Article pubs.acs.org/crystal
Control of Crystal Size during Oiling Out Crystallization of an API Masahiro Takasuga*,†,‡ and Hiroshi Ooshima‡ †
Chemical Development Laboratories, CMC Center, Takeda Pharmaceutical Company Limited, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka, 532-8686, Japan ‡ Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan ABSTRACT: The oiling out crystallization of a pharmaceutical compound API-T dissolved in acetone/water was conducted using a 100 mL crystallizer, and the effect of oil droplets on the resulting crystal size was investigated. The size of oil droplets was controlled by varying agitation speed, and the resulting crystal size decreased with an increase in the size of oil droplets, namely, with a decrease in agitation speed. The observed phenomenon could be explained by the difference of nucleation rate in large and small droplets. The generation of small crystals was caused by the ease of primary and secondary nucleation in large droplets. A decrease in oil droplet size restrained the primary nucleation and also the secondary nucleation. In this case, the small amount of crystals that were released to the continuous phase grew by absorbing small droplets. On the other hand, in single-phase crystallization without liquid−liquid phase separation, the crystal size did not depend on agitation speed. crystallization is found to occur.9 However, Derdour14 has shown a methodology to acquire primary crystals that can be used as seed crystals for pharmaceutical active pharmaceutical ingredients (APIs) that tend to oil out and are hard to crystallize. There are also a few cases where the oiling out phenomenon is actively utilized to control the characteristics of the crystals, and Veesler et al.15 have taken advantage of oiling out to acquire spherical crystals. In the present study, we have performed oiling out crystallization of an API in an acetone/water solvent system and investigated the effect of oiling out on the final size of the crystals obtained. We show that the number of crystals can be controlled by controlling the size of the oil droplets. Thus, we believe the oiling out phenomenon can be actively utilized for the control of crystal size.
1. INTRODUCTION Crystallization is one of the most important separation technologies in the pharmaceutical industry. The bioavailability of drugs is strongly affected by the physical properties of crystals such as crystal size, polymorphism, and shape, which depend on the method used for the crystallization. It is also required to have consistent bioavailability for different manufacturing lots, so it is crucial to reproducibly control the characteristics of crystals. In crystallization using a mixed solvent of water and watermiscible organic solvents, a liquid−liquid phase separation phenomenon is often observed during cooling or on adding the poor solvents, which is known as oiling out crystallization. The phenomenon has been reported, for instance, in the crystallization of proteins.1−4 In studies on oiling out crystallization of low molecular weight compounds, the phase diagram is often considered to understand the whole picture of the liquid−liquid phase separation.5,6 A comprehensive understanding of the phase diagram is helpful for understanding oiling out crystallization, in particular, such complicated cases as those involving polymorphism.7,8 Lu et al.9 succeeded to prevent oiling out and improve the quality of idebenone by determining the phase diagram, and there are some reports showing the usefulness of in situ monitoring to understand the mechanism of oiling out crystallization.10−13 However, there are few reports about the oiling out crystallization of low molecular weight compounds, presumably due to the usual idea that oiling out crystallization is undesirable and should be avoided whenever possible in industrial processes. For instance, the nucleation rate in oiling out crystallization is usually slow, in spite of the high supersaturation ratio,10−13 and there are many cases where no © 2014 American Chemical Society
2. EXPERIMENTAL SECTION 2.1. Materials. The pharmaceutical compound used in this study was API-T, produced by Takeda Pharmaceutical Co., Ltd. with a purity of 99.0 wt %. Acetone was of reagent grade, and distilled water was obtained from Wako Pure Chemical Industries. 2.2. Solubility and Cloud Point. The measurements of the solubility and the cloud point of API-T in acetone/water mixed solvent were carried out in a temperature range of 10−40 °C using a Chemistation, Tokyo Rikakikai Co., Ltd., with five flasks, a temperature control unit, and a magnetic stirrer for each flask. The solubility was measured by dissolving as much as possible of an excess amount of crystals. The API-T concentration was analyzed using a high performance liquid chromatograph (HPLC), LC-2010, from Received: August 8, 2014 Revised: September 30, 2014 Published: October 1, 2014 6006
