Purpose: Light exposure is an important stress condition for protein pharmaceuticals. During light-exposure, photo-sensitive amino acids such as Trp, Cys, Tyr and Phe convert into oxidation products. Recently, light-induced, non-conventional chemical and especially oxidative modifications of mAbs have been recognized to play a potentially critical role for immunogenicity. Thus, a series of IgG4-Fc derivatives with N-glycosylation at Asn297 and/or O-glycosylation at Ser375 were utilized as model systems to identify light-induced non-conventional oxidation products, and to investigate the correlation of photooxidation with glycosylation.
Methods: High mannose IgG4-Fc (HM, GlcNAc2-Man(8+n) (n= 0-4)) was prepared by yeast expression, followed by protein G and hydrophobic interaction chromatography purification. Man5 (GlcNAc2-Man5) and GlcNAc1 were prepared through an in vitro enzymatic approach. Nonglycoform N297Q, and several other site-directed mutants, W381F, W381A (O-mannosylation enriched), and H435R/Y436F, were produced. Photoirradiation was performed in a Rayonet® UV irradiator equipped with four lamps emitting at either λ =254 nm or λmax = 305 nm. Chemical modifications were analyzed by peptide mapping and HPLC-MS/MS. Conformational stability of glycoforms (HM, Man5, GlcNAc1, N297Q, and O-mannosylation) was examined with differential scanning calorimetry (DSC) and intrinsic fluorescence over a temperature range of 10-90°C.
Results: The photoirradiation of IgG4-Fc in air-saturated solutions revealed multiple photo-products originating from Tyr 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. Product formation showed either no (cleavage of Tyr373) or significant (cleavage of Tyr436) inverse product solvent isotope effects (SIE), indicating a role for proton transfer in the cleavage mechanism of Tyr436. The role of electron transfer in the cleavage of Tyr436 was investigated through mutation of an adjacent Trp381, W381F and W381A. 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.
Conclusion: Light-induced Tyr side chain fragmentation is reported in IgG4-Fc. Tyr side chain fragmentation products may be relevant for light-induced inactivation, aggregation, fragmentation or immunogenicity in protein therapeutics, and therefore, need to be carefully characterized and monitored. Studies regarding to how glycosylation affects protein pharmaceuticals, and their correlation with physico-chemical stability, are of significance to build a bridge between glyco-engineering and the development of therapeutic proteins.