DOI: 10.1021/cg900492j
Protein Crystallization by Forced Flow through Glass Capillaries: Enhanced Lysozyme Crystal Growth
2010, Vol. 10 1074–1083
Michael M. Roberts,* Jerry Y. Y. Heng, and Daryl R. Williams Department of Chemical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom Received May 5, 2009; Revised Manuscript Received November 23, 2009
ABSTRACT: The growth of protein crystals in quiescent mode depends on the supply of protein in a closed vessel, where crystals grow to a size limited by the mass of protein in solution in contact with the crystal. Here, we show that by inducing solution flow into a lower temperature environment, protein crystals grow in capillaries faster and larger than those obtained under quiescent conditions in sealed capillaries. The crystal size distribution is also more regular under capillary flow. In 27 h, lysozyme crystals grow up to 0.37 mm under flow conditions and 0.16 mm under quiescent conditions. A 75-fold concentration of crystal mass relative to a sealed batch crystallization was attained within an equivalent capillary space using this flow technique with a greater volume of protein solution. We have exploited this feature to develop a circulatory system to maximize the yield of crystals obtained by capillary flow. Recirculating the protein solution through each capillary for 40 h increases the crystal yield to 80% of total protein, close to that of a sealed capillary batch control crystallization. This method offers promise for growing large protein crystals for use in structure determinations and for the mass preparation of protein therapeutics.
1. Introduction The primary aim of protein crystal growth is for the determination of protein structures to atomic resolution by crystallography using X-ray diffraction data. In many cases, the crystals obtained are insufficiently large for X-ray diffraction studies. This is especially true for large assemblies such as virus structures, where a minimum number of unit cells in the crystal are required to give X-ray data to solve the molecular structure, requiring crystals > 0.1 mm. Neutron diffraction studies of proteins require even larger crystals to locate the hydrogen atoms.1 The growth of protein crystals which are an order of magnitude larger than that required for X-ray crystallography makes it possible to perform numerous other physical studies on proteins due to their high packing density in the crystal, including spectroscopy, thermal, mechanical, and optical measurements.2 There are other increasingly important needs for protein crystallization: the industrial mass production of protein crystals as a means of purification, for biocatalysis, and as an effective formulation of protein therapeutics for the pharmaceutical industry.3 Most proteins are purified by standard liquid column chromatographic methods, which often involve more than one stage, are both time-consuming and expensive, and do not always guarantee native and active protein. Insulin is a good, but rare, example of a therapeutic protein commercially purified by crystallization.4 Large crystals with a narrow size distribution are important for effective purification from the supernatant.5 Also, in the pharmaceutical industry, a narrow crystal size distribution is often desirable in order to guarantee the best drug delivery performance.5 However, for protein therapeutics, crystal sizes below 0.1 mm are required. We have chosen to work with chicken egg-white lysozyme (CEWL) in these studies, since it is a well-characterized protein in terms of its solubility and crystallization behavior.2,6-38 *To whom correspondence should be addressed. Phone: þ44-(0)207-5945565. Fax: þ44-(0)207-594-5700. E-mail:
[email protected]. Web: www.imperial.ac.uk/spel. pubs.acs.org/crystal
Published on Web 02/04/2010
Tetragonal CEWL crystals were grown for our studies, since this is the predominating crystal form in the conditions used here2 (see Experimental Methods and Figures 3, 4, 6, 8, and 9). The effects of protein solution flow on CEWL crystal growth have also been studied extensively,7,15,17-19,21-25,28,29,32,33,35-37 and this work provides a solid base on which to investigate the optimum conditions for protein crystallization by continuous flow methods. The earliest studies reported that protein solution flow resulted in cessation of CEWL crystal growth.17-19 Later work showed these conditions to produce fewer, larger CEWL crystals as a result of reduced nucleation and increased rate of supply of protein to the centers of crystal growth.22-25,28,32,35 The discrepancy between these two sets of findings could be explained in some cases by the state of purity of the protein used. It was found that forced solution flow promoted CEWL crystal growth in cases where the samples were pure, but the presence of impurities resulted in cessation of CEWL crystal growth.18,29,33,36 This effect of impurities has been identified as a cause in the cessation of crystal growth in solution flow for proteins in general.39 Other proteins have demonstrated improved crystal growth under solution flow, such as adenosine deaminase40 