N‐glycan analysis of IgG by enzymatic digestion with Remove‐iT® Endo S and PNGase F (1004.1)

Objective: The objective of this study was to compare the release of N‐glycans from different sources of IgG using a novel endoglycosidase with a chitin‐binding domain tag (Remove‐iT Endo S) and PNGase F. Biotechnology companies are currently developing a growing number of monoclonal IgG antibodies as therapeutic agents. Critical for the structure and biological activity of the molecule is the N‐glycan moiety attached to the asparagine 297 residue in the constant domain of the antibody. There are many variables in cell culture systems that can greatly influence the heterogeneity of the glycans on IgG. For this reason it has become increasingly important to monitor the glycosylation profiles of these biotherapeutics in the production process. Methods: Remove‐iT Endo S (also known as Endoglycosidase S) was cloned from Streptococcus pyogenes and overexpressed in E. coli as a fusion to the chitin‐binding domain. IgG samples were enzymatically deglycosylated under native conditions using Remove‐iT Endo S and PNGase F Glycerol Free. The deglycosylated IgG samples were then analyzed by SDS‐PAGE gel shift analysis and ESI‐TOF MS to estimate the degree of deglycosylation. In addition, the glycans released from IgG following deglycosylation by both enzymes were analyzed using nano LC‐TOF MS. Results: Remove‐iT Endo S has a high specificity for removing the N‐glycan moiety of IgG after the first N‐acetylglucosamine (GlcNAc) residue, leaving only the GlcNAc with or without a core fucose residue. However, PNGase F cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from N‐linked glycoproteins. Conclusion: Remove‐iT Endo S is a more robust enzyme for the method described herein as it completely removes the sugar residues from IgG. Conversely, the PNGase F glycerol free digest does not result in a complete digestion under native conditions.