Rituximab Glycosylation Activity Assay

Activity on fully glycosylated Rituximab was measured using 5 µM Rituximab with 1 µM AlfC_WT or AlfC_E274A in PBS for 1 month at 25 °C. Activity on partially deglycosylated Rituximab was measured using 5 µM Rituximab with 50 nM EndoS2 and 1 µM AlfC_WT in PBS overnight at 25 °C. EndoS2 rapidly hydrolyzes antibody glycans between the first and second N-acetylglucosamine residues, leaving one N-acetylglucosamine residue and fucose attached to each heavy chain of the antibody. After the designated incubation, 10 μl aliquots of each reactions were quenched by the addition of 1.1 μl trifluoroacetic acid. The quenched reactions were then mixed with 50 mM TCEP, and analyzed by LC–MS using an Accela LC System attached to an LXQ linear ion trap mass spectrometer (ThermoScientific, Waltham, MA), as previously described^{39 (link)}. Relative amounts of substrate and hydrolysis products were quantified after deconvolution of the raw data using BioWorks (ThermoScientific, Waltham, MA). Relative intensities by mass were then exported to GraphPad Prism and replotted, overlayed, and annotated.