and human triosephosphate isomerase.41 This includes the use of microfluidics for thaumatin, which also produced larger crystals.42 The improvements in crystal growth under solution flow are not necessarily evident simply in crystal size but also in the improved quality of X-ray diffraction data.21,23,36,37,40,41 These flow experiments were conducted in recirculatory systems quite different from ours and did not demonstrate any overall increase in protein crystal mass as a result of forced flow. Much depends on the vessel in which the crystallization is taking place. CEWL crystals have a strong tendency to adhere to glass.43 In fixed positions, the crystals would have an advantage of protein supply for crystal growth under forced flow over crystals in suspension that flow with the solution. In addition, the curvature of the glass capillaries provides better surface contact with the crystals, strengthening the adhesiveness. Studies of nucleation rates at a given level of CEWL supersaturation show that heterogeneous r 2010 American Chemical Society
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nucleation on the glass walls of a vessel predominates over homogeneous nucleation in solution.28,43-45 The intrinsic ability of a protein to crystallize depends on its level of supersaturation. This is typically measured as ln(c/csat),12,16 where c is the actual concentration of protein in solution and csat is the concentration at which the protein is saturated in solution. Significant nucleation and crystal growth of CEWL usually occurs only when ln(c/csat) > 2.12,16,38,43-45 This can be achieved by lowering the solution temperature and this technique has been used to grow CEWL crystals in capillaries.38 If the supersaturation level is too low, crystals take much longer to grow, or may not grow at all. In this study, using temperature reduction, we have increased the rate of continuous capillary flow to maximize protein crystal production significantly beyond the amount expected and observed here for quiescent crystallization in capillaries. 2. Experimental Methods Preparation of Lysozyme Solutions. Sodium acetate (0.1 M, from Sigma-Aldrich) was prepared with deionized water and adjusted to pH 4.8 with glacial acetic acid (from BDH Chemicals) to give 0.17 M acetate buffer. Three times recrystallized CEWL (99.96% pure by HPLC analysis) from Sigma-Aldrich (lot no. 1343474) was dissolved in this buffer to a concentration of 30 mg/mL. The precipitation buffer was prepared by dissolving NaCl (from BDH Chemicals) to a concentration of 1.1 M in 0.17 M pH 4.8 acetate buffer. All solutions were passed through a 0.2 μm filter (Corning Inc.). Lysozyme Crystallization. At 20-22 °C, the CEWL solution was mixed with an equal volume of precipitation buffer and immediately set up for crystallization in either 50 mm 1 mm I.D. borosilicate glass capillaries (The Technical Glass Company) or 100 mm 1 mm I.D. and 160 mm 2 mm I.D. soda glass capillaries (Bilbate Ltd.) by drawing the solution mixture into these capillaries at different flow rates using either a 2-channel Reglo ISM 795C or an 8-channel IPC-N peristaltic pump (Ismatec). Flow crystallization on a larger scale was investigated using 30 cm 6 mm O.D. methyl-silanized glass tubes (Analytical Columns) of 2 and 4 mm I.D. For each experiment, a quiescent crystallization control in a sealed capillary/ tube was set up under the same conditions as the capillaries/tubes under solution flow. To induce crystallization, the capillaries were immersed in an ice/water bath or cooled in a flow cell of water circulated at 0 °C. This temperature was specifically chosen to achieve the maximum possible supersaturation as a driving force for crystal growth within the time allocated for each experiment. In this way, repeated flow runs could be made in the shortest possible time to collect as much data as possible. This is also to maximize yield, emphasizing the differences between flow and quiescent conditions. At the same time, 0 °C would be just above the cloud point for lysozyme8 in these solution conditions, so avoiding any excessive protein precipitation. Images of crystal growth were recorded to a magnification of 150 at different time points using a Micro-Scopeman MS 500B video microscope (Moritex) and Intellicam image capture software (Matrox Electronic Systems). The width of CEWL crystals was measured across the parallel {110} faces46 on the digital images of the crystal photographs, where the crystal orientation in the glass capillary allows. Flow System Setup. This layout is illustrated in Figure 1. The flow circuit resembles earlier systems used to study CEWL crystal growth under flow conditions,18,21,36 although this study is the first report of the use of glass capillaries in such a system. The glass capillaries in a horizontal orientation in ice/water or embedded in a flow cell of water circulated at 0 °C (Figure 1b) are joined to the flow system at each end with 1 mm I.D. silicone tubing. These were joined to sections of 0.6 mm I.D./1.6 mm O.D. Teflon tubing to draw from the bottom of the protein solution container or to the Tygon pump tubing at the other end via adaptors in cases where the fit of silicone to Tygon tubing was incompatible. Different pump flow rates were achieved simultaneously on the 8-channel pump using different diameters of Tygon pump tubing (0.38, 0.57, 0.89, 1.09, 1.3, 1.65,
