One-pot enzymatic glycan remodeling of a therapeutic monoclonal antibody by endoglycosidase S (Endo-S) from Streptococcus pyogenes

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.

Mass spectrometric analysis of core fucosylation and sequence variation in a human–camelid monoclonal antibody

“…In order to retain the core GlcNAc and fucose residues, the purified protein was bound to immobilized endoglycosidase S (deGlyIT, Genovis, Cambridge MA) to hydrolyze the b-1,4 linkage between the variable glycans and the core GlcNAc residue. 42,43 One preliminary sample of EG2-hFc from another experiment 39 was prepared from media containing 40 mM 2-fluoroperacetylated fucose (2FF), a competitive inhibitor of the FUT8 enzyme that normally adds the fucose to the core GlcNAc. 44 SDS-PAGE gels were routinely run to ascertain purity and recovery of the protein at each stage of protein preparation.…”

Chemoenzymatic Methods for the Synthesis of Glycoproteins

“…Later on it was verified that the wild type Endo-S catalyzed transglycosylation with Man 3 GlcNAc oxazoline ended up with very low yield, partly due to quick hydrolysis of the Man 3 GlcNAc oxazoline by the wild type enzyme. 264 At about the same time, Wang and co-workers reported the first example of glycosynthases from the GH18 family ENGases. An array of mutants, including EndoS-D233A and D233Q, were generated by site-directed mutagenesis of Endo-S, an ENGase from Streptococcus pyogenes.…”

Glycan Remodeling of Glycoproteins Using ENGases

“…In particular, they focused on the operational stability, reusability and high reactivity of the immobilized enzyme. Furthermore, Wang et al (53) reported the transglycosylation of wild type Endo S using high mannose or hybrid type N-glycan oxazolines, which could not hydrolyze the corresponding product as substrate. This is also one solution, that the synthesized glycoprotein has high stability under the presence of Endo S.…”