A Study on Nucleation for Protein Crystallization in Mixed Vessels Stephan Tait,†,‡ Edward T. White,† and James D. Litster*,†,§ Chemical Engineering, The UniVersity of Queensland, Brisbane, Queensland, 4072, Australia ReceiVed August 20, 2008; ReVised Manuscript ReceiVed February 5, 2009
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ABSTRACT: This study investigated mechanisms of secondary nucleation for the crystallization of the protein hen egg white lysozyme. A parametric study of the secondary nucleation of lysozyme in a stirred tank crystallizer was performed, testing the influence of stirrer speed, the size and volume of added seed crystals, solution temperature, supersaturation, and sodium chloride concentration on measured nucleation rates. The measured rates were highly variable, which made assessment of the influence of experimental parameters difficult. Only stirrer speed exhibited significant influence over experimental variability (10-fold increase in nucleation rates with a 50% increase in stirring speed). These results were interpreted by comparison with parameter influence that is commonly observed with secondary nucleation of nonproteins. A novel tubular crystallizer was used to assess the influence on secondary nucleation of fluid shear forces acting on lysozyme crystals. It was observed that fluid shear forces acting on mounted lysozyme crystals do not notably influence secondary nucleation. Overall, the results suggest that attrition is an important source of secondary nuclei in lysozyme crystallization and that secondary nucleation caused solely by fluid shear forces is not. Introduction Secondary nucleation, which is crystal birth caused by “parent crystals” in a nucleating solution, strongly influences the crystal size distribution (CSD) in many industrial crystallizers1 (CSD is typically a key product quality). This is also true for the stirred crystallization of biological macromolecules such as proteins.2-6 Research on secondary nucleation is ubiquitous in the literature on the crystallization of nonproteins, but despite its practical importance to protein systems, secondary nucleation in protein crystallization has not received much research attention. There are no reports of dedicated studies on the mechanisms of secondary nucleation in protein crystallization. This is the topic of the study presented in this paper. If the kinetics of nucleation in nucleating crystallizers is to be controlled, a quantitative understanding of nucleation behavior and of the influence of process parameters is required. The influence of process parameters on nucleation rates is commonly expressed according to empirical power law expressions of the form:7
B ) kBN pM qS r
(1)
where kB is a nucleation rate coefficient, N (stirring speed) captures the effects of mixing, M (crystal concentration) represents the effects of crystal collisions, S (supersaturation) accounts for influence of solution thermodynamics, and p, q, and r are power law exponents. The dominant mechanism by which secondary nucleation occurs can determine the sensitivity of a nucleating system to certain process parameters. For instance, secondary nucleation of the nonprotein magnesium sulfate heptahydrate (MgSO4 · 7H2O) is much more sensitive to changes in supersaturation when nucleation is only caused by fluid shear forces acting on a “parent crystal” (fluid shear nucleation, r ≈ 12),8 rather than by crystal collisions (contact nucleation, r ) 0.5-2.5).9 Conversely, the sensitivity of a nucleating system to process parameters may provide empirical * To whom correspondence should be addressed. E-mail:
[email protected]. † The University of Queensland. ‡ Present address: Gutteridge Haskins and Davey Pty Ltd., 201 Charlotte Street, Brisbane, QLD, 4000 Australia. § Present address: Chemical Engineering/School of Pharmacy, Purdue University, Indiana 47906.
