Experimental Study of the Effect of Process Parameters in the

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Experimental Study of the Effect of Process Parameters in the Recrystallization of an Organic Compound Using Compressed Carbon Dioxide as Antisolvent Martin Mu 1 ller and Ulrich Meier Novartis Pharma AG, CH-4002 Basel, Switzerland

Alwin Kessler† and Marco Mazzotti* ETH Zu¨ rich, Institut fu¨ r Verfahrenstechnik, Sonneggstrasse 3, CH-8092 Zu¨ rich, Switzerland

A difficult-to-comminute organic pharmaceutical substance was precipitated successfully through a carbon dioxide gas antisolvent (GAS) recrystallization process. Several experimental runs were performed, changing key process parameters such as the rate of addition of carbon dioxide and temperature, which was varied between 5 and 50 °C. Mean particle size of the precipitated product could be reproducibly adjusted between 0.2 and 10 µm. Particle size distribution was unimodal and rather narrow for very fast or very slow antisolvent addition rates but was bimodal for intermediate rates. The product was obtained in amorphous, partially agglomerated spheres if precipitated from ethanol, whereas pure crystals were formed from acetone or acetonitrile under otherwise identical operating conditions. These experimental results, particularly the key role of the carbon dioxide addition rate, are discussed and explained in the light of conventional crystallization theory as well as of the theoretical understanding of the GAS recrystallization process. In fact, it is shown how three kinetic phenomena, namely nucleation, particle growth, and supersaturation buildup, compete. The rate of the last one is determined by the carbon dioxide addition rate, which can thus be exploited to tune the average particle size and the particle size distribution of the final product. 1. Introduction and Background Industry rather often faces the need for the manufacturing of micrometer or submicrometer size solid particles in the production of different compounds, such as dyes, polymers, explosives, salts, and pharmaceuticals, including proteins. In the last case microparticles are intended for controlled drug release applications, for drug delivery by inhalation and for enhancing biovailability of poorly soluble compounds. In some cases these are drug-loaded polymer microparticles (with an average size below 50 µm) which undergo degradation or erosion by body fluids over time, thus minimizing drug concentration variations in the blood. In other cases they are just drug microparticles, possibly proteins, with an average particle size of less than 5 µm.1 The conventional particle reduction techniques include mechanical comminution (through milling, crushing, and grinding), lyophilization, and recrystallization of the solute particles from solution (through solventantisolvent techniques, spray drying, and freeze-drying). All these techniques suffer from one or more disadvantages, such as thermal and/or chemical degradation, high solvent requirements or difficult removal of solvent traces from the final product. Therefore, there is increasing interest to develope technologies which, particularly in the case of pharmaceuticals, allow one to produce microparticles with controlled particle size * To whom correspondence should be addressed. Telephone: ++41-1-6322456. Fax: ++41-1-6321141. E-mail: [email protected]. † Present address: Sulzer Chemtech, P.O. Box 65, CH-8404 Winterthur, Switzerland.

distribution and product quality (crystallinity, purity, morphology) under mild and inert conditions. Supercritical fluid technology, particularly when using carbon dioxide, offers different possibilities to tackle the above-mentioned challenge. These include the processes called RESS (rapid expansion of supercritical solutions), PCA (precipitation with compressed antisolvent, sometimes referred to as SAS, i.e., supercritical antisolvent process, or ASES, i.e., aerosol spray extraction system) and GAS (gas antisolvent) recrystallization.2-4 Several applications of PCA and GAS recrystallization to the formation of microparticles of various materials, such as polymers, pharmaceutical and organic compounds, protein powders, superconductor and catalyst precursors, and explosives have been reported and recently reviewed.2-4 Most of these contributions are of the proof of concept type, and only a very few theoretical studies have been reported, mainly focusing on the thermodynamics of the systems involved.5-7 Contrary to RESS, PCA and GAS recrystallization exploit the low solubility of pharmaceutical compounds in supercritical solvents. The latter, particularly carbon dioxide, are used as antisolvents for the solute, which is initially solubilized in a conventional solvent (completely miscible with carbon dioxide). When the solution is mixed with carbon dioxide, a reduction of the solvent power of the solution occurs and the solute precipitates. This can be accomplished in two different ways. In the PCA process the initial solution is sprayed through a nozzle into compressed carbon dioxide. In the GAS process, which constitutes the focus of this work, the initial solution is kept in a vessel and expanded more

