Bulk crystallization is used industrially for the recovery and purification of many inorganic and organic materials. However, very little is reported on the application of bulk crystallization for proteins. In this work, ovalbumin was selected as a model protein in order to investigate the feasibility of using bulk crystallization for the recovery and purification of proteins.
A stirred 1 litre seeded batch crystallizer was used to obtain crystal growth kinetics of ovalbumin from ammonium sulphate solutions at 30 ° C. Ovalbumin solubility data obtained by previous workers was confirmed in this study and a correlation was developed that predicts ovalbumin solubility as a function of ammonium sulphate concentration, pH and temperature with in a error of ± 5 %.
Like many other materials, ovalbumin was also found to exhibit primary and secondary nucleation thresholds. The width of the metastable region, where crystal growth can occur without nucleation, is equivalent to about 20 times the solubility concentration. Laboratory crystallization experiments were undertaken within this range (initial ovalbumin concentrations were less than 10 times the solubility concentration), in the absence of nucleation. The ovalbumin concentration in solution was measured by UV absorbance and checked by crystal content measurement. Crystal size distributions were measured both by using a Malvern Mastersizer and by the counting and measurement of crystals.
This crystallization study focused on pH values greater than the isoelectric point, where solubility data was available. In this region the crystals grew as elongated prisms (monoclinic). This led to a problem of crystal breakage at higher stirrer speeds (stirrer Reynolds number >10,000). To avoid this, small seed crystals and a low stirrer speed (35 rpm) with a special low shear, angled blade impeller were used. The stirrer speed was still sufficient to easily and fully suspend the crystals. A second problem which is probably facilitated by the needle shape of the crystals, is the tendency of the crystals (due to an apparent hydrophobic attract ion) to form loose aggregates of small numbers of crystals. These aggregates interfere with the crystal sizing. Despite a number of actions, this effect could not be modified or overcome.
The crystal growth was found to be size independent and growth cessation at large crystal sizes was not observed. The crystal growth rate shows a second order dependence upon the ovalbumin supersaturation. While there is a slight effect of ammonium sulphate concentration on the growth rate, there is a ten fold increase in the growth rate constant, kGσ with pH, over the rather small pH range 4.6 to 5.4. A correlation was developed that predicts the growth rate constant, kGσ, as a function of ammonium sulphate concentration and pH at 30°C within an error of ± 12%.
To demonstrate the degree of purification which can be achieved by bulk crystallization, ovalbumin (45,000 Da) was crystallized from a solution containing conalbumin (80,000 Da) and lysozyme (14,600 Da). After one crystallization and a crystal wash, ovalbum in crystals were produced with an estimated protein purity greater than 99%. No contamination by either protein was observed when using over loaded SDS-PAGE gels stained with Coomassie blue stain and only trace amounts of lysozyme were observed when using a silver stain. The presence of these other proteins in solution did not effect the crystal growth rate constant or crystal shape.
The crystal growth mechanisms and crystal growth rates found for ovalbumin are comparable to that found for other proteins. Although ovalbumin exhibits crystallization behaviour similar to small molecule materials, the supersaturations required for ovalbumin to obtain crystal growth rates similar to small molecule materials is much higher. Using the kinetic data obtained in this study a continuous crystallizer design is presented as an example of a protein crystallizer.
The study demonstrates the feasibility of using bulk crystallization for the recovery and purification of ovalbumin. It should be readily applicable to other protein systems.