Quantitative Evaluation of Protein Solubility in Aqueous Solutions by PEG-Induced Liquid–Liquid Phase Separation
This chapter describes an experimental method to quantitatively evaluate the solubility of proteins in aqueous solutions. Measurement of protein solubility can be challenging because low solubility can be manifested through various pathways (e.g., crystallization, aggregation, gelation, and liquid–liquid phase separation), some of which may occur over long periods of time. In the method described here, a nonionic polymer, polyethylene glycol (PEG), is added to a protein solution of interest to induce instantaneous formation of protein-rich liquid droplets. After incubation at a given temperature, the samples are centrifuged. The protein concentration in the supernatant is measured at various PEG concentrations to calculate an equilibrium binding free energy, which provides a measure of protein solubility. Based on the first principles of thermodynamics, this method is highly reproducible and applicable to various proteins and buffer conditions.
Key wordsProtein solubility Colloidal stability Aggregation Crystallization Liquid–liquid phase separation PEG Gelation Precipitation
- 9.Thompson RW Jr, Latypov RF, Wang Y, Lomakin A, Meyer JA, Vunnum S, Benedek GB (2016) Evaluation of effects of pH and ionic strength on colloidal stability of IgG solutions by PEG-induced liquid-liquid phase separation. J Chem Phys 145(18):185101. https://doi.org/10.1063/1.4966708CrossRefPubMedGoogle Scholar
- 13.Banks DD, Latypov RF, Ketchem RR, Woodard J, Scavezze JL, Siska CC (2012) Razinkov VI native-state solubility and transfer free energy as predictive tools for selecting excipients to include in protein formulation development studies. J Pharm Sci 101(8):2720–2732. https://doi.org/10.1002/jps.23219CrossRefPubMedGoogle Scholar