10.1021/ie990828y CCC: $19.00 © 2000 American Chemical Society Published on Web 06/13/2000

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Figure 1. Saturation line and critical supersaturation line as a function of volumetric expansion by carbon dioxide of the organic solvent/solute solution (isothermal conditions). The qualitative evolution of the system during three GAS experiments performed at different carbon dioxide addition rates, i.e., different volumetric expansion rates, is represented by curves A (fast expansion), B (intermediate expansion), and C (slow expansion). In all cases the starting point is R within the stable solution region, then the system reaches point β at the boundary between the metastable and the nucleation region, and finally it ends up in point ω, which is the same for all experiments under the assumption that the initial amount of solution is the same (in fact maximum expansion is determined by the vessel volume and the initial amount of solution).

or less gradually by adding compressed carbon dioxide (subcritical or supercritical).8 The reduction of solubility due to volumetric expansion of the solution is qualitatively illustrated in Figure 1 where the saturation concentration (in terms of mass of solute per unit mass of organic solvent) is plotted as a function of volumetric expansion. Accordingly, the critical supersaturation decreases when the solution expands, as is also shown in the figure. It is worth recalling that the saturation and critical supersaturation lines divide the plane in Figure 1 in three regions. Below the saturation line the solution is stable, and solid particles, if added to the solution, will dissolve. Between the two lines, there is the metastable region, where existing solid particles grow; however, in the case of a perfectly homogeneous solution, with no particles, primary nucleation of new particles is so slow to be negligible in practice. Above the critical supersaturation line, the rate of nucleation of new particles becomes extremely high, and the phenomenon called catastrophic nucleation occurs. It is worth noting that Figure 1 is drawn at a given temperature; in most cases both lines would be shifted upward if temperature were increased. The intersection of the saturation line with the vertical axis gives the solubility of the solute in the organic solvent at the given temperature. Figure 1 provides a suitable framework to describe GAS recrystallization at least qualitatively and to discuss its features. The following discussion follows a similar one proposed earlier by Gallagher et al.9 In the GAS process, the vessel is first loaded with a certain amount of the initial solution at a solute concentration below saturation (this state is represented by point R in Figure 1). Then, carbon dioxide is added at a constant flow rate, so as to expand the solution. In the diagram of Figure 1, this stage of the process is represented by a horizontal line, since the carbon dioxide free solute concentration does not change. Assuming no solid particles are initially present in the solution, nucleation may occur only beyond the critical supersaturation limit,

i.e., on the right-hand side of point β. Beyond this point, two phenomena compete: on one hand carbon dioxide addition induces supersaturation, thus driving the point representing the state of the system toward the right; on the other hand formation of nuclei of the solid-phase reduces supersaturation by consuming the available solute and reducing its concentration in the solution, i.e., driving the representative point in Figure 1 downward. The former phenomenon is controlled by the carbon dioxide addition rate, whereas the kinetics of the latter depends on different parameters, such as temperature and physical properties of the system. Two limit situations may be envisaged. In the first situation (represented by curve A in Figure 1), the carbon dioxide addition rate is very large and the system is driven toward larger values of supersaturation where the nucleation rate becomes very large and catastrophic nucleation occurs. This involves the formation of a large number of fast growing nuclei, which consume the solute almost completely and reduce the supersaturation below the critical supersaturation limit, where solid particles only can grow but no more nuclei are generated. In the second limit case, corresponding to curve C in Figure 1, the addition of carbon dioxide is so slow that as soon as supersaturation is created, i.e., just beyond point β, nucleation occurs, thus producing a smaller number of nuclei than in the previous case; however, these grow before further nucleation occurs. Therefore, the system is soon driven below the critical supersaturation line. It is worth noting that, in the metastable region between the two lines, there is competition between particle growth and carbon dioxide addition. When the latter phenomenon is slow, the few nuclei created previously grow fast enough to keep the system below the critical supersaturation limit. Summarizing, in the first case, i.e., fast carbon dioxide addition, the formation of a large number of small particles with a narrow particle size distribution can be expected. In the second case, i.e., slow addition, a smaller number of larger particles are produced, though again with a narrow particle size distribution. Between these two limit behaviors, intermediate situations occur. Among these, one is illustrated in Figure 1 (see curve B). In this case, the competition between supersaturation buildup through carbon dioxide addition and supersaturation depletion due to crystal growth is such that the system crosses the critical supersaturation line twice during the whole process. In this case, a bimodal particle size distribution is expected, the larger and smaller particles being formed during the first and the second bursts of nucleation, respectively. It can be concluded that in general these intermediate situations associated with intermediate values of the carbon dioxide addition rate yield a product with a multimodal particle size distribution.9 This analysis leads to the conclusion that in the GAS recrystallization process the rate of addition of carbon dioxide is expected to be a key parameter for controlling the particle size distribution of the product. So far only a small number of reports has addressed this issue.10,11 In the last contribution in particular, the carbon dioxide induced GAS recrystallizations of L-asparagine and ascorbic acid from ethanol were studied. In agreement with the above-presented theory, a smaller carbon dioxide additon rate and a larger carbon dioxide addition rate yield larger and smaller average particle sizes, respectively.11

