Heterogeneity of Protein Environments in Frozen Solutions and in the Dried State

Freezing and freeze-drying of protein formulations often produce multiple populations of protein molecules, with different local environments and different stabilities. Such heterogeneity is commonly related to formation of ice, and includes concentration gradients created due to difference in the diffusion coefficients of proteins and other solutions, redistribution of the charged species and electric potential on the ice/solution interface, and solution inclusions by ice crystals. Also, one should expect heterogeneity to exist in freeze-concentrated solution even if ice-related inhomogeneity cases are eliminated, as heterogeneity is a fundamental property of amorphous systems. As a result of heterogeneity, shelf life of a pharmaceutical protein formulation would be limited by the most unstable population of protein molecules, which may represent a relatively minor fraction. Identifying this least stable portion of protein molecules and targeting formulation development efforts on this fraction, rather than going after the main (and potentially the most stable) part, would allow a formulator to optimize stabilization and formulation development efforts.