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

Abstract

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.

Purification of proteins from solutions containing residual host cell proteins via preparative crystallization

“…Previous crystallization experiments of pure lysozyme showed that best crystallization yield and kinetics were obtained at a pH of 4.0 (Hebel et al 2013b). As mentioned, the spiked HCP of the impure lysozyme solution would precipitate at this low pH.…”

“…Stirred batch and fed-batch crystallization experiments were performed using a parallelized unbaffled 5 ml stirredtank crystallization system with 12 vessels (Hebel et al 2013b;Smejkal et al 2013b). The spiking with HCP was carried out before the initiation of the crystallization.…”

“…Applications of ATPE for the purification of proteins have been described for some time (Kula et al 1982;Asenjo and Andrews 2012). The successful scale-up of preparative protein crystallization in stirred-tank crystallizers has also been reported (Harrison et al 2003;Hebel et al 2013b;Smejkal et al 2013b;Hekmat 2015). Therefore, integrated purification processes comprising a combination of ATPE and preparative chromatography have been described (Azevedo et al 2009).…”

Current Trends and Advances in Bulk Crystallization and Freeze-Drying of Biopharmaceuticals

“…Hekmat and coworkers describe the successful transfer of vapor diffusion-based crystallization to agitated batch crystallization at milliliter scale using lysozyme as a model protein [19]. The same research group also describes a stirred-tank crystallization-based approach [53] towards successful scale-up of batch crystallization conditions using lysozyme, lipase [18], and therapeutic mAbs [17,52] as model proteins. Smejkal et al describe a maximum local energy dissipation-based scale-up approach for stirred crystallization using lysozyme and Canakinumab Fab-fragment as model proteins.…”

Solvent-freeze-out (SFO) technology: A controlled crystallization process—Case study of jack bean urease

“…Proteins, especially crystalline proteins, are increasing in demand in areas as medical applications, chemical and food industries (Motoki and Seguro, 1998;Hebel et al, 2013). In most of the cases, proteins cannot be used without further purification (Weber et al, 2008a).…”