Article pubs.acs.org/crystal
Controlled Nucleation and Growth of Protein Crystals by Solvent Freeze-Out Bo Hyun Ryu and Joachim Ulrich* Martin-Luther-Universität Halle-Wittenberg, Zentrum für Ingenieurwissenschaften, Verfahrenstechnik/TVT, D-06099 Halle, Germany ABSTRACT: Solvent freeze-out technology has been developed as a new concept in the field of protein crystallization. This technology allows the separation of the nucleation and growth steps but requires an understanding of the thermodynamics of the complex mixture of protein, solvent, salt, and buffer at temperatures for which few accurate data currently exist. The phase diagram of the given protein system was systematically investigated and confirmed for the identification of optimal crystallization conditions for zone I, which is the best region of protein crystallization, by employing a preliminary screening. As an initial necessity for protein crystallization, the delicate balance between repulsive and attractive forces in the given protein system was found at pH 4.4 and 5 wt % NaCl. The precise value of the supersaturation level (S) of zone I was estimated to be 9.56 ≤ S ≤ 29.34 after a statistical analysis of the initial screening by a Linbro test. Next, the supersaturation levels for the metastable zone were identified to be 7.0 ≤ S < 17.1 from a statistical analysis of all of the experimental results from both the Linbro test and the individual crystal growth measurements. Protein crystallization in zone I by the freeze-out technology was carried out and evaluated. The key process variable levels (KPVLs) were operated within the boundary of the phase diagram that was confirmed by the preliminary screening. With a NaCl concentration of 5 wt % at pH 4.4, quite a good quality of tetragonal hen egg-white lysozyme (HEWL) crystals was produced as a result of proper tuning of the net surface charge of HEWL, even with a very low value of the initial protein concentration. The number of tetragonal HEWL crystals was increased by increasing the ice mass, since the cooling rate applied to the system determines the ice growth rate and therefore the nucleation and growth rates of the protein crystals. Hence, nucleation of the given protein system can be controlled by moderate adjustment of the ice growth rate. ature and pH.5 Proteins are stable only over a narrow range of crystallization conditions, as most protein lose their enzymatic activity at temperatures above 40 °C.6 By far, they are generally crystallized by salting-out or drowning-out processes, as solvent removal by evaporation is unsuitable because the majority of proteins denature at temperatures significantly above physiological levels. Furthermore, these methods demand a very large quantity of salt (at least 60 wt % salt concentration7), which is significantly environment-unfriendly. Nonetheless, the efficiency and the reproducibility these processes exhibit are quite low.8 In most protein crystal growth processes currently in use, the nucleation step within the growth solution (mother liquor) is achieved under poorly understood conditions, and the growth proceeds in a more-or-less uncontrolled manner.9 Consequently, nucleation in this field has hardly been controlled, which is still a major difficulty for industrial applications of protein crystallization technology. Hence, new strategies have been sought to provide a better understanding of crystallization
1. INTRODUCTION Both therapeutic proteins and industrial enzymes are currently experiencing a period of rapid growth in terms of worldwide demands.1 Traditionally, standard formulations of those proteins have been aqueous solutions and amorphous precipitated lyophilizates. However, those methodologies have exhibited certain disadvantages, such as low product concentration and purity, inadequate shelf life, and deficiencies with regard to handling. Crystallized proteins may offer superior properties compared with standard formulations since they usually have a higher purity and longer shelf life.2 Furthermore, it has been shown that crystalline pharmaceutical proteins exhibit the advantage of controlled slow release of therapeutic activity.3 Crystallization has shown great potential in downstream processing of the proteins.4 To provide the required amounts of crystalline proteins, efficient and reproducible methods for the crystallization of various kinds of proteins on a large scale are required. However, because of the complexity of the proteins, such methods have not yet been found. In fact, many proteins are difficult to crystallize or seem not to crystallize at all. A major difficulty in crystallizing proteins is their general sensitivity and instability toward the crystallization conditions to which they are subjected, in particular temper© 2012 American Chemical Society