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Figure 1. Solubility and cloud point of API-T (a) in a mixed solvent of 50 wt % acetone and (b) at 20 °C. Region I is where the solution is unsaturated, region II is where the solution is saturated and single phase, and region III is where the solution is saturated and liquid−liquid phase separation occurs. Shimadzu Co., Ltd. For the cloud point measurement, a given concentration of API-T solution was prepared with a given acetone composition of mixed solvent, and the solution was gradually cooled until liquid−liquid phase separation was observed. The cloud point was determined as the temperature at which the phase separation occurred. The same experiments were carried out by changing the API-T concentration and the acetone composition in the range of 0.014−1.05 g/g-solvent and 45−75 wt %, respectively. 2.3. Oiling Out Crystallization. Oiling out crystallization was carried out at 20 °C using a 100 mL three-necked/semiellipsebottomed glass crystallizer equipped with a three-retreating-blades agitator and a temperature probe, with the temperature being controlled by a thermostatic bath. In the selection of the experimental conditions, care was taken to prevent the aggregation of oil droplets during the crystallization and its observation. As a result, the experimental condition at which the total volume of oil phase became smaller than that of the continuous phase was chosen. For example, 2 g of API-T was placed in the crystallizer and dissolved with 14.1 g of acetone at 20 °C. Then 14.1 g of water preadjusted to 20 °C was added to the solution, resulting in oiling out of the solution at 50 wt % acetone. The initial supersaturation ratio for that condition was 4.1, and under agitation, the oil phase consisted of numerous small droplets. The agitation speed was controlled at a speed in the range of 50−200 rpm. The compositions of both phases were analyzed, as follows. A sample containing both the oil and continuous phases was withdrawn from the mixture before nucleation, and the oil and continuous phases were separated by centrifugation before being weighed. The API-T concentration and the acetone composition of each phase were analyzed using HPLC and gas chromatography, GC-2014, Shimadzu Co., Ltd., respectively, and the water composition was calculated by mass balance. As a control experiment, single-phase crystallization was conducted by increasing acetone composition to 53 wt %, using a mixed solvent of 14.9 g of acetone and 13.3 g of water for the same amount of API-T at 20 °C. The initial supersaturation ratio at that condition was 2.5. 2.4. Measurements of the Size of Oil Droplets and Crystals. An aliquot of the mixture was withdrawn from the oiling out crystallization mixture and placed on a slide glass. Oil droplets were quickly observed with an optical microscope equipped with a digital camera, VHX-600, Keyence Corp., before crystallization started. Then, the diameters of more than 400 droplets for each sample were measured on the digital images saved as graphic files. The numberweighted median diameter was determined as the representative of the droplet size. Crystals obtained after 8 h crystallization were also observed by the microscope. The length and breadth of platelike crystals were measured for more than 600 crystals. In order to convert the size to
the equivalent volume diameter, a representative thickness and breadth of crystals were determined by using scanning electron microscope images, VE-9800, Keyence Corp. The crystal size distribution was provided for the equivalent volume diameter, and the mass-weighted median size was used as the representative of the crystal size.
3. RESULTS AND DISCUSSION 3.1. Phase Diagram. The solubility curves are presented as a function of temperature or acetone composition as shown in Figure 1, with an example of the cloud point curve at 50 wt % acetone shown in Figure 1a. From the cloud point curves obtained by changing the acetone composition, another cloud point curve was determined as a function of acetone composition at 20 °C, as shown in Figure 1b. Region I is where the solution is unsaturated, region II is where the solution is saturated and single phase, and region III represents where the solution is saturated and liquid−liquid phase separation occurs. Figure 2 shows both the homogeneous crystallization solution and a liquid/liquid-phase separated solution in a static condition. 3.2. Compositions of Continuous and Oil Phases in Oiling Out Crystallization. In the oiling out crystallization, the continuous phase composition and oil phase composition were determined as shown in Table 1. More than 60% of the API-T was distributed to the oil phase, although the mass was only about one tenth of the total liquid mass. The acetone
Figure 2. A single-phase crystallization solution of API-T dissolved in acetone/water (left) and an oiling out crystallization solution (right): continuous phase (top) and oil phase (bottom). 6007
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Table 1. Composition of the Two Phases in Oiling out Crystallization phase
weight (g)
API-T concentration (g/g-solvent)
API-T distribution (wt %)
acetone composition (wt %)
solubilitya (g/g-solvent)
supersaturation ratio (−)
continuous oil total charge
27.1 3.1 30.2
0.028 0.680 0.070
37 63 100
48.5 70.8 50.0
0.013 0.315 0.017
2.2 2.2 4.1
a
The solubility curve is presented in Figure 1b.