Figure 1. (a) Schematic diagram of the flow system. Arrows indicate directions of solution flow. (b) The flow cell used to cool the CEWL solution flowing through the capillaries simultaneously at different flow rates and a capillary with a quiescent control solution. Cooling water runs outside the capillaries in counter-current mode at 8.5 L/min, giving 34 changes/min for a 250 mL cell. A constant temperature in the cell was monitored with a mercury thermometer. 2.06, 2.79 mm I.D.) with a roller speed setting to give a calculated flow rate of 15.9 μL/min for the 1.09 mm I.D. tube. Real flow rates were checked experimentally by measuring the mass of water passed out of each tube over 100 min. The same flow rate of 50 μL/min was applied to both 30 cm 6 mm tubes, the different internal flow velocities being achieved by the differences in the tube internal diameters (Table 1). The protein solution was pulled rather than pushed into the capillaries to ensure a smooth flow of solution not subject to pulsations from the pump. The protein solution outflow was recirculated back into each container, taking care that it was directed against the side of the vessel above the liquid level to avoid splashing, a technique previously used to avoid the denaturation of CEWL.21 This also ensured that all the original CEWL solution in each container was used before the recirculated solution was drawn into the capillaries without mixing. In this way, the solution was repeatedly recirculated through the capillaries to ensure that as much protein as possible can crystallize out of solution by temperature reduction. Similar experiments were performed without recirculation of protein to determine the yield of crystals that could be produced in one pass of the solution through each capillary. The capillaries were initially monitored with CEWL under these flow conditions at 20 °C for 19 h. No measurable crystal growth was seen, demonstrating that a temperature reduction was necessary for crystallization to occur. The temperature reduction of water flowing into cooled 2mm glass capillaries in the flow cell was measured using a Fluke digital thermometer with a type K thermocouple probe (Table 2). The thermal properties of water should be similar to the CEWL solution. Nonetheless, all crystal images and size measurements
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Table 1. Effect of Solution Flow on CEWL Crystal Yield in Glass Tubes (a) A Single Passage of Protein Solution Drawn through Tubes without Recirculation tube type 50 mm 1 mm borosilicate glassa (39.3 μL) 30 cm 6 mm methyl-silanized glassa 2 mm I.D. (0.94 mL) 4 mm I.D. (3.78 mL) 100 mm 1 mm soda glassa (78.5 μL)
flow velocity (mm min-1)
crystal mass (mg)
yield of total protein (%)
incubation time (h)
0.44 32.9
74.4 11.0
32 32
3, 4 3, 4
78.0 12.4 75.1 24.9
22 22 22 22
8 8 8 8
52.0 12.9 7.0 3.4 2.3 90.0
22 22 22 22 22 98
6 6 6 6 6
sealed 12.8 sealed 16 sealed 4
10.6 89.4 40.6 179.1
sealed 12.7 25.65 60.3 106.8 sealedb
0.6 25.4 30.1 32.35 38.2 1.1
reference to figures
(b) With Recirculation of Protein Solution tube type 100 mm 1 mm soda glass (78.5 μL) with recirculation of 4 mL of protein solution through ice/water bath see Figure 9
140 mm 2 mm soda glass (440 μL) with recirculation of 4 mL of protein solution through flow cell cooled to 0 °C
flow velocity (mm min-1)
crystal mass (mg)
yield of total protein (%)
incubation time (h)
sealed 3.0c 6.0 13.2 19.7 27.4 42.5 63.0 107.5
0.7 13.8 18.3 20.6 21.3 21.5 24.0 26.9 21.5
68.6 25.1 31.7 35.8 37.0 37.3 41.5 46.7 37.2
29 29 29 29 29 29 29 29 29
sealed 5.6 12.6 18.3 25.4 38.4 56.9
2.0d 49.2 49.5 49.0 50.1 49.7 50.1
81.4 78.8 79.2 78.5 80.3 79.6 80.2
40 40 40 40 40 40 40
recirculation frequency 0 0.95 1.9 4.2 6.2 8.7 13.5 20.0 33.9 0 10.6 23.7 34.5 47.9 72.5 107.3
a In ice/water bath. b In water flow cell cooled to 0 °C. c At this flow velocity, only 3.8 mL was drawn into the capillary, so the protein solution was not recirculated. d This is the weight of CEWL crystals in the 5 cm length of solution in the sealed capillary. The entire capillary therefore has the capacity for 5.6 mg of CEWL crystals.
were recorded at least 2 cm from the entry point of protein solution into the cooled cell. Calculation of Crystal Yield. Using the pump flow, the CEWL crystals were initially rinsed in situ with ice-cold precipitation buffer to remove protein supernatant. This was followed by 0.17 M pH 4.8 acetate buffer at 22 °C, which dissolved all the crystals from the capillary. The concentrations of the resulting CEWL solutions were estimated from the extinction coefficient, ε280 = 2.64 mL/(mg cm)26 by measuring the UV absorbance at 280 nm. The mass of CEWL crystals and their yield of total protein were calculated from the volumes of redissolved crystals in solution or the remaining protein supernatant.
