Article pubs.acs.org/crystal
Development and Scale up of High-Yield Crystallization Processes of Lysozyme and Lipase Using Additives Dirk Hebel, Mark Ü rdingen,‡ Dariusch Hekmat,* and Dirk Weuster-Botz †
Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, Boltzmannstrasse 15, 85748 Garching, Germany Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany
‡
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.
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99%. 10 Fungal lipases were crystallized from clarified concentrated fermentation broth on a 225 mL scale11 and in 500 mL bottles on a vibrating platform using seed crystals.12 Schmidt et al. reported the separation of two isoforms of an undisclosed protein in 2, 10, and 100 mL stirred tanks with the stirrer operating at different speeds.13 The crystal growth kinetics were found to be nearly independent of the stirrer speed. However, a lower mean stirrer energy input and lower shear forces were favorable for the formation of larger crystals. A three-step crystallization process in a 100 L stirred tank for the purification of L-methionine γ-lyase from a pretreated crude enzyme solution was reported by Takakura et al. with an 87% yield and an overall process duration of approximately 3 days.14 The crystals were stable for several months with no significant loss of activity. Weber et al. reported the successful purification of jack bean urease by a combination of an extraction and a crystallization step on a 50 mL scale. The cooling crystallization was performed by placing a nonagitated vessel in a refrigerator at 4 °C for two days.15 Matthews and Bean reported the crystallization of a recombinant therapeutic protein on a 1 L scale and proposed to scale-up the process to 800 L.16 The crystal growth kinetics were improved using controlled temperature decrease and equilibrium was reached after 8.5 h. Astier and Veesler have also reported the use of temperature alterations in order to control protein crystallization but only
INTRODUCTION The worldwide demand for biotechnologically produced proteins is expected to grow significantly in the future. In order to satisfy this demand, the production processes for proteins, especially for therapeutic proteins, have been optimized with regard to higher protein concentrations. However, this development created a bottleneck in the subsequent downstream processing, which currently heavily relies on costly preparative chromatography steps. Hence, alternative purification technologies need to be established. Current research on alternative purification technologies follows different approaches (e.g., aqueous two-phase extraction,1 magnetic adsorbent particles,2 membrane-based techniques,3 and protein crystallization). As crystalline proteins offer advantages regarding product activity and product stability,4,5 protein crystallization can also overcome drawbacks of the current protein formulations, primarily aqueous solutions or amorphous precipitated lyophilizates.6,7 In addition, significant cost reductions can be expected compared to conventional preparative chromatography methods. While protein crystallization is best known for its use in protein structure determination, the technical-scale application for protein purification has been studied in a limited number of research projects over the past years, the most prominent example being stirred batch crystallization of the polypeptide insulin with volumes up to 500 L.8,9 For larger biomolecules, early research work demonstrated the crystallization of ovalbumin from a solution containing two protein contaminants in 1 L stirred batch crystallizers with a purity greater than © XXXX American Chemical Society
Received: February 4, 2013 Revised: April 8, 2013
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́ Borbón and Ulrich reported a on a small scale of 15 μL.17 Diaz solvent freeze-out crystallization of lysozyme from a lysozyme− ovalbumin mixture in a nonagitated beaker with a crystallization yield of 69%.18 The initial volume of the protein solution was 60 mL. The lysozyme crystals preserved 94% of the original enzymatic activity, and a mean crystal size of 77.8 μm was observed after a crystallization time of 15.1 h. The aforementioned results demonstrate the feasibility of protein purification by large-scale crystallization. However, only a few of these studies were performed in well-defined, scalable stirred vessels.19 In addition, low yields and rather long process durations were often observed. A straightforward prediction of crystallization conditions for proteins (e.g., based on the second virial coefficient B22) is not yet feasible.20 Therefore, further work is necessary in order to establish protein crystallization as a viable alternative to common purification techniques, with the focus on the scalability of the crystallization processes. One way to accelerate protein crystal growth kinetics is the addition of water-miscible ionic liquids to the crystallization agent mixture.21 Ionic liquids (i.e., organic salts which are liquid at room temperature) were already proven to be useful as alternative solvents