A Study on Nucleation for Protein Crystallization in Mixed Vessels

Abstract

This study investigated mechanisms of secondary nucleation for the crystallization of the protein hen egg white lysozyme. A parametric study of the secondary nucleation of lysozyme in a stirred tank crystallizer was performed, testing the influence of stirrer speed, the size and volume of added seed crystals, solution temperature, supersaturation, and sodium chloride concentration on measured nucleation rates. The measured rates were highly variable, which made assessment of the influence of experimental parameters difficult. Only stirrer speed exhibited significant influence over experimental variability (10-fold increase in nucleation rates with a 50% increase in stirring speed). These results were interpreted by comparison with parameter influence that is commonly observed with secondary nucleation of nonproteins. A novel tubular crystallizer was used to assess the influence on secondary nucleation of fluid shear forces acting on lysozyme crystals. It was observed that fluid shear forces acting on mounted lysozyme crystals do not notably influence secondary nucleation. Overall, the results suggest that attrition is an important source of secondary nuclei in lysozyme crystallization and that secondary nucleation caused solely by fluid shear forces is not.