smaller the size of oil droplets, the larger the resulting crystals become. The effect of agitation on the size of crystals was also examined for the single-phase crystallization conducted under 53 wt % acetone (without liquid−liquid phase separation), and the result is also presented in Figure 6. In the single-phase crystallization, the crystal size did not change by varying the agitation speed. 3.4. Microscopic Observations of Nucleation and Crystal Growth in Oiling Out Crystallization. Nucleation and crystal growth in oiling out crystallization were observed on a slide glass as shown in Figure 8. Panels a and b show a case where a large crystal contacts an oil droplet and consumes the API-T contained in the oil, leaving the oil droplet noticeably smaller. The process will involve the release of acetone from the droplet to the continuous phase. On the other hand, panels c and d show a crystal growing in a droplet, and then the droplet has disappeared, respectively. Although these phenomena were observed on a slide glass, they suggest that oil droplets are consumed by the growth of crystals formed inside droplets and by crystals attaching to droplets. 3.5. Mechanisms of Generation of Small or Large Crystals in Oiling Out Crystallization. In order to understand the relationship between oil droplet size and the crystal size shown in Figure 7, microscopic observation of oil droplets was carried out during an oiling out crystallization. Some oil droplets are presented in Figure 9, where panel a shows an oil droplet containing many fine crystals inside. Panel b shows that large oil droplets contain more crystals inside than small oil droplets and that there is no crystal in small droplets. Figure 9 suggests that the nucleation rate is affected by the size of the oil droplet. Oil droplets containing crystals were numbered from the photographic data taken at given time intervals, and the ratio to the total number of droplets was determined. The data were taken for different sizes of oil droplets by controlling the agitation speed, as explained above (Figure 4). Figure 10a,b presents the percentage of oil droplets containing crystals as a function of droplet size and as a function of crystallization time, respectively. The percentage of oil droplets containing fine crystals increased with both the droplet size and the crystallization time. For instance, the percentage of droplets containing crystals was 72% for droplets in the size range of 100−200 μm at 7 min after the oiling out crystallization started, whereas in the case of droplets in the size range of 20−40 μm, it was only 7%, and oil droplets of less than 20 μm showed no nucleation within 15 min. The rapid fall in the increasing rate of the percentage of droplets containing crystals observed after 7 min for all size range of droplets is due to the disappearance of the oil droplets by crystal growth. From these results, we conclude that the primary nucleation is easier in large oil droplets. Gong et al.16 have presented a similar observation in the colloidal crystallization of poly-N-isopropylacrylamide, noting that the probability of two independent nucleation chain events occurring in a single droplet decreases as the
composition and the API-T concentration in the oil phase were higher than those in the continuous phase. However, the supersaturation ratios of both phases were the same level in this case due to the solubility of API-T increasing as the acetone content increased. 3.3. Effect of Oiling Out on Crystal Size. Oil droplets formed under agitation at 75 rpm were larger than those at 200 rpm, as can be seen in Figure 3, which implies that the oil
Figure 3. Micrographs of oil droplets obtained during oiling out crystallization at (a) 75 rpm (coefficient of variation; 1.21) and (b) 200 rpm (coefficient of variation; 0.93).
droplet size is controllable by varying the agitation speed. The experimental result is presented in Figure 4, showing that the size of oil droplets decreases as the agitation speed increases.
Figure 4. Relationship between agitation speed and oil droplet size in oiling out crystallization.
Oiling out crystallization was carried out at 20 °C using 50 wt % acetone as solvent, with agitation at 75 and 200 rpm. Crystals obtained at 75 rpm were clearly smaller than those obtained at 200 rpm, as shown in Figure 5. The effect of agitation on the size of the recovered crystals is presented in Figure 6, and it can be seen that the size of crystals increased with an increase in agitation speed. The result suggested that the crystal size depends on the oil droplet size, and the effect of oil droplet size, measured at the beginning of crystallization, on the resulting crystal size is presented in Figure 7. It can be seen that the 6008
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Figure 5. Crystals obtained at (a) 75 rpm (coefficient of variation; 0.44) and (b) 200 rpm (coefficient of variation; 0.37) in oiling out crystallization.