in many cases.22,23 The positive effects of ionic liquids on protein crystallization, including faster crystal growth kinetics, a more compact crystal morphology, and an increased protein stability were reported for small-scale vapor diffusion and microbatch experiments.21,24−26 For example, ionic liquids were shown to have an influence on different crystal modifications and to reduce precipitate formation for lysozyme and a lipase even at higher crystallization agent concentrations.27−29 Additionally, a study focusing on the transfer of crystallization conditions obtained in vapor diffusion experiments to milliliter-scale stirred-tank crystallizers was performed.30 However, for protein crystallization, water-soluble protic ionic liquids are usually used at rather low concentrations in contrast to other applications with water-immiscible ionic liquids acting as sole solvent or as a secondary phase.31,32 When used at low concentrations, most ionic liquids were shown to have no detrimental effect on the protein structure.26 The ionic network which can be formed by ionic liquids at high concentrations or in pure solutions breaks up at low concentrations.33 Weaver et al. suggested that the ionic liquid choline dihydrogenphosphate should be considered as an osmolyte similar to other water-soluble compounds, such as sugars and salts when used at low concentrations.34 Therefore, it remains unclear whether the positive effects of ionic liquids on protein crystallization are caused by the unique characteristics of ionic liquids or rather by the properties of individual dissociated ions. Furthermore, the crystallization buffer was shown to be included in the lysozyme crystals.35 Little knowledge exists with regard to the removability of these ionic liquids from the protein crystals prior to formulation. As a consequence, the present work deals with (a) the improvement of the crystal growth kinetics and the crystal yield for lysozyme and lipase with the addition of ionic liquids in microbatch experiments and in milliliter-scale stirred-tank experiments as a starting point for the subsequent scale-up, (b) the identification of the ions responsible for the effects of the ionic liquids on the crystallization, (c) scale-up studies of the crystallization processes for lysozyme and lipase to a 1 L stirred tank, (d) the characterization of crystal morphologies and crystal size distributions, and (e) the evaluation of the removability of unwanted substances from the crystals (e.g., additives, salts).
Article
EXPERIMENTAL SECTION
Lysozyme from Gallus gallus was purchased from Sigma-Aldrich, Taufkirchen, Germany (No. 62971). Lipolase type 100 L EX was purchased from Novozymes A/S, Bagsvaerd, Denmark. This product contained approximately 2% (w/w) of a fungal lipase from Thermomyces lanuginosus, which was produced by fermentation of a genetically modified Aspergillus oryzae. In addition, lipolase contained approximately 0.5% (w/w) CaCl2 and 25% (w/w) propylene glycol for stabilization purposes. The ionic liquids, 2-hydroxyethylammonium formate and choline dihydrogenphosphate, were kindly provided by Merck KGaA, Darmstadt, Germany. Choline chloride and choline dihydrogencitrate were purchased from Sigma-Aldrich, Taufkirchen, Germany. All other chemicals were analytical grade purchased from Carl Roth, Karlsruhe, Germany. Preparation of the Lipase Solution. The stabilizing components had to be removed from the lipase solution prior to crystallization, as described previously.28 In order to purify larger volumes of lipase solution in one step, cross-flow ultrafiltration was used instead of dialysis and subsequent concentration by centrifugation. The filtration equipment was purchased from Sartorius Stedim, Gö ttingen, Germany. A polyethersulfone membrane (0.1 m2, 5 kDa MWCO, no. 3051462901E-SG) was used in a Sartocon Slice holder (no. 17521002). The system was operated at 2 bar using a SartoJet membrane pump (no. 17521-11). First, 5 L of lipase solution was concentrated by ultrafiltration to a volume of 1 L. Subsequently, the stabilizing components of the lipase solution were removed by continuous diafiltration against 5 L of Tris buffer (25 mM, pH = 9.0). Finally, the lipase solution was concentrated to 130 g L−1 by a second ultrafiltration step to a final volume of 150 mL. Microbatch Crystallization. Ten microliter-scale microbatch crystallization experiments were carried out in 72-well Terasaki plates (Greiner, Frickenhausen, Germany). The crystallization conditions used in this work are based on screening experiments described in previous work.27,28,30 Since this paper focuses on the scalability of protein crystallization, the result of the preliminary screening is not detailed. The protein concentrations were adjusted to 25 g L−1 for lysozyme and 100 g L−1 for lipase. The pH was adjusted to 4.0, using a 25 mM sodium acetate buffer. For each crystallization condition, protein and crystallization agent stock solutions were prepared separately. The drops were prepared by gently mixing the protein and crystallization agent stock solutions in the wells. The Terasaki plate was sealed with 