EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans.

Enzymes that affect glycoproteins of the human immune system, and thereby modulate defense responses, are abundant among bacterial pathogens. Two endoglycosidases from the human pathogen Streptococcus pyogenes, EndoS and EndoS2, have recently been shown to hydrolyze N-linked glycans of human IgG. However, detailed characterization and comparison of the hydrolyzing activities have not been performed. In the present study we set out to characterize the enzymes by comparing the activities of EndoS and EndoS2 on a selection of therapeutic monoclonal antibodies (mAbs), cetuximab, adalimumab, panitumumab, and denosumab. By analyzing the glycans hydrolyzed by EndoS and EndoS2 from the antibodies using MALDI-TOF, we found that both enzymes cleaved complex glycans, and that EndoS2 hydrolyzed hybrid and oligomannose structures to a greater extent compared to EndoS. A comparison of UHPLC profiles of the glycan pool of cetuximab hydrolyzed with EndoS and EndoS2 showed that EndoS2 hydrolyzed hybrid and oligomannose glycans whereas these peaks were missing in the EndoS chromatogram. We utilized this difference in glycoform selectivity and developed a liquid chromatography separation method to quantify high-mannose content in the Fc fragments of the selected mAbs. We conclude that EndoS and EndoS2 hydrolyze different glycoforms from the Fc-glycosylation site on therapeutic mAbs, and that this can be used for rapid quantification of high-mannose content.