Physicochemical stability and effector function of IgG4-Fc: impact of photo-induced chemical modification and glycosylation

Immunoglobulin gamma monoclonal antibodies are glycoproteins that have emerged as powerful and promising protein therapeutics. During the process of production, storage and transportation, exposure to ambient light is inevitable, which can cause protein physical and chemical degradation. For mechanistic studies of photo-degradation, we have exposed IgG4-Fc to UV light. The photoirradiation of IgG4-Fc with monochromatic UVC light at λ = 254 nm and UVB light with λmax = 305 nm in air-saturated solutions revealed multiple photo-products originating from tyrosine side chain fragmentation at Tyr300, Tyr373 and Tyr436. Tyr side chain fragmentation yielded either Gly or various backbone cleavage products, including glyoxal amide derivatives. A mechanism is proposed involving intermediate Tyr radical cation formation, either through direct light absorption of Tyr or through electron transfer to an initial Trp radical cation, followed by elimination of quinone methide. In previous studies we discovered that the exposure of IgG4-Fc and IgG1 to UV light resulted in the side chain cleavage of specific Tyr and Trp residues, converting these amino acids into a series of products, including Gly (Haywood et al. Mol. Pharm. 2013, 10, 1146-1150; Kang et al. Mol. Pharm. 2019, 16, 258-272). In order to evaluate the physico-chemical consequences of such photochemical transformations, we prepared a series of IgG4-Fc mutants, in which Trp and Tyr residues were mutated to Gly, i.e., Y300G, Y373G, Y436G, W381G, and W381A for biophysical studies. Among these mutants, Y373G displayed significantly lower melting temperatures compared to wild-type IgG4-Fc, as analyzed by differential scanning calorimetry and fluorescence spectroscopy, indicating a decrease of thermal stability of both the CH2 and CH3 domains. In contrast, W381G and W381A showed no thermal transitions, indicating a significant loss of overall thermal stability. Both, W381A and Y300G IgG4-Fc displayed ca. 10-fold reduction of binding affinity to FcγRIIIA as compared to wild-type IgG4-Fc. Interestingly, W381A and W381G IgG4-Fc did not only contain N-linked glycans but also high levels of O-mannose ( 60%) at Ser375. Furthermore, a series of well-defined N-glycosylated IgG4-Fc variants were utilized as model to investigate the effect of glycan structure on the physico-chemical properties (conformational stability and photostability) and interaction with FcɣRIIIA. 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 presence of HM and Man5 reveals increased affinity for FcɣRIIIA by at least 14.7-fold, as compared to GlcNAc1 IgG4-Fc. N297Q does not present a detectable affinity to FcɣRIIIA