3. Results and Discussion Although CEWL at 30 mg/mL in 0.17 M pH 4.8 acetate buffer is below the saturation level at 20 °C, mixing this with an equal volume of the precipitant, 1.1 M NaCl in the same buffer, at this temperature causes the mixture (15mg/ml CEWL) to be supersaturated to a level of c/csat = 1.5 (ln(c/csat) = 0.4) since CEWL solubility in this mixture at 20 °C is ∼10 mg/mL.6,8,9,14,38 This level of supersaturation is far too low to produce any measurable crystal growth within a week.12 Consequently, it is not surprising that the capillaries filled with this mixture for 19 h at 20 °C in quiescent mode and
with the flow rates used here produced no visible crystals. Reducing the temperature of the capillaries in an ice/water bath reduces CEWL solubility in the crystallization mixture to ∼1.5 mg/mL.6,8,9 This gives ln(c/csat) = 2.3, which is above the ln(c/csat) = 2 limit for significant crystal growth,12,16,38,43-45 and this produced crystals in a few hours. This was aided by the selection of the buffer pH as 4.8, which has been established as a solubility minimum for tetragonal CEWL crystals.6 In the sealed unsilanized tubes, batch crystallization without flow gives a crystal yield of total protein at 0 °C which increases with incubation time from 22 h (52%) to 98 h (90%) (Figure 2). This yield corresponds to that expected from the solubility of CEWL at this temperature. If all the CEWL (15mg/ml) were to crystallize to the point at which the supernatant reached the solubility limit of 1.5 mg/mL at this temperature, then the crystal yield would also be expected to be 90%. Clearly the crystallization is almost complete when the crystals were harvested at 4 days incubation. A plot of the five yield values in Table 1 (Figure 2) shows that the growth rate of the crystals tails off with time and plateaus toward the maximum yield asymptotically. This behavior is characteristic of the growth of CEWL crystals in undisturbed solutions7 since the rate of protein depletion from solution is proportional to the supersaturation. The time course of the increase in protein
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Figure 2. Plot of CEWL crystallization yield as a percentage of total protein in solution with incubation time at 0 °C in unsilanized glass capillaries under quiescent conditions at 15 mg/mL CEWL in 0.17 M pH 4.8 acetate buffer containing 0.55 M sodium chloride. Data taken from Table 1.
Figure 4. Progress of CEWL crystal growth in horizontal 5 cm 1 mm borosilicate glass capillaries at different time points during incubation in ice/water in (a) sealed capillary, (b) capillary with 12.8 mm/min flow velocity containing 15 mg/mL CEWL in 0.17 M pH 4.8 acetate buffer with 0.55 M sodium chloride. Protein solution was not recirculated. Arrows indicate the locations of the parallel {110} faces for sizing.
Figure 3. CEWL crystal growth. Horizontal 5 cm 1 mm I.D. borosilicate glass capillaries in ice/water after 32 h incubation with 15 mg/mL CEWL in 0.17 M pH 4.8 acetate buffer containing 0.55 M sodium chloride in (a) sealed capillary, (b) capillary with 12.8 mm/min solution flow velocity. Protein solution was not recirculated. Arrows indicate the locations of the parallel {110} faces for sizing.
