Article pubs.acs.org/OPRD
Robust Crystallization Process Development for the Metastable δ‑form of Pyrazinamide Martin Wijaya Hermanto,*,† Alvin Yeoh,† Beatrice Soh,† Pui Shan Chow,† and Reginald B. H. Tan*,†,‡ †
Institute of Chemical and Engineering Sciences Limited, 1 Pesek Road, Jurong Island, Singapore 627833 Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore
‡
S Supporting Information *
ABSTRACT: The α-form of the first-line antituberculosis drug pyrazinamide (PZA), which is the thermodynamically stable polymorph at ambient conditions, exhibits a fine needle shape that easily forms a mesh-like structure, making it undesirable for downstream processing. This study implements the Quality by Design (QbD) approach to develop a robust design space for isolating the metastable δ-form of PZA. A solvent mixture of methanol and 1,4-dioxane is selected for the process. Five process parameters are considered in the development of the design space: cooling rate, seeding temperature, seed loading, solvent composition, and seed preparation technique. The robustness of the established design space is verified by model uncertainty evaluation and demonstrated through experiments.
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INTRODUCTION Crystallization is an important separation and purification process in pharmaceutical, food, and specialty chemical industries. The crystal product quality is typically characterized by its purity, shape, size distribution, and polymorphic form.1−4 Achieving robust product quality is critical, especially in the pharmaceutical industry, where a slight change in the product physicochemical property may affect the efficacy of the drug, which in turn may adversely affect patient safety. Controlling polymorphism has been of interest recently, and its significance was highlighted by a high profile case involving the unexpected appearance of a more thermodynamically stable form (Form II) of ritonavir5 (Norvir, Abbott Laboratories, protease inhibitor for the treatment of HIV), with different dissolution properties from the intended commercial Form I. As Form II is much less soluble than Form I, the observed dissolution rate is lower, hence adversely affecting its therapeutic properties and resulting in withdrawal of the drug from the market. This example serves to show that polymorphism has to be considered an important attribute for the application of new drug substances.6 The introduction of current good manufacturing practices (cGMP) for the 21st century by U.S. Food and Drug Administration (FDA)7 has generated interest in Quality by Design (QbD) among pharmaceutical industries, research institutes, and universities.8−20 Through this initiative, FDA encourages pharmaceutical companies to build quality, safety, and efficacy into new products based on sound science and risk management. The QbD approach, by which quality is built-in rather than tested into the product, is described in published guidelines (ICH Q8, ICH Q9, and ICH Q10).21−23 One important aspect of QbD is the development of a design space, which is defined in ICH Q8 as “the multidimensional combination and interaction of input variables (e.g., material attributes) and process parameters that have been demonstrated to provide assurance of quality”. Defining a design space © XXXX American Chemical Society
improves process understanding, reduces risk, and at the same time may alleviate the burden of regulatory refiling, as working within the design space is not considered a change.21 Various literatures have studied design space development for the crystallization of stable polymorphs.10,11,16,17 However, developing a design space to isolate the metastable polymorph can be important for some compounds, such as when the pure stable form cannot be obtained12 or when the stable form has an undesirable morphology.14 In the current study, QbD is implemented to develop a design space for polymorphic crystallization of pyrazinamide (PZA). PZA (Figure 1) is a frontline antituberculosis drug and
Figure 1. Chemical structure of pyrazinamide.
is on the World Health Organization (WHO) model list of essential medicines.24 It is usually used in combination with rifampicin and isoniazid for tuberculosis treatment. Previous studies indicate that PZA is known to exist in four packing polymorphic forms: α, β, γ, and δ.25,26 The stability order under ambient conditions is found to be α > δ > γ > β, where the stable α form is used commercially. However, published studies25,26 and our preliminary results have shown that the fine needle shape of the α-form easily forms a mesh-like-structure, making it undesirable for downstream processing, as it may cause inefficient filtration and drying, as well as poor flowability. To avoid such problems, suitable alternative polymorphs can be investigated. Under storage conditions of 15−40 °C and 30− Received: July 22, 2015
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DOI: 10.1021/acs.oprd.5b00234 Org. Process Res. Dev. XXXX, XXX, XXX−XXX
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Currently, commercially used PZA is the stable α-form. Cherukuvada et al.26 studied the stability of different polymorphs of PZA at 15−40 °C and 30−70%RH and found that the δ-form remains stable after one year of storage. The γform remained stable up to 6 months, but converted to the αform upon prolonged storage. The findings imply that the alternative polymorphic forms (δ and γ-forms) are rather stable, with the δ-form being more stable. According to Biopharmaceutics Classification System (BCS), PZA is classified as a borderline BCS Class 3/1 drug,28 which means the drug is highly soluble, but its absorption is limited. The dissolution profiles (which relate to the therapeutic efficacy of the drug) are also reported in the literature,26 with the intrinsic dissolution rates of α, δ, and γ being 2.07, 2.05, and 2.78 mg cm−2, respectively. These studies indicate that the δ and γ forms are both possible alternative to the commercially used α-form. Though all three polymorphic forms are suited to be used as drug compound from the stability and therapeutic efficacy point of view, processability is also an important factor to consider when selecting polymorphic form. Morphology is one of the important considerations of processability. As reported in the literature25,26 and verified by our preliminary study, the α-, δ-, and γ-forms of PZA have distinct morphologies (Figure 2a).
