Advanced analyses of kinetic stabilities of iggs modified by mutations and glycosylation.

The stability of Immunoglobulin G (IgG) affects production, storage and usability, especially in the clinic. The complex thermal and isothermal transitions of IgGs, especially their irreversibilities, pose a challenge to the proper determination of parameters describing their thermodynamic and kinetic stability. Here, we present a reliable mathematical model to study the irreversible thermal denaturations of antibody variants. The model was applied to two unrelated IgGs and their variants with stabilizing mutations as well as corresponding non-glycosylated forms of IgGs and Fab fragments. Thermal denaturations of IgGs were analyzed with three transitions, one reversible transition corresponding to C(H)2 domain unfolding followed by two consecutive irreversible transitions corresponding to Fab and C(H)3 domains, respectively. The parameters obtained allowed us to examine the effects of these mutations on the stabilities of individual domains within the full-length IgG. We found that the kinetic stability of the individual Fab fragment is significantly lowered within the IgG context, possibly because of intramolecular aggregation upon heating, while the stabilizing mutations have an especially beneficial effect. Thermal denaturations of non-glycosylated variants of IgG consist of more than three transitions and could not be analyzed by our model. However, isothermal denaturations demonstrated that the lack of glycosylation affects the stability of all and not just of the C(H)2 domain, suggesting that the partially unfolded domains may interact with each other during unfolding. Investigating thermal denaturation of IgGs according to our model provides a valuable tool for detecting subtle changes in thermodynamic and/or kinetic stabilities of individual domains.