crystal yield in Figure 2 is therefore independent of capillary diameter. Laminar flow has been observed with the movement of particulate matter through the capillaries. This is supported by the fact that the Reynolds number Re = Fνd/μ is < 2 at the highest flow velocity (ν) used in this work for capillary diameter d = 1 mm, using density (F) and viscosity (μ) values for water. Therefore, the flow velocity at the perimeter of the tube will be slower than toward its center. Consequently, there will be a temperature gradient perpendicular to the direction of flow from the perimeter of the tube where it is coldest at the glass surface to the center of the tube as the CEWL solution
flows through the cooled cell. The nucleating particles of CEWL that form on the inside glass surface as a result of increased supersaturation by cooling will be fed by protein from the warmer solution flowing through the central part of the tube to deposit onto the growing crystals. The protein solution crystallized soon after entering the cooled capillaries, and the reduced protein concentration of the supernatant following crystal deposition and exit from the cell prevented any further crystal deposition elsewhere as it rewarmed to room temperature. As a result, crystal growth by forced flow was generally observed throughout the length of the cooled sections of the tubes but not elsewhere. To estimate how quickly the CEWL solution entering the capillaries cools to reach the required degree of supersaturation for the crystal growth observed, we have experimentally determined the cooling profiles of water passing through the 2 mm tubes at different flow velocities through the cooled flow cell using a temperature probe placed at fixed distances from the entry into the cell (Table 2). This measures the average temperature of the flow water rather than any cross-sectional variation in temperature at any given position along the length of the tube. At the highest flow velocity used in our experiments with 2 mm tubes (56.9 mm/min), cooling to about 1 °C would be achieved 1 cm into the flow cell and close to 0 °C at
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2 cm into the flow cell, from an analysis of the cooling profiles of flow velocities closest to this value. This represents the most extreme case of temperature lag in our work. At lower flow velocities, the rate of cooling with distance increases. For a given flow velocity at a specified entry distance into the cooled section of the tube, the temperature difference between the coolant and liquid flowing through the tube will increase exponentially with the square of the tube diameter, based on a plug liquid flow approximation.47 This implies that for 1 mm diameter tubes cooling to below 0.1 °C would be achieved 1 cm along the tube into the flow cell even at the highest flow velocity we have used (107.5 mm/min). Consequently, a high degree of supersaturation for nucleation and crystal growth would be quickly attained for the CEWL solution entering the cooled tubes. Under flow conditions at low temperature, where protein crystallized from solution is continually replenished, crystal growth is increased up to 0.37 mm in 27-32 h across the {110} faces46 compared to crystal growth up to 0.16 mm under quiescent conditions (Figures 3, 4, 6, 8, and 9) and this is reflected in the increase in crystal mass measured by UV absorbance (Table 1 and Figures 7 and 10). However, within the first few hours, crystallization in the flow through capillaries appears to be no more advanced than crystallization in the sealed capillaries (Figure 4), although in both cases the crystals are still quite small (63 mm/min), the inhibitory effects of these impurities on crystal growth, coupled with the shear effects and temperature lag due to shorter residence time in the tube, delaying crystallization, will result in a lower crystal yield. These may be the reasons for the lower crystal yield observed at a flow velocity of 107.5 mm/min (Figure 10). Clearly, more measurements between 63 and 107.5 mm/min are needed to establish the optimal flow velocity for maximum crystal yield. This may depend on the number of times the solution is recirculated and hence the solution volume and run time and also the capillary diameter. Each of these factors will lower the protein concentration in the supernatant to a different degree as crystals grow out of the flowing solution. The maximum growth rate of CEWL crystals obtained by recirculated forced flow through polypropylene hollow fiber membranes 1.5 mm in diameter was determined to be 66 mm/ min,36 close to the flow velocity for our maximum crystal yield under recirculation conditions. They also used three times recrystallized CEWL from Sigma (20 mg/mL) at 5 °C in 0.1 M pH 4.6 acetate buffer. The CEWL supersaturation ln(c/csat) = 2.5, also close to our value. These conditions correspond to a quiescent CEWL crystal growth rate of 0.002 μm/s,11 a similar value for our conditions. This suggests that the higher quiescent crystal growth rates, which can be estimated from CEWL solution temperature and supersaturation values,11 increase the optimum flow velocity for maximum crystal growth and yield. However, their crystal growth rate and size (5% w/v) combined with forced flow.36 The properties of the membrane may also affect crystal growth rate and habit, such as surface porosity, altered flow dynamics, and vapor diffusion. An optimum flow velocity for maximum crystal growth rate and yield can be explained by the two control processes in crystal growth: kinetic and transport control.29,35,51 In quiescent solutions, there is a depletion in protein concentration at the surface of the crystal due to the incorporation of protein from solution to crystal.48,49 In this situation, transport control of protein to the crystal by diffusion or convection is the rate-limiting step. Therefore, the application of a solution flow will increase the rate of supply of protein to the crystal to a point where the kinetic control becomes the rate-limiting step in the crystal growth rate. Flow velocities higher than this optimal value will not increase the crystal growth rate any
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further. In our studies, the maximum crystal yield is determined by the crystal growth rate. An inspection of the crystals from fresh (Figure 6) and recirculated protein (Figure 9) suggests the crystal size but not number increases with flow velocity. This may explain the close agreement between the flow velocities for maximum CEWL crystal growth rate36 and yield in our study. The crystal habit appears to be of a better quality at the lower flow velocities. At the higher flow velocities the crystals are more distorted and appear aggregated due to excessive flow effects, particularly for recirculated protein (Figure 9). The likelihood of CEWL crystal chunks being detached from glass tubes during flow to give secondary nucleation by fluid shear forces has been eliminated in a separate study,52 so this cannot be a reason for reduced crystal growth at higher than optimum recirculating flow velocities. One would also expect a more consistent crystal growth rate under constant flow conditions where irregular convective and diffusion effects are minimized. The results of this are seen in Figure 5, where a more regular CSD is obtained under flow conditions. The other important point to consider is the residence time of the solution flowing through the cooled capillary tube; longer residence times will allow the cooled