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Figure 2. GAS recrystallization experimental setup.

It is the objective of this work to verify this theoretical conjecture through an extensive experimental investigation. To this aim, the precipitation of an organic pharmaceutical substance from an organic solution using carbon dioxide as antisolvent is considered as a model system. (The name and chemical formula of the organic pharmaceutical are protected by a nondisclosure agreement.) The effect of different operating parameters, particularly the carbon dioxide addition rate and the operating temperature, on the particle size distribution of the final product is analyzed. Moreover, the role of the organic solvent in determining the degree of crystallinity of the microparticles is shown. The results obtained in the experimental setup described in section 2 are presented in the third section of the paper and discussed in the light of the present understanding of the GAS process, as summarized in this introduction. 2. Experimental Section

Figure 3. Solubility of the organic solid compound in ethanol as a function of temperature.

2.1. Experimental Setup. A scheme of the experimental equipment is drawn in Figure 2; it consisted of a 300 mL pressure vessel (B1) equipped with magnetic stirrer (S), safety valve (SV), and rupture disk (RD). Crystallizer temperature was controlled by means of a heating jacket fed by circulating water from a thermostat (H1, TIC2). Carbon dioxide was drawn from a dip tube cylinder (CO2), subcooled by a cryostat (C1, TIC1) and pumped to the pressure vessel by either a Gilson HPLC pump (P2, FIC1, PI3) for low flow rates or a Haskel pneumatic piston pump (P1) for high flow rates. Before entering the crystallizer, the liquid carbon dioxide was preheated in a coiled piece of tubing (PH) immersed in the water bath. A micrometering valve (MV) served to maintain the pumping pressure in order to avoid cavitation. The carbon dioxide reached the liquid phase inside the crystallizer via a cylindrical stainless steel frit immersed in the solution. A similar frit was connected to the outlet tube in order to avoid entrainment of particles. Fluid leaving the vessel was heated to approximately 80 °C in an oil bath (H2, TIC3) in order to avoid blockage of the pressure release control valve (CV, PIC1) by dry ice, solvent, and/or solute. A Videal video system (CCD) with high pressure optical system and stroboscopic lighting made it possible to directly observe solid precipitation in the crystallizer.

Temperature and pressure inside the vessel were measured with a thermocouple (TI4) and an electronic pressure transducer (PI2) and registered manually. 2.2. Materials and Analytical Methods. Carbon dioxide was purchased from Carba (99.9%) and used without further purification. Ethanol, acetonitrile, and acetone were purchased from Fluka (analytical grade) and used without further purification. The solid organic solute was prepared through crystallization and jet-milling. Its molecular mass and melting point are approximately 600 g/mol and 200 °C, respectively. The solubility of the substance in ethanol as a function of temperature is illustrated in Figure 3. At 25 °C its solubility in both acetone and acetonitrile is 3 g/L, i.e., about six times smaller than in ethanol. The solute exhibits very poor water solubility and is virtually insoluble in supercritical carbon dioxide. The volumetric expansion of the three solvents involved in this study when contacted with carbon dioxide at about room temperature has already been studied in the literature. In particular, the following data are available: acetone at 27 °C;9 acetonitrile at 25 °C;12 ethanol at 25 °C.12 It can be readily observed that both subcritical and supercritical carbon dioxide can produce a rather large volumetric expansion of all three solvents.