70%RH, Cherukuvada et al.26 has shown that δ- and γ-forms are stable up to 1 year and 6 months, respectively, rendering both polymorphic forms suitable alternatives. Hence, the current study aims to develop a design space to isolate one of the metastable forms of PZA with better morphology (plates for δ-form or prismatic for γ-form). We were not able to isolate the β-form due to its transient nature. In any case, the highly unstable nature of the β-form renders it unsuitable from a manufacturing and quality point of view. In the next section, the experimental setup for our current study is discussed, followed by the description of polymorphic composition quantification. Then, polymorph and solvent selections are elaborated, followed by the development and verification of a design space to produce the desired polymorph. Finally, the findings of this study are concluded.
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EXPERIMENTAL SETUP PZA (α-form) was purchased from Hangzhou Dayangchem and used as is throughout all experiments. Methanol (HPLC) was purchased from J. T. Baker, 1,4-dioxane (ACS, ISO) was purchased from Merck Millipore, and ethanol (HPLC) and nitromethane (GR) were purchased from Fisher Chemical. Experiments for polymorphic accessibility study, onset time of polymorphic transformation study, and design space development were performed in a 75 mL jacketed glass vessel, where a magnetic stirrer (IKAMAG RCT Basic) was utilized for mixing. For the design space verification experiments, a 1 L jacketed flat-bottomed glass crystallizer with an inner diameter of 100 mm was used. It has four baffles which help to enhance mixing properties. A stainless-steel marine-type impeller with a diameter of 42 mm driven by a variable speed overhead stirrer motor was utilized to agitate the system at 300 rpm. The temperature of each experimental setup was controlled by a water circulator (Julabo FP50) equipped with a Pt100 thermocouple. After each experiment, the product crystals were filtered and dried overnight in an oven (∼45 °C). The dried crystals were then analyzed using powder X-ray diffraction (powder-XRD) (scan region: 5−50°, step size: 0.00835°, 0.1 s/step, scan time: 10 min), and the polymorphic composition was calculated from the XRD pattern based on least-squares method,27 except that improvements were made to take into account variability in sample preparation and to emphasize important regions in the spectra. The improved least-squares method is described in the Supporting Information. Crystal size distribution (CSD) measurements for seeds were obtained using Malvern Mastersizer 2000 equipped with Hydro 2000S, and ethanol saturated with PZA was used as the dispersant. All microscope images were captured using Olympus microscope (BX51) equipped with Sony CCD camera (SSCDC58P). Solubility measurements were conducted using the Crystalline (Avantium) high-throughput crystallization platform. The Crystalline system comprises 8 parallel reactors (1−5 mL working volume), which can be independently programmed for different heating and cooling cycles.
Figure 2. Microscope images of (a) α, (b) δ, and (c) γ-PZA.
The commercially used α-form has a fine needle-like morphology (Figure 2a). During filtration, the crystals tend to form a mesh-like structure (Figure 3), which potentially
Figure 3. Filtered α-form of PZA obtained from cooling crystallization in water. The crystals form a mesh-like structure that is undesirable for downstream processing.
causes long filtration time and may lead to inefficiency in downstream processing due to poor flowability. As shown in Figure 2b and 2c, δ- and γ-forms have more desirable morphologies (plate and prismatic, respectively) than the αform and therefore are preferable for downstream processing. Solvent Selection. Solvent Range. In this study, a range of solvents from the ICH Class 2/3 table was selected.29 The solvents were then categorized by their polarity and Hansen Solubility Parameters (HSP). HSP assigns a value to a solvent’s dispersion, polar, and hydrogen bonding potential, which gives information about interactions between solvent and solute. HSP also allows us to make rational solvent substitutions and selection of solvents on the premise that solvents with similar
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POLYMORPH AND SOLVENT SELECTIONS Polymorph Consideration. The polymorph selection of any active pharmaceutical ingredient (API) is largely influenced by the stability and therapeutic efficacy of the crystal form. B
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Table 2. Solubility Values of α-PZA in Different Solvents
HSP values will behave in a similar manner. Hence, to represent different solvent behavior, five solvents with nonsimilar HSP values were selected as shown in Table 1.