supersaturated solution to nucleate and crystallize more effectively in the capillary tube. Higher flow velocities will increase crystal growth, but there comes a point where there is a trade-off in terms of less time for the solution to cool, nucleate, and crystallize in the glass capillary tube, as a result of temperature lag. This may contribute to the reduction in crystal yield beyond a critical flow velocity. This is minimized as far as possible by the high flow rate of cooled water through the flow cell. The statistics on the crystal size distributions were gathered at the slowest flow velocities in the narrowest tubes where the temperature lag is minimized. For unrecirculated protein, crystal yield decreases with increasing flow velocity, because there is likely to be a core of warmer protein solution flowing along the middle of the tube that has too low a level of supersaturation to deposit crystals in the time it takes to flow through the tube. This is circumvented by recirculation, where any uncrystallized protein can be crystallized on repeated passes through the cooled tube. The temperature lag of liquid flowing into the cooled glass capillary from room temperature was shown to be insignificant (Table 2) but does increase with flow velocity, which reduces the residence time and therefore the crystal yield. Temperature lag will also increase with tube diameter. However, crystal growth recorded in the 2 mm and 4 mm 30 cm long flow tubes after 22 h at positions 5, 10, 15, 20, or 25 cm from the tube entry point showed more crystal growth closer to the point of entry of protein solution into the tubes probably due to depletion of protein with the deposition of crystals onto the glass walls of the tube as it moves downstream (data not shown). This supports the theory that the initial nucleation and crystal growth under flow take place primarily in the solution close to the inner glass surface. This is further supported by the finding that the differences in the forced flow crystal yields between the 2 and 4 mm tubes are proportional to the inner glass surface area rather than the internal volume. Homogeneous nucleation in the bulk solution would occur by cooling further downstream in the tube and be removed by flow. One final consideration is capillary capacity. The yield of crystals, 11% of total protein passed through the 50 mm 1 mm capillary at a flow velocity of 12.8 mm/min, is slightly lower than the crystal yield in the 100 mm 1 mm capillary
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tube with a 12.7 mm/min flow velocity (12.9%). This could be due to the reduced capacity for further crystal growth, since the experiment was conducted for a longer time in a smaller capillary and the crystal mass/tube volume ratio is much higher and may be close to its saturation limit. The run time for the protein recirculation experiment was extended to 40 h using capillaries of a larger capacity (140 mm 2 mm) to accommodate the additional crystal growth (Table 1b). This improved the crystal yields to 79-80% of total protein for all flow rates, close to that of the quiescent control in the sealed tube (81.4%). Therefore, capillary flow has achieved the same crystallization yield with 9 times the solution volume into an equivalent capillary space. 4. Conclusion We have increased the growth of CEWL crystals in continuous flow capillaries by lowering the temperature to achieve a supersaturation level that produces crystal mass within a defined space that is up to 75 higher than that obtained with quiescent crystallization from a larger volume of protein solution. A sampling of one experiment shows a more regular CSD under capillary flow. Maximum crystal yield has been observed at a flow rate of 63 mm/min in a recirculating system with a specially designed flow cell. An optimum flow velocity for crystal growth needs to be explored beyond this value and with different flow conditions and levels of supersaturation and will be the subject of future investigations. This technique has the advantage of concentrating crystal growth from larger bulk volumes into a single tube rather than from several batch crystallization tubes. It is also more compatible with the continuous flow procedures used in industry. We have also shown that this method of crystal production can be scaled up into larger size tubes, where the effect of tube surface methylation has also been tested. Much larger volumes of protein solution can be brought to the same crystallization yield as a quiescent solution using capillary flow. In view of this, it should be possible to scale this process up to pilot plant production. It remains to be seen what effect impurities will have on this crystallization process. It will be important to overcome this challenge to develop this technique for large-scale protein purification. Also, crystal columns could be built this way with protein enzymes for biocatalysis. This new technology for growing larger size CEWL crystals should be applicable to other proteins, which would make it possible to conduct a range of physical studies on proteins in the crystalline state that were previously not possible due to current limitations in crystal size. Protein crystals of a specified size required for X-ray or even neutron diffraction studies can now be grown in a much shorter time. It takes a week to grow CEWL crystals by conventional nonflow methods to a size of 0.24 mm across the {110} faces12 in similar solution conditions to the ones used in our study, whereas our flow method can achieve 0.37 mm in a day. This time saving factor will be important to reduce operation costs in the pharmaceutical and biotechnology industries. These flow methods could be adapted to produce more protein therapeutics in crystalline form as improved formulations with better bioavailability.3 Although there may be some loss of protein in the supernatant, the protein incorporated into a crystal for drug formulation is likely to be in the native state and therefore active. The crystalline form of a protein therapeutic will therefore contain the active protein in a purer and more concentrated state than would exist in a solution formulation,
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where there could be significant amounts of inactive forms of the protein. In this sense, crystallization can be considered to be the final purification step to remove degraded, aggregated, or misfolded forms of the protein. This, together with the improved efficiency of flow crystallization in downstream processing, should more than compensate for any loss of supernatant protein. The attachment of the crystals to the insides of the tubes has an advantage over stirred bulk crystallizers, since this more easily enables removal of impurities and washing to be performed. This feature could also be used as a means to make affinity purification columns with the crystals as the stationary phase. The larger crystal sizes reported here cannot be administered as a therapeutic protein but could serve as a more stable storage form of purer concentrated protein for subsequent solubilization and injection. More medically relevant proteins could give more suitable crystal size distributions under forced capillary flow. Acknowledgment. This work was financially supported by the BBSRC (Grant BB/F004990/1) and the Bioprocessing Industry Research Club. We wish to thank Marc Schaepertoens and Wei Kheng for their temperature equilibration studies on the flow cell to estimate the degree of cooling during forced capillary flow.