Received: August 29, 2012 Revised: October 12, 2012 Published: November 8, 2012 6126
dx.doi.org/10.1021/cg301258t | Cryst. Growth Des. 2012, 12, 6126−6133
Crystal Growth & Design
Article
processes and to improve them in order to avoid the use of large amounts of salts as crystallizing agents. For those purposes, the “solvent freeze-out technology” for crystallizing proteins is introduced here as an advanced crystallization technology that provides efficient yields for industrial applications and a high reproducibility of production processes due to controlled nucleation, which is a major key factor for a large scale-up of the processes for industrial application. It shows the possibility of isolated and controlled nucleation for optimal protein crystallization separately from the subsequent growth phase.10,11 The freeze-out technique can control the nucleation stage by having a localized cold surface within the growth medium. Hence, the localized supersaturation is controlled by a temperature strategy. Nucleation in the solvent freeze-out process is confined to a small volume of the solution. This new technique in protein crystallization involves freezing out the solvent (i.e., layer crystallization of the solvent, known from melt crystallization) in order to enrich and therefore supersaturate the protein solution. At low process temperatures, the proteins are sufficiently stable, and crystallization by freezing out the solvent is a feasible alternative to salting out the crystals. It has the advantage that the absolute amounts of protein and precipitant (sodium chloride is commonly used in the case of lysozyme8) can be reduced, as the removal of the solvent increases the concentration of the solutes in the course of the process. The technique is wellknown as a method for crystallization and purification and as a means of concentrating aqueous solutions.12−14 In the case of freeze-out crystallization, the temperature and the cooling rate applied to the system determine the rate at which the ice grows. As a consequence, these parameters also determine the rate at which supersaturation of the protein solution is obtained and thus govern the nucleation and subsequent protein crystal growth. As an initial model protein, hen egg-white lysozyme (HEWL) was chosen. Here, the water was frozen by means of a simple tube-and-shell heat exchanger (“cold finger”), which acts as heat exchanger while at the same time supplying the surface upon which the ice grows. The process thus involves an initial solid-layer melt crystallization technique followed by a second solution crystallization step generating the protein crystals. Problems and advantages of the technology are discussed on the basis of experimental results. The generic protein phase diagram was reported in 1997 by Muschol and Rosenberger15 (Figure 1). The phase diagram demonstrates a solid−liquid equilibrium (solubility line) as well as a metastable liquid−liquid equilibrium. Crystals grown under the limiting crystallization conditions of zone I are usually of high quality. To identify the optimal crystallization conditions for the zone I region, which is the best region for protein crystallization, a preliminary screening can be performed employing a Linbro test as a high-throughput screening system along with individual crystal growth measurements by timelapse video microscopy. Related key process variable levels (KPVLs) for protein crystallization are pH, temperature, and the concentrations of the crystallizing agent (salt, often mentioned as the precipitant) and protein.16
Figure 1. The generic protein phase diagram.15 Zone I is a region of supersaturation where well-formed lysozyme crystals form. Zone II is a region where the lysozyme solution undergoes rapid liquid−liquid phase separation, with the resulting concentrated lysozyme phase quickly transforming to the more stable crystalline form. Crystals formed in zone II are of poor quality. Zone III is a region characterized by gel formation and is unsuitable for crystal growth.
