Fig. 5: Remodeling of IgG-Fc N-glycans on trastuzumab using Sx-GTs.
From: A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans
a Schematic of bioenzymatic routes to hybrid- and complex-type N-glycan structures linked to asparagine 297 (N297) of the trastuzumab antibody. Trastuzumab bearing Man5GlcNAc2 glycan (M5; glycan 11) derived from glycoengineered HEK293F lacking GnTI activity was used as a glycan primer. Subsequent cell-free glycan remodeling reactions yielded the following N-glycan structures: 12 (M5+GlcNAc); 2 (G0-GlcNAc); 3 (G0); 4 (G2); and 6 (G2S2). Glycan notation follows IgG glycan short naming system. For a complete glycan list with chemical structures, see Supplementary Table 2. SIMPLEx-reformatted GTs and glycosidase for each synthesis step are provided above reaction arrow. b Deconvoluted LC-MS spectra in 140–160 kDa range using intact antibody analysis of trastuzumab bearing glycan 11 as starting material and enzymatically derived product glycans 2–4, 6, and 12. Structures of anticipated N-glycan products are provided in each spectrum. Full MS spectra (0–200 kDa) for all structures are provided in Supplementary Fig. 13.