Figure 6. Relationship between agitation speed and crystal size, for oiling out crystallization (circles) and single-phase crystallization (triangles). Figure 8. Crystals and oil droplets observed on a slide glass. (a) A crystal contacting an oil droplet and (b) its change at 5 min later, (c) a crystal growing inside an oil droplet, and (d) its change at 1 min later.
Figure 9. Oil droplets observed during oiling out crystallization; (a) a large oil droplet containing many fine crystals after 7 min of oiling out crystallization at 75 rpm and (b) after 15 min. Figure 7. Effect of the initial size of oil droplets on the size of crystals finally obtained.
further understand the crystallization behavior in the oil phase, an experiment was conducted, in which a single-phase crystallization was carried out with a large volume of API-T solution having the same composition as that of the oil phase, as shown in Table 1 (12.3 g of API-T, 12.7 g of acetone and 5.2 g of water). The crystallization was started by adding water to the API-T/acetone solution at 20 °C under agitation at 200 rpm. The size of crystals obtained from the API-T solution was small, as shown in Figure 11, and similar to the size of crystals shown in Figure 5a. The result suggested that the small crystals shown in Figure 5a must be generated through a secondary nucleation in large droplets, and the same situation can also be
droplet volume is reduced. In other words, primary nucleation easily occurs in large droplets that have a large nucleation volume. Kadam et al.17 showed that the metastable zone width in the cooling crystallization of paracetamol depends on the size of the crystallizer for small crystallizers (e.g., 1 cm3), with the MSZW being varied for each experiment. The result also indicated that the nucleation frequency is influenced by the volume. Once the primary nucleation occurs in a droplet, the secondary nucleation can begin in the same droplet. In order to 6009
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Figure 10. Percentage of the oil droplets containing at least one crystal against (a) oil droplet size for different elapsed times, and (b) elapsed time for different ranges of oil droplet size.
Figure 8c,d. The released crystals grow by absorbing small droplets that remain without any nucleation, as shown in Figure 8a,b. The number of released crystals is small because it is hard to nucleate inside small oil droplets. As a result, crystals generated in small droplets will grow to a large size. This is the reason why large crystals are produced when the droplet size is small. 3.6. Control of Crystal Size in Oiling Out Crystallization. Generally, oiling out crystallization has been recognized as an undesirable operation in industry, because it is hard to control the nucleation and a buildup of scale may occur in the crystallizer. However, it was found in the present study that oiling out crystallization may spread the range of crystal-size control (see Figure 6), even if control is difficult in single-phase crystallization. Figure 12 shows three different crystal size distributions attained by changing the droplet size. When large crystals are desired, we should control the droplet size to be small.
Figure 11. Crystals obtained in a single-phase crystallization at 20 °C and 200 rpm from an API-T solution having the same composition as that of the oil phase shown in Table 1.
