Development and Scale up of High-Yield Crystallization Processes of Lysozyme and Lipase Using Additives

Compared to standard protein formulations like aqueous Solutions, crystalline proteins may offer superior properties higher. purity and reduced storage costs, and enhanced shelf life. In this work, crystallization conditions for lysozyme from Gallus gallus and a lipase from Thermomyces lanuginosus were characterized in microbatch experiments. The previously described positive effects of water-soluble substituted alkylammonium-based ionic liquids as additives on the crystallization of these enzymes (e.g., faster crystal, growth kinetics and the formation of larger, sturdier crystals), was confirmed. With the use of optimized conditions, the crystallization process were transferred into parallel-operated stirred crystallizers on a 5 mL scale. A higher yield and faster crystal growth kinetics were observed when using additives. For lysozyme a yield of 97% was obtained within 2 h. For lipase, a yield of 95% was obtained within 2 h by stepwise addition of 50 g L-1 PEG 10000. The crystallization processes were successfully scaled-up into geometrically similar stirred crystallizers on a 100 mL and 1 L scale respectively. Favorable crystal morphologies and adequate crystal size distributions were obtained. Unfavorable substances were removed from the crystals by washing.