mixing appropriate amounts of sodium acetate and acetic acid to reach the required pH. The sodium chloride solution was prepared by adding the appropriate amount of NaCl to the buffer. 2.2. Methods. 2.2.1. Linbro Test. A 96-well Linbro plate (BRAND, Wertheim, Germany) was used. An overview of experimental procedure was described by Carter.18 A protein stock solution with a suitable concentration was prepared in distilled water and diluted appropriately with buffer solution to achieve the desired concentrations of protein and NaCl. The experiments were performed over the temperature range from −7 to 4 °C. The resulting crystallization state was observed and evaluated using optical microscopy (BH-2, Olympus). 2.2.2. Individual Crystal Growth Measurements. A time-lapse optical microscope (BH-2, Olympus) was employed for the individual crystal growth measurements. An overview of the experimental procedure was described previously.19,20 The growth cell had a diameter of 3.5 cm and a height of 0.5 cm, and the total volume of the solution was 4 mL. 2.2.3. Freeze-Out Crystallization. The experimental setup is shown in Figure 2. It consisted of a double-walled beaker and a single tubeand-shell heat exchanger (cold finger). The heat exchanger and the beaker were cooled by independent thermostats. A temperature gradient was applied across the solution; the temperature of the beaker was set to a constant 1 °C, whereas the heat exchanger was cooled to a temperature sufficient to freeze out the solvent. Experiments were carried out at pH values between 4.0 and 5.2 and NaCl concentrations between 1 and 10 wt %. The initial protein concentration ranged between 0.3 and 15 mg mL−1. The protein concentration and the specific activity of HEWL in the ice, the mother liquor, and the crystals after centrifugation (4 °C, 9000 rpm, 5 min) were analyzed using UV spectrophotometry and turbidimetric measurements21 of the turbidity decay of a Micrococcus lysodeikticus suspension in the presence of lysozyme, respectively. The effect of the cooling rate on the protein content of the ice was investigated for cooling rates of 0.05, 0.1, and 0.4 °C min−1. The total protein content was measured by sampling cross sections of the ice layer. The protein concentration gradient over the diameter of the ice layer and the specific activity with each layer were assessed by melting the ice and taking samples at different times.
2. MATERIALS AND METHODS 2.1. Materials. As an initial model protein, HEWL was purchased from Fluka (product no. 62971) and used as received. HEWL is a globular protein with a molecular mass of 14 400 Da containing 129 amino acid residues.17 Its shape is roughly ellipsoidal, with dimensions 45 Å × 30 Å × 30 Å. The 0.1 M sodium acetate buffer was prepared by 6127
dx.doi.org/10.1021/cg301258t | Cryst. Growth Des. 2012, 12, 6126−6133
Crystal Growth & Design
Article
Table 1. Solubility Equations for HEWL over Different Temperature Ranges Determined by Nonlinear Regression (c∞,NaCl = 5 wt %, pH 4.4) temperature range (°C)
a
fit equationa
4 to 20
f = 0.3331 + 0.1087x − 0.0102x 2 + 0.0004x 3
−10 to 20
f = 0.3310 + 0.0311x − 0.0018x 2 + (4.0559 × 10−5)x 3
f = c/(mg/mL); x = T/°C.
3.2. Identification of a Supersaturation Level for a Metastable Zone in Zone I. The precise value of the supersaturation level for a metastable zone in zone I should be identified for the strategy of Wiencek.25 For this purpose, individual crystal growth measurements were performed. The details of the experimental conditions are given in Table 2. The Figure 2. Freeze-out crystallization experimental setup: (1) lysozyme solution containing buffer and NaCl; (2) double-walled beaker; (3) cold finger; (4) thermometer; (5) thermostats; (6) insulation layer.
Table 2. Experimental Conditions of KPVLs for Individual Measurements by Time-Lapse Optical Microscopy KPVL value
pH 4.4
ci,pr (mg/mL)a 2−15
c∞, NaCl (wt %)b 5
T range (°C) −7 to 4
a Initial concentration of the protein. bNaCl concentration of the bulk solution.
3. RESULTS AND DISCUSSION 3.1. Estimation of the Solubility of HEWL. In this study, the level of supersaturation (S) was expressed as S = c/ceq, where c is the bulk initial protein concentration and ceq is the equilibrium concentration (i.e., the solubility). Solubility determinations are fundamental for the identification of supersaturation levels. Solubility data for HEWL at low temperatures (
