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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2016-10, Vol.1032, p.139-143
• Main Authors: Szigeti, Marton, Bondar, Judit, Gjerde, Douglas, Keresztessy, Zsolt, Szekrenyes, Akos, Guttman, Andras
• Format: Article
• Language: English
• Subjects: Animals; Capillary electrophoresis; Electrophoresis, Capillary - methods; Enzyme immobilization; Enzymes, Immobilized - chemistry; Escherichia coli - enzymology; Fetuins - chemistry; Glycoproteins - chemistry; Immunoglobulin G - chemistry; N-glycosylation; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - chemistry; PNGase F; Polysaccharides - isolation & purification; Recombinant Fusion Proteins - chemistry; Ribonucleases - chemistry
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2016.02.006

•Immobilized recombinant glutathione-S-transferase (GST) tagged PNGase F enzyme microcolumns have been developed.•Rapid and efficient removal of N-linked carbohydrates from glycoproteins has been proved.•The method was tested with all major N-linked glycoprotein types of: (i) neutral (IgG), (ii) highly sialylated (fetuin), and (iii) high mannose (ribonuclease B) carbohydrate containing glycoprotein standards. 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 clones should be analyzed quickly from limited amounts of samples. On the other hand, identification of disease related 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 enzyme microcolumns for rapid and efficient removal of N-linked carbohydrates from glycoproteins. Digestion speed and efficiency were compared to conventional in-solution based protocols. The use of PNGase F functionalized microcolumns resulted in efficient N-glycan removal in 10min from all major N-linked glycoprotein types of: (i) neutral (IgG), (ii) highly sialylated (fetuin), and (iii) high mannose (ribonuclease B) carbohydrate containing glycoprotein standards. The approach can be readily applied to automated sample preparation systems, such as liquid handling robots.