Effects of Glycan Structure on the Stability and Receptor Binding of an IgG4-Fc

A series of well-defined N-glycosylated IgG4-Fc variants were utilized to investigate the effect of glycan structure on their physicochemical properties (conformational stability and photostability) and interactions with an Fc γ receptor IIIA (FcγRIIIA). High mannose (HM, GlcNAc₂Man_(8+n) [n = 0-4]), Man₅ (GlcNAc₂Man₅), GlcNAc₁, and N297Q IgG4-Fc were prepared in good quality. The physical stability of these IgG4-Fc variants was examined with differential scanning calorimetry and intrinsic fluorescence spectroscopy. Photostability was assessed after photoirradiation 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 FcγRIIIA by at least 14.7-fold compared with 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 nonglycosylated N297Q IgG4-Fc did not present measurable affinity to FcγRIIIA.