A stepwise mechanism for aqueous two-phase system formation in concentrated antibody solutions

Significance Concentrated liquid formulations of therapeutic antibodies are susceptible to colloidal instability, including liquid–liquid phase separation (LLPS). Herein, we measure the kinetics of aqueous two-phase system (ATPS) formation, the macroscopic stage of LLPS, along a linear temperature gradient device. These studies indicate that ATPS formation proceeds by the growth of protein-rich droplets and their subsequent coalescence. Droplet growth accelerates as the solution is cooled because protein–protein interactions become increasingly free energetically favorable. At lower temperatures, however, ATPS formation is rate-limited by a small barrier to droplet coalescence. A gel can form at the coldest temperatures, which arrest phase separation. We propose a simple 2-step model that relates these results to a reaction coordinate diagram for ATPS formation.