References (1) Niimura, N.; Bau, R. Acta Crystallogr., Sect. A 2008, 64, 12–22. (2) Ataka, M.; Tanaka, S. Biopolymers 1986, 25, 337-350 and references therein. (3) Basu, S. K.; Govardhan, C. P.; Jung, C. W.; Margolin, A. L. Exp. Opin. Biol. Ther. 2004, 4, 301-317 and references therein. (4) Brange, J.; Volund, B. Adv. Drug Delivery Rev. 1999, 35, 307–335. (5) Shi, D.; Mhaskar, P.; El-Farra, N. H.; Christofides, P. D. Nanotechnology 2005, 16, 5562-5574 and references therein. (6) Cacioppo, E.; Pusey, M. L. J. Cryst. Growth 1991, 114, 286–292. (7) Forsythe, E.; Ewing, F.; Pusey, M. L. Acta Crystallogr., Sect. D 1994, 50, 614–619. (8) Lu, J.; Carpenter, K.; Li, R.-J.; Wang, X.-J.; Ching, C.-B. Biophys. Chem. 2004, 109, 105–112. (9) Forsythe, E. L.; Pusey, M. L. J. Cryst. Growth 1996, 168, 112–117. (10) Sazaki, G.; Kurihara, K.; Nakada, T.; Miyashita, S.; Komatsu, H. J. Cryst. Growth 1996, 169, 355–360. (11) Nadarajah, A.; Forsythe, E. L.; Pusey, M. L. J. Cryst. Growth 1995, 151, 163–172. (12) Judge, R. A.; Jacobs, R. S.; Frazier, T.; Snell, E. H.; Pusey, M. L. Biophys. J. 1999, 77, 1585–1593. (13) Chayen, N. E.; Radcliffe, J. W.; Blow, D. M. Protein Sci. 1993, 2, 113–118. (14) Forsythe, E. L.; Judge, R. A.; Pusey, M. L. J. Chem. Eng. Data 1999, 44, 637–640. (15) Durbin, S. D.; Feher, G. J. Cryst. Growth 1986, 76, 583–592. (16) Forsythe, E.; Pusey, M. L. J. Cryst. Growth 1994, 139, 89–94. (17) Nyce, T. A.; Rosenberger, F. J. Cryst. Growth 1991, 110, 52–59. (18) Vekilov, P. G.; Rosenberger, F. J. Cryst. Growth 1998, 186, 251–261. (19) Pusey, M.; Witherow, W.; Naumann, R. J. Cryst. Growth 1988, 90, 105–111. (20) Hirschler, J.; Charon, M. H.; Fontecilla-Camps, J. C. Protein Sci. 1995, 4, 2573–2577.