Ind. Eng. Chem. Res., Vol. 39, No. 7, 2000 2263 Table 1. Operating Conditions of the GAS Experiments and Properties of the Microparticles Produceda

run

solvent

temp (°C)

E05A E05B E25A E25B E25C E25D E25E E25F E25G E50A E50B A25A N25A

EtOH EtOH EtOH EtOH EtOH EtOH EtOH EtOH EtOH EtOH EtOH acetone AN

5 5 25 25 25 25 25 25 25 50 50 25 25

specific CO2 addition rate (min-1)

initial solution vol (mL)

run time (min)

av particle size (µm)

0.04 4.4 0.04 0.12 0.1 0.2 0.5 2.9 4.4 0.04 6.6 0.03 0.03

50 50 50 50 20 50 50 50 50 50 50 70 70

142 1 130 55 155 28 13 1.5 1 120 0.7 140 130

3.2 0.24 8.1 1/5 2/6 0.5/5 0.3/5 0.65 0.36 10 3.2 n.a. n.a.

agglomeration extent + + ++ + ++ + + ++ + +++ +++ n.a. n.a.

morphology A A A A A A A A A A A C C

figure no. 9 10 4 5 6 8 11

a

Within the run’s label the first letter and the digits indicate the solvent (E for ethanol, A for acetone, and N for acetonitrile) and the operating temperature in °C, respectively. The specific carbon dioxide addition rate is defined as the carbon dioxide feed flow rate divided by the initial solution volume. The run time represents the duration of the experiment, which is measured at the time when the precipitation vessel is full of liquid. Estimation of particle size and agglomeration extent are obtained by microscopy quantitative analysis of the SEM photomicrographs (see the last column of the Table); about one hundred particles for each sample are used to obtain the corresponding statistics. In the case of particle size when two numbers are reported they indicate the average size of the small and large particles, respectively; the agglomeration extent is qualitatively indicated as small (+), intermediate (++) and large (+++). The results of X-ray powder diffraction analysis are simply reported as A (amorphous microparticles) and C (crystalline microparticles). In the case of precipitation from acetone and acetonitrile, polydisperse crystals have been obtained; hence, no indication about particle size has been given (n.a., i.e., not applicable, in the table).

The specifications of the final product, i.e., the particle population, were determined by standard off-line analytical tools: light and scanning electron microscopy (SEM) for shape and size; X-ray powder diffraction analysis for crystallinity. The sample for SEM analysis was prepared by gold sputtering. Estimation of particle size and agglomeration extent was obtained by microscopic quantitative analysis of the SEM photomicrographs; about 100 particles for each sample were used to obtain the corresponding statistics. 2.3. Experimental Procedure. A batch of solute in the organic solvent was prepared (between 20 and 70 mL) at a concentration equal to 80% of the solubility at the process temperature (see Figure 3) and filled into the crystallization vessel. As soon as the system temperature was stable, feeding of carbon dioxide was started with the outlet valve (OV) closed, the liquidphase being mixed with the magnetic stirrer. A constant feed flow rate (between 10 and 220 mL/min) was maintained until the vessel was filled completely with the expanded liquid phase containing the precipitated solid. The final liquid expansion ratio ∆V/V0 reached values between 120 and 1200%, depending on the initial volume. After full expansion the feed was shut off and stirring continued for half an hour. Then the outlet shutoff valve was opened and feeding of carbon dioxide resumed at a flow rate of 3 mL/min. Rinsing was continued overnight with pressure kept constant by means of the outlet control valve (CV). Finally, the carbon dioxide supply was stopped and the entire fluid content of the vessel released until reaching atmospheric pressure. The crystallizer was then opened and the dry solid content collected and analyzed. 3. Experimental Results The operating conditions of the GAS recrystallization experimental runs and the properties of the microparticles formed are reported in Table 1. Each run is characterized by a label, which carries information about the organic solvent (first letter, where E indicates ethanol, A acetone, and N acetonitrile) and the operating temperature (the two digits). It is worth noting that temperature was monitored during the experiments.