8 mL paraffin oil to avoid evaporation. The plates were stored at 20 °C in a refrigerating incubator (Binder, Germany). After 4 days, the results were evaluated qualitatively by microscopy. The outcome was categorized in crystallization, precipitation, and clear drops. A phase diagram for the crystallization of lysozyme in sodium acetate buffer at pH = 4 was created from qualitative data available in the literature30,36,37 (Figure 1). For lipase, no phase diagram was recorded due to the very narrow nucleation zone. Agitated Batch Crystallization. Stirred tank reactors have proven to be simple and efficient for the scale-up of many bioprocesses. Hence, three geometrically similar unbaffled stirred tanks were used in order to study the agitated batch crystallization of the two enzymes on 1 L and 100 and 5 mL scales (Figure 2). Pitched-blade impellers were used for gentle mixing of the crystal slurry, as this type of impeller was known to exert low shear forces on the fluid.38 The impellers of all three stirred tanks were operated in the scoping mode (upward flow near the stirrer axis). The 1 L stirred tank was built using commercially available components (IUL Instruments, Königswinter, Germany). It featured a fill height to inner diameter ratio of 1.0 and an agitator diameter to tank inner diameter ratio of 0.5. The glass vessel had a cooling jacket for temperature control and ports in the tank head allowed for sampling and feeding of crystallization agent solutions. Both smaller stirred tanks were custom-built geometrically similar scaled-down versions of the 1 L stirred tank. Standard round-bottom centrifuge tubes were used as glass vessels which were immersed in a refrigerating circulator for temperature control. B
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Table 1. Dimensions of the Three Stirred-Tank Crystallizersa V (mL) H, mm D, mm d, mm H/D, − d/D, − P/V, W m−3 parallel reactors a
Figure 1. Phase diagram for the crystallization of lysozyme in sodium acetate buffer at pH = 4 based on qualitative data available in the literature30,36 (●, crystal growth; ×, precipitate formation). The solubility data represented by the dashed line was taken from Howard et al.37
1000 120 120 60 1.0 0.5 150 min−1:19 250 min−1:46 1
100 58.9 48.8 24.8 1.2 0.51 − 2
5 20.0 22.0 11.3 0.9 0.51 − 12
P/V values from Smejkal et al.19
volume of 150 μL crystals were sedimented by centrifugation for 2 min at 16.200g. The supernatant was removed, and the crystals were resuspended in a 1.5 mL wash solution. After 5 min, the crystals were sedimented again by centrifugation and subsequently dissolved in 1.5 mL of water for analysis. The protein concentration was measured by UV spectroscopy at 280 nm. For the lipase crystals, the PEG 10000 content was determined using a modified Dragendorff reagent method.39 Acetate, citrate, ethanol, formate, phosphate, and 2hydroxyethylammonium were quantified using common HPLC methods. The NaCl content was determined using a chloride assay purchased from Merck KGaA, Darmstadt, Germany (no. 1.14897.0001). Functional Lipase Assay. A modified 4-nitrophenyl palmitate based assay40,41 was used for the determination of lipase activity in 300 μL 96-well plates (Nunc, Thermo Electron LED GmbH, Langenselbold, Germany). The reaction mixture with a total volume of 270 μL, consisting of 0.25 g L−1 4-nitrophenyl palmitate, 0.3 g L−1 gum arabic, 2.9 g L−1 Triton X-100, and 1.45 M Tris-HCl at pH = 7.5, was generated by mixing 22.5 μL of solution A, 157.5 μL of solution B, and 90 μL of the lipase sample. Prior to the addition of the sample, the baseline absorbance at 410 nm was measured for 10 min at 37 °C. Subsequently, the kinetics were determined under the same conditions
The crystallization was started by mixing protein and crystallization agent stock solutions in the stirred tanks. In order to monitor the progress of the crystallization process, samples of approximately 40 μL were taken from the tanks at variable time intervals. Crystals were sedimented for 2 min at 16.200g in a refrigerated centrifuge (4 °C). Subsequently, the supernatant was diluted, and the protein concentration in solution was determined by UV spectroscopy at 280 nm. Additionally, a second sample was taken for the evaluation of the crystal morphologies. The crystal suspension was directly transferred from the crystal suspension onto a microscope slide without a centrifugation step. The crystal size distributions were characterized by the area densities calculated from measured crystal cross section areas from microphotographs, as described previously.30 Washing of Protein Crystals and Analysis of the Removed Substances. Protein crystals were washed in order to deplete unwanted substances from the solvent channels. First, approximately a
Figure 2. Geometrically similar unbaffled stirred tanks used for the agitated batch crystallization on 1 L and 100 and 5 mL scales (see Table 1). C
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for 20 min. Solution A: 3 g L−1 4-nitrophenyl palmitate in n-propanol. Solution B: 1 g L−1 gum arabic, 2.5 M Tris-HCl at pH = 7.5, and 5 g L−1 Triton X-100.