seen in Figure 9a. There still remains a question about the possibility of secondary nucleation inside oil droplets, because the inside of oil droplets is not vigorously agitated. However, it is well-known that there is inner flow within droplets in liquid− liquid two-phase flow systems,18,19 so it is reasonable to surmise that the secondary nucleation inside droplets is induced by such inner flow. Furthermore, there have been some investigations concerning secondary nucleation in stagnant solutions,16,20,21 in which Ooshima et al.21 proposed a new mechanism for secondary nucleation involving the directional diffusion of substances caused by growth of existing crystals. According to their mechanism, the secondary nucleation in oil droplets could be induced by directional diffusion of the API-T molecules to a crystal generated by primary nucleation, even if there is no convection flow inside the oil droplets. From these results and discussion, we can conclude that small crystals are generated in large oil droplets through secondary nucleation, and it can be noted that crystals generated in the continuous phase are similarly small, as shown in Figure 6. On the other hand, the crystals shown in Figure 5b were large. The large crystals are generated when oil droplets are small, and their mechanism of generation can be explained as follows. For small oil droplets, crystals generated by primary nucleation inside can be released to continuous phase without inducing significant secondary nucleation, because the nucleation volume is insufficient. Such a situation is shown in
4. CONCLUSIONS Oiling out crystallization of API-T was carried out in an acetone/water solvent system, where the solubility and cloud point curves were measured as a function of temperature and solvent composition. More than 60% of the API-T was distributed to the oil phase, although the mass was only
Figure 12. Comparison of the crystal size distribution. 6010
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about one tenth of the total liquid mass. The size of product crystals was inversely proportional to the oil droplet size. Thus, the larger the oil droplet size (or the lower the agitation speed), the smaller was the crystal size obtained. The generation of small crystals could be explained by the fact that the primary and secondary nucleation frequency increased in the droplet size. The generation of large crystals could be explained by the growth of a small amount of crystals generated in small droplets through the consumption of API-T in the remaining crystalvacant droplets. The results showed that the crystal size could be controlled by changing the agitation speed in the oiling out crystallization.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +81-3-6300-6437. Fax: +81-3-6300-6154. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The author would like to acknowledge managers in Takeda Pharmaceutical Co., Ltd. for advice and support during preparation of this paper.
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REFERENCES
(1) Gliko, O.; Neumaier, N.; Pan, W.; Haase, I.; Fischer, M.; Bacher, A.; Weinkauf, S.; Vekilov, P. G. J. Am. Chem. Soc. 2005, 127, 3433− 3438. (2) Lu, J.; Carpenter, K.; Li, R.; Wang, X.; Ching, C. Biophys. Chem. 2004, 109 (1), 105−112. (3) Berland, C. R.; Thurston, G. M.; Kondo, M.; Broide, M. L.; Pande, J.; Ogun, O.; Benedek, G. B. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 1214−1218. (4) Thomson, J. A.; Schurtenberger, P.; Thurston, G. M.; Benedek, G. B. Proc. Natl. Acad. Sci. U. S. A. 1987, 84, 7079−7083. (5) Lai, S. M.; Yuen, M. Y.; Siu, L. K. S.; Ng, K. M.; Wibowo, C. AIChE J. 2007, 53 (6), 1608−1619. (6) Lafferrere, L.; Hoff, C.; Veesler, S. J. Cryst. Growth 2004, 269, 550−557. (7) Lafferrere, L.; Hoff, C.; Veesler, S. Eng. Life Sci. 2003, 3, 127− 131. (8) Groen, H.; Roberts, K. J. J. Phys. Chem. B 2001, 105, 10723− 10730. (9) Lu, J. Sep. Purif. Technol. 2012, 96, 1−6. (10) Duffy, D. Chem. Eng. Sci. 2012, 77, 112−121. (11) Bonnett, P. E.; Carpenter, K. J.; Dawson, S.; Davey, R. J. Chem. Commun. 2003, 698−699. (12) Deneau, E.; Steele, G. Org. Process Res. Dev. 2005, 9, 943−950. (13) Veesler, S.; Lafferrere, L.; Garcia, E.; Hoff, C. Org. Process Res. Dev. 2003, 7, 983−989. (14) Derdour, L. Chem. Eng. Res. Des. 2010, 88, 1174−1181. (15) Veesler, S.; Revalor, E.; Bottini, O.; Hoff, C. Org. Process Res. Dev. 2006, 10, 841−845. (16) Gong, T.; Shen, J.; Hu, Z.; Marquez, M.; Cheng, Z. Langmuir 2007, 23, 2919−2923. (17) Kadam, S. S.; Kramer, H. J. M.; ter Horst, J. H. Cryst. Growth 2011, 11, 1271−1277. (18) Yamamoto, Y.; Yamauchi, M.; Uemura, T. J. Comput. Sci. Technol. 2008, 2, 246−257. (19) Yamauchi, M.; Uemura, T.; Ozawa, M. Jpn. J. Multiphase Flow 2000, 14, 466−472. (20) Denk, E. D., Jr.; Botsaris, G. D. J. Cryst. Growth 1972, 13−14, 493−499. (21) Ooshima, H.; Igarashi, K.; Iwasa, H.; Yamamoto, R. J. Cryst. Growth 2013, 373, 2−6. 6011
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