficiency were compared to conventional in-solution based protocols. The use of PNGase Ffunctionalized microcolumns resulted in efficient N-glycan removal in 10 min from all major N-linkedglycoprotein types of: (i) neutral (IgG), (ii) highly sialylated (fetuin), and (iii) high mannose (ribonucleaseB) carbohydrate containing glycoprotein standards. The approach can be readily applied to automatedsample preparation systems, such as liquid handling robots.

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