Aqueous solutions of macromolecules are susceptible to condensations, such as crystallization, aggregation, and phase separation, which are all driven by attractive intermolecular interactions. Protein condensations are of particular importance due to their role in the instability of biopharmaceutical drug formulations and the assembly of membrane-less organelles in biology. Despite their ubiquity, the underlying mechanisms of protein condensations are still not fully understood. Here, we studied the thermodynamics and kinetics of liquid-liquid phase separation (LLPS) in concentrated protein solutions, the process by which a homogenous solution macroscopically separates into a lower, protein-rich phase and an upper, protein-poor phase. The separations were measured over time by light scattering obtained via dark-field microscopy along a temperature gradient microfluidic device. Our analysis of the temperature-dependent kinetics of LLPS provided evidence for a two-stage mechanism, involving the nucleation and subsequent coalescence of LLPS droplets into a macroscopic protein-rich phase. By modulating the concentration of protein and a crowding agent, polyethylene glycol, we revealed the relationship of this mechanism to the thermodynamic regions of a colloidal phase diagram. This work provides a general mechanistic framework for metastability, spinodal decomposition, and gelation in concentrated solutions of macromolecules.

Exploring the mechanisms of liquid-liquid phase separation in concentrated protein solutions

254th National Meeting (2017)

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