Only slight deviations with respect to the nominal, average temperature were observed, which depended on the feed flow rate, as expected. At 25 °C these were (0.6, (1.5, and (3.0 °C at the small, intermediate, and large feed flow rates, respectively. The fourth and fifth columns report the specific carbon dioxide addition rate, which is given by the carbon dioxide addition rate divided by the initial solution volume, and the initial volume of the solution itself. The run time represents the duration of the experiment, which is measured at the time when the precipitation vessel is full of liquid. Estimation of particle size and agglomeration extent are obtained by microscopy quantitative analysis of the SEM photomicrographs; about one hundred particles for each sample are used to obtain the corresponding statistics. When bimodal particle distributions are observed, these are characterized by the values of the average particle size of the two sets of microparticles in which the whole distribution can be partitioned (see runs E25B, E25C, E25D, and E25E). The extent of agglomeration is qualitatively evaluated to be small (+), intermediate (++), or large (+++). The residual organic solvent content in the produced particles has been measured and found to be always below 0.01 wt %. The experimental investigation has focused on ethanol as organic solvent, because this offers the largest solubility values and is a rather good choice from the viewpoint of the other steps of the process. For the sake of comparison, a couple of runs with acetone and acetonitrile have been performed and the results are also reported in Table 1. The parametric analysis has studied the effects of carbon dioxide addition rate, i.e., volume expansion rate, and temperature. The reproducibility of the experimental results has been duly checked. 3.1. Effect of Carbon Dioxide Addition Rate. First, let us focus on the experiments at 25 °C in ethanol, i.e., runs E25A to E25G in Table 1, which have been performed at increasing values of the specific carbon dioxide addition rate. Microparticles formed during run E25A at a low addition rate, i.e., 0.04 min-1, are shown in Figure 4; primary particles have a rather

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Figure 4. SEM photomicrograph of the microspheres precipitated from ethanol in experiment E25A: temperature 25 °C; specific carbon dioxide addition rate ) 0.04 min-1; average particle size ) 8.1 µm; maximum particle size ) 14 µm; standard deviation ) 3.4 µm.

Figure 5. SEM photomicrograph of the microspheres precipitated from ethanol in experiment E25D: temperature 25 °C; specific carbon dioxide addition rate ) 0.20 min-1; average particle size ) 0.79 µm; maximum particle size ) 5.9 µm; standard deviation ) 0.9 µm.

regular spherical shape with an average diameter of 8.1 µm. The particle population is unimodal with standard deviation of 3.4 µm. A fairly large degree of agglomeration is observed, with many bridges between pairs of primary particles. The spherical shape indicates that the structure of the microparticles is amorphous as it was confirmed by X-ray powder diffraction. It is worth noting that all particles produced by precipitation from ethanol are amorphous, as reported in Table 1. When the specific carbon dioxide addition rate is increased to 0.12 min-1 in run E25B, a particle distribution with a rather clear bimodal character is obtained, with average diameters of about 1 and 5 µm. A bimodal distribution with fairly the same average particle size

(2 and 6 µm) has been observed by repeating the experiment with a different initial volume of solution (run E25C), to check the reproducibility of the phenomenon. Bimodal microparticle populations have been obtained by further increasing the specific carbon dioxide addition rate up to 0.2 and 0.5 min-1 in runs E25D and E25E, respectively. The particles formed during the former experiment are shown in Figure 5, where the bimodal character of the distribution is apparent. It can also be observed that the small particles have a submicrometer size and that the degree of agglomeration is moderate. By a further increase in the specific carbon dioxide addition rate up to 2.9 and 4.4 min-1 in runs E25F and

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Figure 6. SEM photomicrograph of the microspheres precipitated from ethanol in experiment E25G: temperature 25 °C; specific carbon dioxide addition rate ) 4.4 min-1; average particle size ) 0.36 µm; maximum particle size ) 0.61 µm; standard deviation ) 0.11 µm.

Figure 7. GAS recrystallization from ethanol. Effect of specific carbon dioxide addition rate on average particle size at different operating temperatures: (∇) 5 °C; (b) 25 °C; (0) 50 °C. Operating conditions as in Table 1. Bimodal distributions are represented by two circles, corresponding to the average values reported in Table 1, linked by a vertical line. For the sake of clarity dashed lines connect the experiments at minimum and maximum expansion rate at each temperature; these lines have no other physical meaning.