evidence for the occurrence of a particular mode of secondary nucleation.7 For example, a linear dependence of nucleation kinetics on crystal concentration is indicative of the occurrence of nucleation by crystals colliding with a stirrer or crystallizer, rather than by crystals colliding with one another (expected second order or higher order effect).7 Another approach to study mechanisms of secondary nucleation is to employ experiments which only allow one mode of nucleation to occur. For instance, Jaganathan et al.10 studied fluid shear nucleation of MgSO4 · 7H2O by subjecting a mounted crystal to fluid shear forces from an impinging jet of solution. In this way they avoided contact nucleation. Further, to avoid fluid shear nucleation in a study on contact nucleation of MgSO4 · 7H2O, Ness and White9 operated a stirred crystallizer at solution conditions where fluid shear nucleation was not observed. They established these conditions in a scope study with a mounted crystal subjected to fluid shear forces at various levels of supersaturation. The techniques above should be applicable to any crystallizing system, including proteins. However, proteins do exhibit some special characteristics. They are generally more fragile than crystals of inorganic and small organic molecules. Tait et al.11 measured the mechanical properties of single protein crystals and in this way showed that they are highly likely to fracture during collision with each other or the crystallization equipment. Protein crystals also have a lattice of very large molecules so that the resulting large interstices between molecules are porous to solvent molecules and salt ions and in solution typically contain large amounts of unbound solvent. Consequently, the properties of protein crystals are very sensitive to environmental conditions12 such as salt gradients or drying. The high solvent content of protein crystals also influences their secondary nucleation behavior since the apparent density varies with the solvent density and the resulting density difference between the crystal and the solvent is typically small. These effects influence the inertia of protein crystals in a stirred crystallizer and their tendency to undergo collisions and secondary nucleate upon contact. Consequently, special care is needed when examining nucleation for protein systems. Studies on bulk protein crystallization report an approximately linear influence of crystal content on nucleation kinetics (q ) 0.7 for the protein lipase;4 q ) 0.8 for the protein insulin,
10.1021/cg8009145 CCC: $40.75 2009 American Chemical Society Published on Web 03/16/2009
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determined by correlating data in the literature5). This effect indicates the occurrence of contact nucleation by crystals colliding with a stirrer or crystallizer. The effect of supersaturation appears to be strong with secondary nucleation in protein systems (r ) 1.6 for the protein subtilisin,6 r ) 2.3 for insulin, determined by correlating the nucleation rate data in the literature5). Cherdrungsi2 also observed an increase in the number of lysozyme nuclei generated when larger lysozyme seed crystals (80 µm) were swirled at the bottom of glass shaker flasks for “about 1 hour”. Secondary nucleation kinetics of the protein glucose isomerase have also been correlated to the fourth moment of crystal size,3 which assumes that larger parent crystals generate nuclei more effectively by crystal collisions than smaller ones. The resulting power law exponent of dependency on the fourth moment of the crystal size distribution was 1.5.3 This paper presents the first dedicated study on secondary nucleation in protein crystallization. The investigation is performed on hen egg white lysozyme (HEWL), a protein that is commonly selected in studies on the fundamentals of protein crystallization. Experiments are conducted in a stirred tank crystallizer as a parametric study attempting to clarify the influence of some process parameters on secondary nucleation of lysozyme, and also in a novel tubular crystallizer to investigate the influence of fluid shear forces on secondary nucleation. Results are interpreted by comparison with the commonly observed effects of process parameters on secondary nucleation of nonprotein systems. Experimental Procedures Materials. HEWL was sourced from DSM (Servian, France) and prior to use in the experiments this protein was recrystallized to a final protein purity >99%, by salting out with sodium chloride. The enzyme activity of this recrystallized lysozyme was tested on Micrococcus lysodeikticus (Sigma, St. Louis, MO, USA) and was found to be comparable with the enzyme activity of a highly active lysozyme standard (100 000 standard units/mg, Fluka, Poole, UK). Deionized water and analytical reagent grade inorganic chemicals were used for the preparation of stock solutions. Prior to use, stock solutions were always filtered through 0.2 µm PES or nitrocellulose membranes to remove insoluble impurities. Lysozyme seed crystals were prepared by salting out with sodium chloride. These crystals were wet sieved into two selected size fractions, 105-177 µm (Figure 1) and 177-210 µm. These sieved fractions were stored in sieving solution at 19-23 °C prior to use (within a month). The sieving solution contained 80 g L-1 sodium chloride and 0.1 M sodium acetate (pH 4.6) and was saturated with respect to lysozyme. It was filtered through a 0.2 µm nitrocellulose membrane prior to use. Any small crystal fragments generated by abrasion of the seed crystals during storage and handling were screened out with a 105 µm sieve immediately prior to adding the seed crystals to crystallizers in the experiments. Apparatus. Crystallizers used in the experiments consisted of one of the following two configurations: (a) One liter flat bottom cylindrical glass vessels stirred with 6-blade stainless steel disk turbines (Rushton turbines). Swirling was prevented with four stainless steel baffles mounted 2 mm above the bottom of the vessel and 2 mm away from the vessel walls to avoid the collection of crystals. The shape, size, and positioning of this crystallizer setup (Figure 2a) were geometrically similar to a system characterized by Bates et al.13 Power input (P) was measured for this apparatus while agitating water and was correlated using the dimensionless impellor Power number (NP):
