One-pot enzymatic glycan remodeling of a therapeutic monoclonal antibody by endoglycosidase S (Endo-S) from Streptococcus pyogenes |
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Authors: | Xin Tong Tiezheng Li Jared Orwenyo Christian Toonstra Lai-Xi Wang |
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Institution: | Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA |
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Abstract: | A facile, one-pot enzymatic glycan remodeling of antibody rituximab to produce homogeneous high-mannose and hybrid type antibody glycoforms is described. This method was based on the unique substrate specificity of the endoglycosidase S (Endo-S) from Streptococcus pyogenes. While Endo-S efficiently hydrolyzes the bi-antennary complex type IgG Fc N-glycans, we found that Endo-S did not hydrolyze the “ground state” high-mannose or hybrid glycoforms, and only slowly hydrolyzed the highly activated high-mannose or hybrid N-glycan oxazolines. Moreover, we found that wild-type Endo-S could efficiently use high-mannose or hybrid glycan oxazolines for transglycosylation without product hydrolysis. The combination of the remarkable difference in substrate specificity of Endo-S allows the deglycosylation of heterogeneous rituximab and the transglycosylation with glycan oxazoline to take place in one-pot without the need of isolating the deglycosylated intermediate or changing the enzyme to afford the high-mannose type, hybrid type, and some selectively modified truncated form of antibody glycoforms. |
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Keywords: | Monoclonal antibody Fc glycosylation Glycoengineering Endoglycosidase Transglycosylation Endo S Chemoenzymatic synthesis Rituximab |
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