title: EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans.
abstract: 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 immunoglobulin G. 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 matrix-assisted laser desorption ionization time of flight, we found that both the enzymes cleaved complex glycans and that EndoS2 hydrolyzed hybrid and oligomannose structures to a greater extent compared with EndoS. A comparison of ultra-high-performance liquid chromatography (LC) 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, in combination with the IdeS protease, and developed a LC 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.
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund 221 84, Sweden Genovis AB, Lund 200 07, Sweden jonathan.sjogren@med.lu.se.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Sjögren
authors.affiliation: Waters Corporation, Milford, MA 01757, USA.
authors.forename: Eoin F J
authors.initials: EF
authors.lastname: Cosgrave
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund 221 84, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Allhorn
authors.affiliation: Genovis AB, Lund 200 07, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Nordgren
authors.affiliation: Genovis AB, Lund 200 07, Sweden.
authors.forename: Stephan
authors.initials: S
authors.lastname: Björk
authors.affiliation: Genovis AB, Lund 200 07, Sweden.
authors.forename: Fredrik
authors.initials: F
authors.lastname: Olsson
authors.affiliation: Genovis AB, Lund 200 07, Sweden.
authors.forename: Sarah
authors.initials: S
authors.lastname: Fredriksson
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund 221 84, Sweden.
authors.forename: Mattias
authors.initials: M
authors.lastname: Collin
mesh_terms: D000068879:Adalimumab / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D000068818:Cetuximab / Q000737:chemistry; D000069448:Denosumab / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006868:Hydrolysis; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D008351:Mannans / Q000032:analysis* / Q000737:chemistry; D000077544:Panitumumab; D011134:Polysaccharides / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D007141:Immunoglobulin Fc Fragments; D008351:Mannans; D011134:Polysaccharides; D000069448:Denosumab; D000077544:Panitumumab; D006026:Glycoside Hydrolases; C431325:NDOS protein, Streptococcus pyogenes; D000068879:Adalimumab; D000068818:Cetuximab
keywords: EndoS; IgG; Streptococcus pyogenes; endoglycosidase; glycosylation