NP )
P FN 3D5
(2)
where D is the impellor diameter, N is the impellor speed, and F is the fluid density. The impellor flow was found to be turbulent at stirring
Figure 1. Lysozyme seed crystals with a sieved size of 105-177 µm. speeds above 300 rpm with a corresponding value for NP of 3.8, of similar order to a literature value of 5.0.13 (b) 250 mL borosilicate glass bottles with inlet and outlet ports for circulation of the crystallizing test solution (Figure 2b) in an external circulation loop to and from mounted lysozyme crystals. The external loop (Figure 2c) consisted of silicone tubing, a flow meter, and a mounting device which was a borosilicate glass tube (32 cm, long, 5 mm inner diameter). Test solution was circulated with a peristaltic pump and the contents of the crystallizer were adequately mixed by forced circulation (typical hydraulic retention times of about 10 s). This flow provided the fluid shear for the fluid shear nucleation tests. In all cases the temperatures of the crystallizing test solutions were controlled to within 0.5 °C by immersing the crystallizers in a water bath with a heater circulator and/or refrigeration unit. Submersible magnetic stirrer plates (Variomag, Daytona Beach, FL, USA) were useful when test solutions were to be maintained at uniform temperature while being mixed. Measurements of the temperature of crystallizing test solutions leaving the outlet port and entering the inlet port of the 250 mL crystallizers proved that the temperature of the solution in any part of the external loop did not significantly differ from that of the crystallizer contents. Analytical Methods. The concentration of lysozyme in all the test solutions was determined by measurements of UV light absorbance at a wavelength of 280 nm and using a Beer-Lambert law extinction coefficient of 2.635 mL mg-1 cm-1.14 Solutions were filtered (0.2 µm PES) and diluted as required with deionized water. Light absorbance was measured in acrylic cuvettes with an Ultrospec 2000 spectrophotometer (Pharmacia Biotech, Sweden). The relative error with 95% confidence on lysozyme concentrations measured by this method was estimated to be less than 1%. The level of supersaturation of each test solution was expressed as σ ) (C/C*) - 1 (with dimensionless units), where C is the measured lysozyme concentration of the test solution and C* the solubility concentration at the test conditions, for which values were sourced from the literature.15 In the parametric study (experiments performed in the 1 L stirred vessel), nucleation was evaluated from volume based crystal size distributions with the relationship: Lmax
NT )
fV(L)Vc π 3 L)Lmin L 6
∑
(3)
where NT is the number of crystals per volume of crystallizer slurry, fv(L) is the normalized volume based size distribution of crystals being counted, the size limits of the distribution are Lmax and Lmin, and Vc is
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Figure 2. Apparatus used in the experiments. (a) 1 L crystallizer shown without a lid; (b) 250 mL crystallizer which is also the “crystallizer reservoir” depicted in connection with (c), the other equipment items used in the fluid shear experiments. The photograph image with (c) displays lysozyme protein crystals which were attached to the inside of the discharge end of the glass tube. the crystal content of the slurry being analyzed. The quantities fv(L) (1-600 µm) and Vc were measured by laser diffraction using a Malvern Mastersizer/E (Worcestershire, UK). The measurements were performed in a small sample cell (20 mL). The sample was stirred during sizing with a magnetic star stirrer. The suspending dispersant was a filtered (0.2 µm) solution saturated with respect to lysozyme, containing 80 g L-1 sodium chloride and 0.1 M sodium acetate buffered at pH 4.6. Successive measurements on lysozyme crystals left stirring for up to 30 min in the sizer sample cell showed that the crystals did not dissolve or break significantly. Crystals with a volume equivalent size of 3 µm (mL of crystallizer contents)-1 min-1] and kB is a nucleation rate constant (min-1). Equation 6 implies that crystals/ nuclei which form induce further nucleation. This is a form of secondary nucleation, not primary nucleation. Given that the number of seed crystals on the wall was small compared to the number of nuclei which circulated in the circulation loop and in the 250 mL reservoir and assuming that all new crystals are inducing further nucleation, the constant kB in eq 6 is also the nucleation rate per crystal in suspension:
B 1 dNT ) ) kB NT NT dt
Figure 5. Immobilized lysozyme crystals on the inside wall of the glass tube mounting device used in the fluid shear experiment, (top) before and (bottom) after an experiment. A particular crystal is indicated (box).