Lysozyme Crystallization on a Stirred 5 mL Scale. The effect of 2-HEAF on the stirred 5 mL scale crystallization was studied, using refined crystallization conditions. These were 25 g L−1 lysozyme, 1.25 M NaCl, 25 mM sodium acetate buffer at pH = 4, and either no ionic liquid or 0.5 M 2-HEAF. Due to the considerable temperature dependency of lysozyme solubility, 37,43 the temperature was reduced to 10 °C and subsequently to 0 °C during the crystallization process (Figure 3).
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RESULTS AND DISCUSSION Identification of Ions Responsible for Effects of Ionic Liquids on Protein Crystallization. For lysozyme, the addition of up to 100 g L−1 (which is equivalent to approximately 0.93 M) 2-hydroxyethylammonium formate (2HEAF) was reported to be the most effective.28 The crystallization was transferred into 10 μL scale microbatch experiments using the same crystallization agent concentrations. In these experiments, a lower 2-HEAF concentration of 0.5 M was shown to be sufficient for efficient crystallization of lysozyme, even when using high NaCl concentrations of up to 1.25 M. At these high NaCl concentrations, precipitation, and/ or the formation of crystal clusters like sea urchin, was observed without the addition of IL. It can be deduced that the IL stabilized the native form of the lysozyme in solution and thus prevented precipitation. Hence, a higher supersaturation level and faster crystal growth kinetics could be achieved. The obtained crystals using these conditions were tetragonal-like, measured an average of 230 μm, and no precipitation was observed. However, the crystals tended to be irregularly shaped at 1.25 M NaCl. The ion responsible for the positive effects of 2-HEAF was not identified, as no experiments were carried out containing only the cation of this ionic liquid due to the toxic character of monoethanolamine. Precipitation was observed in all experiments containing only the formate anion instead of 2HEAF. Hence, 0.5 M 2-HEAF was used in all following experiments. For lipase, the crystallization conditions known from previous sitting-drop experiments using the ionic liquid choline dihydrogenphosphate (CDHP)28 proved to be suitable for the 10 μL scale microbatch crystallization. Further microbatch experiments were carried out in order to determine whether both ions of CDHP needed to be present in the solution in order to achieve these positive effects or whether the anion or the cation alone could facilitate these effects independently. Therefore, lipase was crystallized with 55 mM NaCl and 50 mM sodium acetate buffer at pH = 4 and with 0.275 M of (a) CDHP, (b) monosodium phosphate, (c) choline chloride, (d) choline dihydrogencitrate, and (e) with no additive. The formation of agglomerates of sturdy hexagonal-shaped crystals with an average size of 90 μm was observed only in droplets containing CDHP or monosodium phosphate. Either there was no crystal growth or there was precipitation with the addition of choline chloride or choline dihydrogencitrate. Without any additives, slow formation of clusters of needlelike crystals was observed, as described previously.28 Hence, it was shown that the positive effects observed upon the addition of CDHP were caused by the dihydrogenphosphate anion alone. As watersoluble ionic liquids dissociate in aqueous solution at low concentrations just like any other common salt, the positive effects may be facilitated by just one of the ions of the ionic liquid.34 Since the saturation point is shifted upon the addition of the ionic liquids, these substances rather represent a complementary crystallization agent than an additive. However, salts or ionic liquids with both ions having beneficial, possibly synergistic, effects on protein crystallization are conceivable too.42 For further experiments, monosodium phosphate was used instead of choline dihydrogenphosphate in order to reduce the costs of the crystallization agents.
Figure 3. Crystallization of 25 g L−1 lysozyme on the 5 mL scale, using 1.25 M NaCl and 25 mM sodium acetate buffer at pH = 4, n = 150 min−1. ◇, no ionic liquid; ◆, 0.5 M 2-hydroxyethylammonium formate. Depicted are the decrease of the lysozyme concentration in solution and the temperature time course (dashed line, without IL; solid line, with IL) which was lowered stepwise in order to increase the crystallization yield.
The crystallization started as soon as the protein solution and the crystallization agent solution were mixed in the stirred tank. An instantaneous turbidity of the solution was observed. The induction time could not be measured quantitatively and was estimated to be
