Site-specific immobilization of endoglycosidases for streamlined chemoenzymatic glycan remodeling of antibodies

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.

Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation

“…The crude transfer product was purified with a protein A column (GE Healthcare) and promptly dialyzed against a buffer (PBS, 150 mM, pH 7.4) at 4 °C to give the pure core fucosylated antibody. To characterize the core fucosylated antibody, immobilized EndoS2 40 (0.1 mg/ml) and AlfC (0.5 mg/ml) were added to a solution (PBS, 150 mM, pH 7.4, 20 µl) containing the S0G2F-Herceptin (200 µg, 0.007 µmol). The reaction mixture was incubated at 30 °C for 12 h. The crude hydrolytic product was purified with a protein A column and dialyzed against a buffer (PBS, 150 mM, pH 7.4) at 4 °C to give the pure deglycosylated, defucosylated antibody.…”

Sialylated immunoglobulin G: a promising diagnostic and therapeutic strategy for autoimmune diseases

“…The pair of Endo-S and its mutant (Endo-S-D233Q) showed great efficiency in generating bisialylated mAbs, including rituximab and trastuzumab 25 , 160 , 161 . A glycosylase mutant (Endo-S2-D184M) from Endo-S2 was developed by Wang et al 162 , 163 , with improved efficiency of transglycosylation. Additionally, Endo-S2 can cleave all three types of Fc N -glycans, including hybrid, high mannose and complex types 164 - 166 .…”

Innovative Preparation of Biopharmaceuticals Using Transglycosylation Activity of Microbial Endoglycosidases

“…Li et al reported the preparation of immobilized endoglycosidases of Endo-S2 and its glycosynthase mutant D184M using a recombinant microbial transglutaminase for chemo-enzymatic glycan remodeling of antibodies. 58) Recently, Giddens et al reported the site-selective remodeling of the N- glycans of both Fc (attached to Asn-297 of the heavy chain) and Fab (attached to Asn-88 of the heavy chain) of Cetuximab, a therapeutic mAb used for the treatment of a variety of cancers. 59) This work was completed using the high selectivity of the Endo-F3-D165A glycosynthase mutant, which only glycosylates core-fucosylated GlcNAc residues and is unable to bind glycans to non-fucosylated GlcNAc residues, and the Endo-S-D233A glycosynthase mutant, which only glycosylates GlcNAc residues at Asn-297 of IgGs.…”