E25G, respectively, a unimodal particle distribution is recovered, but with a much smaller average size as compared to run E25A. Figure 6 shows the submicrometer particles precipitated during run E25G. It is worth noting the regular spherical shape of the microparticles, the rather narrow size distribution (average particle size is 0.36 µm, with maximum particle size of 0.61 µm and standard deviation of 0.11 µm), and the moderate agglomeration extent. However, by inspection of Table 1, it appears that no regular trend of the extent of agglomeration with expansion rate exists, at least at 25 °C. These results are illustrated in graphical form in Figure 7, where the average particle size is reported as a function of the specific carbon dioxide addition rate. Bimodal distributions are represented by two circles, corresponding to the average values reported in Table 1, linked by a vertical line. The effect of expansion

rate on particle size distribution discussed above is readily observed. It is remarkable that this agrees well with the theoretical discussion reported in the Introduction, where the possibility of obtaining small and large particles at large and small expansion rates, respectively, was predicted, as well as the existence of an intermediate range of expansion rates yielding a polydisperse product. With reference to Figure 1, runs E25A follows the small carbon dioxide addition rate path C; runs E25B to E25E follow the intermediate addition rate path B, whereas runs E25F and E25G follow the fast addition path A. The same effect of specific carbon dioxide addition rate on the average particle size has been observed at all the investigated operating temperatures. As reported in Table 1, runs E05A and E05B at 5 °C exhibit average particle diameters of 3.2 (with maximum particle size of 4.6 µm and standard deviation of 0.57 µm) and 0.24 µm (with maximum particle size of 0.35 µm and standard deviation of 0.064 µm) at addition rates of 0.04 and 4.4 min-1, respectively. Accordingly, runs E50A and E50B at 50 °C exhibit average particle diameters of 10 and 3.2 µm at expansion rates of 0.04 and 6.6 min-1, respectively. It is worth noting that the particle size distributions obtained at 5 °C are remarkably narrow. With reference to Figure 7, it is worth noting that the effect of the expansion rate on particle size seems to be not as large at 50 °C as at 5 °C. However, the data available are not sufficient to draw conclusions about this issue, and further investigation is required. 3.2. Effect of Temperature. The experiments at different temperatures reported in Table 1 and illustrated in Figure 7 indicate that the larger the temperature the larger the particle size. In fact at a specific carbon dioxide addition rate of 0.04 min-1 microparticles of 3.2, 8.1, and 10 µm average diameter are formed at 5, 25, and 50 °C, respectively (runs E05A, E25A, and E50A). This temperature effect is even larger at large carbon dioxide addition rates, where 0.24 and 0.36 µm average diameters are obtained at 5 and 25 °C (runs E05B and E25G), respectively, but microparticles with an average particle size of only 3.2 µm are formed in

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Figure 8. SEM photomicrograph of the microspheres precipitated from ethanol in experiment E50B: temperature 50 °C; specific carbon dioxide addition rate ) 6.6 min-1; average particle size ) 3.2 µm; maximum particle size ) 6.7 µm; standard deviation ) 1.7 µm.

Figure 9. SEM photomicrograph of the microspheres precipitated from ethanol in experiment E05A: temperature 5 °C; specific carbon dioxide addition rate ) 0.04 min-1; average particle size ) 3.2 µm; maximum particle size ) 4.6 µm; standard deviation ) 0.57 µm.

run E50B at 50 °C. When analyzing these results, it must be kept in mind that changing temperature modifies the thermodynamic features of the system; this implies that operations at the same expansion rate but different temperatures cannot be directly and quantitatively compared, since the shape and position of the saturation and critical supersaturation lines drawn in Figure 1 change with changing temperature (cf. also ref 9). Nevertheless, the experimental results described here show a clear effect of temperature on particle size that deserves further investigation in order to be explained and quantitatively assessed. It is also worth noting that the observed effect is consistent with other investigations carried out in the case of the PCA process. Nanoparticles of yttrium acetate, i.e., a superconductor

precursor, were precipitated from DMSO using supercritical carbon dioxide; increasing temperature from 40 to 60 °C yielded a 50% increase of the average particle size.13 Similarly, methylprednisolone acetate microparticles exhibit a 25% size increase when they are precipitated from THF using carbon dioxide and temperature is increased from -6 to +63 °C.14 As indicated in Table 1, temperature has also a rather important effect on the agglomeration extent. Figure 8 shows the microparticles formed during run E50B at 50 °C and with a fast expansion rate. It can be observed that primary particles are spherical, but they are almost indistinguishable since most of them look as if they were melted together. This effect has been observed also in the SEM photomicrographs of the product obtained in

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Figure 10. SEM photomicrograph of the microspheres precipitated from ethanol in experiment E05B: temperature 5 °C; specific carbon dioxide addition rate ) 4.4 min-1; average particle size ) 0.24 µm; maximum particle size ) 0.35 µm; standard deviation ) 0.064 µm.