Figure 6. Typical nuclei formed during a fluid-shear nucleation experiment, as seen in a cell-counting chamber.
the tube wall (dummy experiments). These observations appear to indicate the occurrence of primary nucleation (mounted seed crystals made no difference). However, the approximately
(7)
Interestingly, the crystal number (NT) data collected when nuclei numbers increased approximately exponentially in four of the experiments (the other two experiments had only one data point available) exhibited an approximately identical value of kB (slope of logarithmic-linear fit with respect to time, Figure 7b). This observation suggested that the mode of nucleation observed during these four experiments was the same. The mean value of kB for the four experiments was 0.073 ((0.011) min-1 crystal-1, where the value in brackets is the estimated 95% confidence interval. That is, the nucleation rate per crystal (excluding crystals on the glass tube) was 7.3 × 10-2 nuclei min-1 crystal-1 or 4 nuclei h-1 crystal-1. One possible source for nucleation observed in the fluid shear experiments could have been abrasion of a small number of primary nuclei within the soft rubber tubing section in the pinch portion of the peristaltic circulation pump. This is an unlikely mechanism of nucleation in the crystallization of nonproteins, but not unexpected for lysozyme, due to its relative mechanical weakness.11 The primary nuclei could have formed during preparation of the test solutions and not been removed by the pretreatment protocol, or by primary nucleation during the tests. It should be pointed out that the rate of primary homogeneous nucleation at the selected test conditions has been previously observed to be essentially zero.29 It is difficult to assess what the contribution of heterogeneous primary nucleation may have been. The rate of nucleation measured in the fluid shear experiments was substantially lower than nucleation rates resulting when crystals of nonproteins undergo collisions with soft bodies (for example, stirrers coated with a soft material; typically 10-150 nuclei crystal-1 min-1).9 This discrepancy is attributed to differences in test geometry (stirrer to crystal contacts in the nonprotein systems vs lysozyme crystals in contact with plastic tubing), and to a lower frequency and intensity of the crystal contacts in the fluid shear experiments. A Student t-test was performed to establish whether the crystals mounted on the inside of the mounting device significantly influenced the observed nucleation. For this purpose the times taken for each of the fluid-shear experiments to reach NT ) 1.5 million nuclei > 3 µm mL-1 were considered. These times were 60, 35, and 37 min for the experiments with crystals on the glass tube and 45, 52, and 64 min for the experiments without crystals on the glass tube. The times for experiments with and without crystals on the glass tube wall were found to be not significantly different at the 95% confidence limit. This
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Figure 7. Number of nuclei in the crystallizer slurry (NT, on a logarithmic scale) plotted against experimental time for all the fluid shear experiments. (a) Primary data. Lines are drawn to guide the eye. (b) Same data displaying the approximately identical slope of logarithmic-linear fits in the later stages.
observation indicated that crystals on the inside of the glass tube wall did not significantly influence the observed nucleation and were not necessary to initiate the substantial nucleation observed in the experiments. Overall, these findings (not significant influence of mounted crystals) together with the not significant influence of supersaturation observed with the 1 L stirred vessel experiments indicated that fluid shear nucleation is not important in lysozyme crystallization. Leading on from these observations the increase in secondary nucleation rates of lysozyme with increasing stirrer speed noted in the 1 L stirred vessel experiments likely resulted from an increase in the frequency and magnitude of crystal collisions and a resulting increase in the rate of attrition/abrasion.
(ii) The influence of supersaturation on secondary nucleation rates for lysozyme crystallization was not significant above experimental variability. The literature typically reports a strong effect of supersaturation on fluid shear nucleation in nonprotein systems. (iii) Secondary nucleation rates for lysozyme crystallization notably increased with increasing stirrer speed (10-fold increase in nucleation rates with 50% increase in stirring speed), an observation that is consistent with a contact/attrition based model of secondary nucleation.
Conclusions
It is concluded that attrition is the important source of secondary nucleation in lysozyme crystallization and that secondary nucleation caused solely by fluid shear forces is not important.
This paper presents the first dedicated study on secondary nucleation in protein crystallization. The study is performed on the protein hen egg white lysozyme. Relevant observations made from the experiments are the following: (i) Fluid shear forces acting on mounted lysozyme crystals did not notably influence the observed secondary nucleation.
Acknowledgment. The Australian Research Council funded this research through an ARC discovery grant in collaboration with the Chemical Engineering Department, the University of Delaware, Newark, Delaware, USA. Mr. Wei Hong Tan is gratefully acknowledged for assistance in the preparation of materials used in the experiments.
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