Modulation of the Kinetic Stability of Immunoglobulin G by Solvent Additives

Abstract

Stability of Immunoglobulin G (IgG) is a crucial parameter that affects important aspects of this multidomain protein such as its production, storage and usability. The complex thermal and isothermal transitions of IgGs as well as their irreversibilities pose a significant challenge to the proper determination of parameters describing the stability of IgGs. Here, we have examined the effects of additives on the kinetic stability of IgGs by differential scanning calorimetry (DSC). The thermal denaturation of full-length IgGs was examined by three-step model, simulating the observed three consecutive transitions (one reversible, two irreversible), while the kinetic stability of the corresponding Fab fragments was analyzed according to the Lumry-Eyring model specifying a single irreversible transition. Ten additives were analyzed: polyols (sorbitol, sucrose and trehalose), salts (sodium perchlorate, chloride and sulfate, including arginine) and methylamines (betaine, sarcosine and trimethylamine oxide). Osmolytes (polyols and methylamines) and salts were found to affect the stability of IgGs differently as seen both in different DSC profiles and in the diverse effects of the additives on parameters characterizing the irreversible step. Our findings highlight the influence of both the additives as well as stabilizing mutations in the Fab fragment on the kinetic stability of the whole IgG. These conclusions could only be reached through proper analyses of thermal denaturations of IgGs using the appropriate model simulating their irreversible thermal transitions, and may help in the development of more predictive strategies for formulation development. Acknowledgements The authors thank Dr. Yuguang Zhao (Wellcome Trust Centre for Human Genetics, Oxford University) for his help in IgG expression and Dr. Peter Gimeson (Malvern Instruments, Uppsala, Sweden) for his help in performing DSC experiments. This work was supported by grants from CELIM (316310) funded by 7FP EU Programs REGPOT and PCUBE.