The application of five water-soluble, halogen-free, alkylammonium-based ionic liquids (ILs) as additives for advanced crystallization of lysozyme was investigated. Their biocompatibility was determined by long-term measurement of the overall mean relative enzyme activities. These were maximally reduced by about 10-15% when up to 200 g IL l(-1) was added. Sitting-drop vapor diffusion crystallization experiments revealed that the addition of some of the ILs led to less crystal polymorphism and precipitation was avoided reliably even at larger NaCl concentrations. The addition of ILs tended to result in larger crystals. The kinetics of lysozyme crystallization were significantly enhanced using ILs as crystallization additives, e.g. by a factor of 5.5 when 100 g ethanolammonium formate l(-1 )was added. ILs with "soft" anions, such as formate or glycolate, were superior to ILs with "hard" anions, like nitrate.

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Compared to standard protein formulations like aqueous solutions, crystalline proteins may offer superior properties (e.g., higher purity and concentration, 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 processes 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.

Crystalline proteins may offer superior properties for drug delivery compared to standard protein formulations such as aqueous solutions or amorphous precipitated lyophilisates. In this study, a new approach using biocompatible, hydrophilic, substituted alkylammonium-based ionic liquids (ILs) as additives for the advanced crystallization of two exemplary proteins, lysozyme and lipase, was investigated. Sitting-drop vapor diffusion crystallization experiments revealed that the addition of some of the ILs resulted in less crystal polymorphism and precipitation was consistently avoided, even at larger concentrations of the conventional crystallization agent. The kinetics of lysozyme crystallization were significantly enhanced by a factor of up to 5.5 using ILs with strongly hydrated anions, i.e., formate or glycolate. In contrast, ILs with weakly hydrated anions, i.e., nitrate, led to undesirable spontaneous precipitation. In addition, lipase was crystallized preferentially using an IL with a strongly hydrated anion, i.e. dihydrogenphosphate. Large, sturdy crystals were formed at rates which were enhanced by a factor of up to 4.