Design of Bulk Protein Crystallization Based on Phase Diagrams Accounting for the Presence of Interfacial Water

An effective procedure for design of protein crystallization was proposed, in which binary and ternary phase diagrams were exploited to evaluate the process performance. A method for the construction of phase diagrams that accounted for the presence of water associated with the protein crystalline phase (interfacial water) was described. The model system. for the study included ovalbumin and lysozyme, which were crystallized from aqueous solutions of ammonium sulfate. The design procedure consisted of the determination of the operating window on the plane of the protein and salt concentrations, quantifying the protein phase behavior within the window, and the construction of the corresponding phase diagrams to select operating conditions that ensure a high yield of the operation. The analysis of the solid phase behavior was supported by differential scanning calorimetry, which was used to evaluate the amount of interfacial water in the protein dense phase, thus, to determine the solid phase composition. The approach proposed was verified by several crystallization experiments.