Figure 11. SEM photomicrograph of the crystals precipitated from acetonitrile in experiment N25A: temperature 25 °C; specific carbon dioxide addition rate ) 0.03 min-1; average particle size ) 44 µm; maximum particle size ) 102 µm; standard deviation ) 23 µm.

run E50A. This is likely due to the effect of temperature on all the elementary phenomena responsible for the ultimate extent of agglomeration achieved. The extent of agglomeration in experimental runs at 5 °C is smaller than in those carried out at both 50 and 25 °C. This can be clearly observed in Figures 9 and 10, where the particles formed at 5 °C in runs E05A and E05B, respectively, are shown. In both cases, the microparticles are spherical with unimodal and narrow size distribution and the extent of agglomeration is from moderate to small. Run E05B represents the best result of the whole series of experiments in terms of agglomeration extent. 3.3. Role of Solvent. A few experiments using acetone and acetonitrile rather than ethanol as organic solvent have been performed in order to check the role of solvent on particle size distribution and product morphology. These have shown a dramatic effect, since

GAS recrystallization from both acetone and acetonitrile reproducibly yields a crystalline product, contrary to what happens in the case of ethanol where amorphous microparticles have been obtained in all experiments. The rather nice elongated crystals formed from acetonitrile are shown in Figure 11; these exhibit a fairly broad crystal size distribution with several interconnected elements. A quantitative confirmation that pure crystals are obtained also from acetone was provided by X-ray powder diffraction. It is known that inclusion of solvent molecules in the solid lattice and/or interaction of the solvent with the growing crystal surface may drastically change crystal morphology.15 Therefore, the results obtained in these last experiments are not surprising even if it is rather difficult to explain and predict them. As an example, Meenan16 predicts aspirin crystal morphology by geometrical and lattice energy model simulations and

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relates these to experimental observations. The important influence of the solvent on product morphology in GAS recrystallization has also been observed and reported in the case of the pharmaceutical product Abecarnil17 and of the explosive compound RDX.9 4. Conclusion The micronization of a pharmaceutical organic compound by GAS recrystallization has been studied, using ethanol, acetone, and acetonitrile as conventional solvents and carbon dioxide as antisolvent. It has been proved that the particle size distribution of the final product can be controlled in a reproducible way through tuning of the specific carbon dioxide addition rate and the temperature. In particular the former parameter has an effect on particle size distribution which is consistent with the present theoretical understanding of the elementary phenomena involved in the GAS process. In fact, large and small addition rates yield a product with unimodal particle size distribution, due to a single crossing of the critical supersaturation line and a unique burst of nucleation during the process, while on the other hand intermediate addition rates yield a particle population which is bimodal or multimodal, due to the occurrence of more than one burst of nucleation during the GAS recrystallization. Moreover, as expected large and small addition rates yield small and large microparticles, respectively with up to 20-fold changes of average particle size. The same behavior is observed at the different temperatures investigated. Moreover, temperature has a rather clear effect on the average particle size of the microparticles and on the extent of their agglomeration. Low temperatures improve the quality of the product in both respects, i.e., by decreasing particle size and reducing agglomeration. These effects are clearly due to the role of temperature in all phenomena involved in the GAS process, from rate of nucleation and growth, to phase equilibria and mass transfer efficiency. However, a quantitative assessment of these requires further investigation and a substantial modeling effort. Ethanol has been used as primary solvent in this study, because of the rather large solubility of the organic compound in it. Recrystallization of the product from ethanol yields amorphous spherical particles. Some experiments with acetone and acetonitrile show the striking though not surprising influence of the type of solvent on the morphology of the precipitate; in fact, in the last two cases the product is fully crystalline. The obtained experimental results motivate further work and provide directions for future experimental and theoretical investigation about the potential of the GAS recrystallization technology. These will necessary deal with the following: (1) how general is the role of carbon dioxide addition rate and temperature in tuning product quality; (2) how well can agglomeration be controlled through operating temperature and stirring rate (which was deliberately kept constant in this study); (3) how effective can particles be harvested in order to achieve satisfactory yield and productivity. Acknowledgment The authors are indebted to Ricardo Schneeberger and Bruno Galli, Novartis Pharma AG, for their continuous, scientific support and to Klaus Schafflu¨tzel, Novartis Pharma AG, for help in running the experiments.

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Received for review November 15, 1999 Revised manuscript received March 30, 2000 Accepted April 18, 2000

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