Effects of glycan structure on the stability and receptor binding of an IgG4-Fc

Abstract A series of well-defined N-glycosylated IgG4-Fc variants were utilized to investigate the effect of glycan structure on their physico-chemical properties (conformational stability and photostability) and interactions with an Fc ɣ receptor IIIA (FcɣRIIIA). High mannosylated (HM, GlcNAc2-Man(8+n) (n= 0-4)), Man5 (GlcNAc2-Man5), GlcNAc1 and N297Q IgG4-Fc were prepared in good quality. The physical stability of these IgG4-Fc variants was examined with differential scanning calorimetry (DSC) and intrinsic fluorescence spectroscopy. Photo-stability was assessed after photo-irradiation between 295 and 340 nm (λ max = 305 nm), and HPLC-MS/MS analysis of specific products was performed. The size of glycans at Asn297 affects the yields of light-induced Tyr side chain fragmentation products, where the yields decreased in the following order: N297Q > GlcNAc1 > Man5 > HM. These yields correlate with the thermal stability of the glycoforms. The HM and Man5 glycoforms display increased affinity for FcɣRIIIA by at least 14.7-fold compared to GlcNAc1 IgG4-Fc. The affinities measured for the HM and Man5 IgG4-Fc (0.39 -0.52 μM) are similar to those measured for fucosylated IgG1. Dependent on the mechanisms of action of IgG4 therapeutics, such glycoforms may need to be carefully monitored. The non-glycosylated N297Q IgG4-Fc did not present measurable affinity to FcɣRIIIA.