Aqueous two-phase system formation is the macroscopic completion of liquid-liquid phase separation, a process by which aqueous solutions demix into two distinct phases. Concentrated liquid formulations of therapeutic antibodies are susceptible to phase separation, which compromises their stability. The mechanism of phase separation, however, has remained elusive because the temperature-dependent kinetics is cumbersome to measure. Herein, we overcome the problem of rapidly obtaining kinetic data by capturing dark-field images of protein-rich droplet suspensions as a function of time along a linear temperature gradient. These studies indicate that the kinetics of aqueous two-phase system formation is largely governed by the ability of proteins to dissociate from protein-rich droplets. We quantify the apparent activation energies for this dissociation and for droplet coalescence. Under certain conditions, we observe the formation of kinetically trapped gels that arrest phase separation. This work proposes a simple two-step mechanism for liquid-liquid phase separation that may help in formulating stable biopharmaceuticals and understanding the phase behavior of biological and colloidal systems.

Two-step model for liquid-liquid phase separation in therapeutic antibody solutions

2019 Fall National Meeting

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