Comparative Study on Seeded and Unseeded Bulk Evaporative Batch Crystallization of Tetragonal Lysozyme

Seeding is highlighted as a promising tool to improve controllability, reproducibility, and quality of the process and of the product in bulk protein crystallization. In this experimental study, hen egg white lysozyme is crystallized from its buffered aqueous solution in a stirred tank by evaporative batch crystallization at low pressure. Experiments with and without the use of lysozyme seed crystals are evaluated and compared in terms of nucleation behavior, supersaturation evolution, and product quality. The supersaturations needed for spontaneous nucleation are shown to vary strongly in between the unseeded experiments, resulting in a large variation of the supersaturation evolutions during the experiments and the final crystal size distributions. In contrast, seeding is proved to lead to controlled and reproducible secondary nucleation. The reproducibility of the product crystal size distribution is demonstrated to be dramatically increased by seeding compared to the unseeded experiments. Moreover it is shown that the supersaturation evolution and the crystal size distribution of the seeded experiments can be governed by the seed quantity and the evaporation rate. The findings are expected to pave the way for the application of seeding in future protein crystallization processes.