Molecular thermodynamics of aqueous two‐phase systems for bioseparations

Aqueous polymer-polymer two-phase systems provide a powerful method for separating biomolecules by extraction. When a complex mixture of biomolecules (e.g., a fermentation broth or a solution of lysed cells) is added to such a system, biomolecules partition uniquely between the two phases, achieving separation. A thermodynamic framework is presented for optimizing extraction performance in biological separations. First, a molecular-thermodynamic model, based on the osmotic virial equation, is proposed to describe phase equilibria for dilute aqueous mixtures containing polymers and protein. Second, experimental phase-equilibrium data (protein partition coefficients) are reported for a number of model proteins including albumin, lysozyme, and α-chymotrypsin. To interpret and correlate the experimental data, Low-Angle Laser-Light Scattering (LALLS) measurements were made to determine osmotic second virial coefficients for aqueous mixtures containing polymers, proteins, salts (KCl, KH2PO4 and K2SO4 at concentrations of 50 and 100 mM) and several combinations of polymer-polymer and polymer-protein pairs. Combined with electrochemical measurements (differences in potential between the two phases and protein net charge), these data provide parameters for the model to calculate the desired phase equilibria. A comparison of calculated and experimental results indicates that the virial-equation model provides good prediction of binodals and a reliable basis for estimating infinite-dilution protein partition coefficients for biotechnical process design.