(198b) Homogeneous Nucleation and Size Distributions of Protein Crystals Nucleated and Grown in Weightlessness

Sportiello, M., University of Colorado
The crystallization of bioproducts (especially proteins) for purification and, more recently and more importantly, for delivery demands smaller and more uniform crystal populations. This can only be achieved by maximizing the number of nucleation sites, which in turn requires that nucleation be homogeneous -- independent of pre-existing particles or surfaces. In traditional reactors, nucleation is achieved by rapid mixing under conditions of homogeneous isotropic turbulence (HIT). Earlier studies found, at first by accident and then deliberately, that protein crystals that nucleated, presumably homogeneously, in orbital space-borne reactors at reduced gravity occurred at higher numbers and greater uniformity than expected. Experiments were performed on the U. S. space shuttle and the Russian Mir space station using several crystallization reactors based on a “Fluids Processing Apparatus” (FPA) developed and provided by BioServe Space Technologies (University of Colorado, Boulder). Hen egg lysozyme, sodium chloride and 0.01 M acetate pH 4.0 were used as protein, precipitant and buffer, respectively. In each sample 1.5 mL of protein was injected into 2.0 mL of precipitant over a period of 1 minute. Identical experiments were performed on orbit and in the laboratory. Crystal size distributions were determined with the aid of ImageJ image analysis software. In all cases coefficients of variation of populations that were crystallized on orbiting spacecraft were less than those of populations crystallized on the ground. On the spacecraft, essentially all crystals nucleated in suspension and not on reactor vessel walls. Crystals formed in low gravity had especially smooth, shiny facets implying very low mosaicity. They were also very stable over time, seeming to remain unchanged for up to 22 years in mother liquor at ambient temperature. Apparently the lack of turbulence and lack of sedimentation in low gravity resulted in minimal crystal motion in any direction until the cessation of growth due to depletion of solute in the diffusion-governed environment. These orbital low-gravity studies suggest that, instead of turbulent mixing, which transports crystals through variable and unpredictable concentrations of additional protein molecules, a quiescent environment for combining protein and precipitant may lead to the enhanced size limitation and uniformity desired to achieve certain purification and/or formulation goals. Research supported by NASA Grants NAGW-1197 and NAG 8-1165.