Abstract
Chemoenzymatic glycan remodeling of antibodies using an endoglycosidase and its mutant is emerging as an attractive approach for producing homogeneous antibody glycoforms. We report in this paper a site-specific covalent immobilization of the endoglycosidases (Endo-S2 and its glycosynthase mutant D184M) using a recombinant microbial transglutaminase (MTG) and evaluation of the immobilized enzymes in deglycosylation and glycosylation of a therapeutic antibody. The site-specific covalent immobilization was achieved by introduction of a Q-tag at the C-terminus of the recombinant enzymes followed by conjugation of the enzymes to a primary amine-containing solid support through MTG-catalyzed transglutamination. Using rituximab as a model system, we found that the Endo-S2 wild-type and D184M glycosynthase mutant immobilized by this approach were efficient in the two step antibody glycan remodeling to generate homogeneous antibody glycoforms. Notably using the covalently immobilized enzymes can efficiently avoid the need of intermediate purification and eliminate the residual contamination of wild type enzyme for product hydrolysis, thus streamlining the chemoenzymatic Fc glycan remodeling of antibodies.