Protein crystallization has found little use as a separation/purification technique in a bioprocess industry despite the high purity levels and good separation factors obtained by crystallization. One of the major drawbacks has been the lack of thermodynamic and kinetic data required for efficient crystallization. Here, the population balance model has been used as a relatively easy but efficient way to obtain the kinetic constants for nucleation and crystal growth. Both nucleation and crystal growth rate constants were obtained as functions of supersaturation by a combination of experimental observations and mathematical modeling at atmospheric pressure (0.1MPa) and at three high pressures (34, 68 and 100MPa). Nucleation and growth rate constants were an order of magnitude lower at the higher pressures. It is hypothesized that the protein molecules are in a state of aggregation under pressure, which is not favorable for both nucleation and crystal growth. Transition state theory was used to evaluate the effects of pressure on nucleation and growth rates. The activation volumes (ΔV*) for nucleation and crystal growth were estimated to be +90 and +40cm3/mol, respectively, though the behavior did not conform well to this model.

Crystallization of lysozyme, initially at a concentration of 40 mg/mL, a pH of 4.5 and 0.8M NaCl was carried out at pressures of 0.1, 50, 100, and 150 MPa (1–1500 bar). Pressurization increased the rate of nucleation but reduced the rate of the subsequent crystal growth. An overall enhancement of the rate of crystallization could be obtained by a period of pressurization followed by growth at atmospheric pressure. Initial durations at elevated pressure of either 10 min or 2 h, followed by growth at atmospheric pressure for a total period (including the time of pressurization) of 12, 24, 30 and 36 h resulted in overall crystallization rates that were 2–3 times higher than obtained without pressurization. Enhancements were not evident when the same procedure was used with initial lysozyme concentrations of 19 and 28 mg/mL. No loss of enzymatic activity was observed in these treatments.