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(21) Kadowaki, A.; Yoshizaki, I.; Rong, L.; Komatsu, H.; Odawara, O.; Yoda, S. J. Synchrotron Rad. 2004, 11, 38–40. (22) Adachi, H.; Takano, K.; Yoshimura, M.; Mori, Y.; Sasaki, T. Jpn. J. Appl. Phys. 2003, 42, L314–L315. (23) Adachi, H.; Takano, K.; Matsumura, H.; Inoue, T.; Mori, Y.; Sasaki, T. J. Synchrotron Rad. 2004, 11, 121–124. (24) Adachi, H.; Takano, K.; Yoshimura, M.; Mori, Y.; Sasaki, T. Jpn. J. Appl. Phys. 2002, 41, L1025–L1027. (25) Maki, S.; Murai, R.; Yoshikawa, H. Y.; Kitatani, T.; Nakata, S.; Kawahara, H.; Hasenaka, H.; Kobayashi, A.; Okada, S.; Sugiyama, S.; Adachi, H.; Matsumura, H.; Takano, K.; Murakami, S.; Inoue, T.; Sasaki, T.; Mori, Y. J. Synchrotron Rad. 2008, 15, 269–272. (26) Sophianopoulos, A. J.; Rhodes, C. K.; Holcomb, D. N.; Van Holde, K. E. J. Biol. Chem. 1962, 237, 1107–1112. (27) Durbin, S. D.; Feher, G. J. Mol. Biol. 1990, 212, 763–774. (28) Nanev, C. N.; Penkova, A.; Chayen, N. Ann. N.Y. Acad. Sci. 2004, 1027, 1–9. (29) Vekilov, P. G. Adv. Space Res. 1999, 24, 1231–1240. (30) Boudjemline, A.; Clarke, D. T.; Freeman, N. J.; Nicholson, J. M.; Jones, G. R. J. Appl. Crystallogr. 2008, 41, 523–530. (31) Thomas, B. R.; Vekilov, P. G.; Rosenberger, F. Acta Crystallogr., Sect. D 1998, 54, 226–236. (32) Penkova, A.; Pan, W.; Hodjaoglu, F.; Vekilov, P. G. Ann. N.Y. Acad. Sci. 2006, 1077, 214–231. (33) Vekilov, P. G.; Thomas, B. R.; Rosenberger, F. J. Phys. Chem. B 1998, 102, 5208–5216. (34) Aune, K. C.; Tanford, C. Biochemistry 1969, 8, 4579–4590. (35) Lin, H.; Rosenberger, F.; Alexander, J. I. D.; Nadarajah, A. J. Cryst. Growth 1995, 151, 153–162. (36) Curcio, E.; Simone, S.; Di Profio, G.; Drioli, E.; Cassetta, A.; Lambda, D. J. Membr. Sci. 2005, 257, 134–143. (37) Kadowaki, A.; Yoshizaki, I.; Adachi, S.; Komatsu, H.; Odawara, O.; Yoda, S. Cryst. Growth Des. 2006, 6, 2398–2403. (38) Rosenberger, F.; Howard, S. B.; Sowers, J. W.; Nyce, T. A. J. Cryst. Growth 1993, 129, 1–12. (39) Grant, M. L.; Saville, D. A. J. Cryst. Growth 1991, 108, 8–18. (40) Adachi, H.; Takano, K.; Niino, A.; Matsumura, H.; Kinoshita, T.; Warizaya, M.; Inoue, T.; Mori, Y.; Sasaki, T. Acta Crystallogr., Sect. D 2005, 61, 759–762. (41) Adachi, H.; Niino, A.; Kinoshita, T.; Warizaya, M.; Maruki, R.; Takano, K.; Matsumura, H.; Inoue, T.; Murakami, S.; T.; Mori, Y.; Sasaki, T. J. Biosci. Bioeng. 2006, 101, 83–86. (42) Chen, D. L.; Gerdts, C. J.; Ismagilov, R. F. J. Am. Chem. Soc. 2005, 127, 9672–9673. (43) Tsekova, D.; Dimitrova, S.; Nanev, C. N. J. Cryst. Growth 1999, 196, 226–233. (44) Galkin, O.; Vekilov, P. G. J. Phys. Chem. B 1999, 103, 10965– 10971. (45) Galkin, O.; Vekilov, P. G. J. Am. Chem. Soc. 2000, 122, 156–163. (46) Nadarajah, A.; Pusey, M. L. Acta Crystallogr., Sect. D 1996, 52, 983–996. (47) Bird, R. B.; Stewart, W. E.; Lightfoot, E. N. Transport Phenomena, Revised 2nd ed.; John Wiley & Sons: New York, 2007. (48) Komatsu, H.; Miyashita, S.; Suzuki, Y. Jpn. J. Appl. Phys. 1993, 32, L1855–L1857. (49) Miyashita, S.; Komatsu, H.; Suzuki, Y.; Nakada, T. J. Cryst. Growth 1994, 141, 419–424. (50) Paxton, T. E.; Sambanis, A.; Rousseau, R. W. Langmuir 2001, 17, 3076–3079. (51) Vekilov, P. G.; Alexander, J. I. D.; Rosenberger, F. Phys. Rev. 1996, 54, 6650–6660. (52) Tait, S.; White, E. T.; Litster, J. D. Cryst. Growth Des. 2009, 9, 2198–2206.