water
Table 1. Hansen Solubility Parameters for Selected Solvents solvent
δD dispersion
δP polar
δH hydrogen bonding
water ethanol methanol nitromethane 1,4-dioxane
15.5 15.8 14.7 7.7 17.5
16 8.8 12.9 9.2 1.8
42.3 19.4 22.3 2.5 9
Solubilities in Candidate Solvents. Once the range of solvents was determined, the solubility of PZA in the solvents was measured using the Avantium Crystalline setup. A known excess of the α-form was added to 4 g of solvent and agitated with a 3-bladed impeller at 750 rpm. This slurry was heated at a rate of 0.5 °C/min. A turbidity sensor built into the Crystalline was then used to detect when the slurry was fully dissolved. The temperature at which the solution became clear was regarded as the dissolution temperature. The solubility measurement is an important step during solvent selection, because it determines the feasibility of the solvent as a crystallization medium, the modes of supersaturation generation, the theoretical yield, and the required solvent per product mass. The resulting solubility measurements for the α-form in selected solvents is shown in Figure 4, and their values are tabulated in Table 2. The curvatures of the solubility plots in Figure 4 justify the choice of cooling as a means to generate supersaturation. Based on the solubility values and the curvatures, the solvent selections were narrowed down into three candidates: methanol, water, and 1,4-dioxane. These solvents result in the highest solubilities and reasonably large curvatures, which imply the least required solvent per product mass and reasonable yields. The solubilities of the three polymorphic forms were also measured in water as shown in Figure 5 (refer to Supporting Information for the solubility measurement method). The solubility values for the δ- and γ-forms in water are tabulated in
methanol
T (°C)
solubility (g/kg)
T (°C)
11.7 17.2 24.1 27.6 32.3 37.4 49.7 70.9
8.1 10.9 15.1 16.7 20.9 25.3 44.4 108.7 ethanol
5.0 11.1 17.3 25.5 30.8 32.9 38.7 48.5
1,4-dioxane
solubility (g/kg)
T (°C)
8.5 17.2 10.9 21.0 12.8 25.6 18.6 33.4 21.9 36.8 23.4 48.0 30.1 41.7 nitromethane
T (°C)
solubility (g/kg)
T (°C)
solubility (g/kg)
42.2 45.0 48.8 52.9
14.7 17.7 19.4 26.8
30.4 44.7 50.0
9.1 16.3 20.0
solubility (g/kg) 9.2 10.8 13.4 15.1 18.6 23.6
Table 3. Results show that the three polymorphic forms have a monotropic relationship within the temperature range studied. Polymorph Accessibility in Candidate Solvents. The next step was to investigate the accessibility of polymorphs in the candidate solvents through homogeneous cooling crystallization. Solution saturated with α-form was cooled from 45 to 5 °C (for experiments conducted with water and methanol) or 45 to 15 °C (for experiments conducted with 1,4-dioxane) at three different cooling rates (0.1, 1.0, and 1.7 °C/min). The results of the experiments were as follows: • In water, only the stable α-form was accessible by cooling crystallization irrespective of the cooling rates. • In methanol, a cooling rate of 0.1 °C/min produced the stable α-form, while a cooling rate of 1.0 °C/min produced a mixture of α- and δ-forms. Only crystallization performed at a cooling rate of 1.7 °C/min produced the pure δ-form. Increasing the cooling rate further by quenching a saturated solution in a cold water bath filled with ice cubes produced a mixture of δ and γforms.
Figure 4. Solubility of α-PZA in different solvents. C
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Figure 5. Solubility of α, δ, and γ-PZA in water.
Table 3. Solubility Values of δ and γ-PZA in Water δ-form
temperature. If transformation happens very quickly (within seconds or minutes), it is likely that the seeding temperature needs to be lowered. Nevertheless, it is expected that the induction time when a magnetic stirrer is used is likely to be shorter due to its grinding effect than when overhead stirrer is used. Hence, the induction times measured are rather conservative. Meanwhile, it was not observed that the magnetic stirrer to be a major factor in causing polymorphic transformation in this study, or else pure δ-form could not be obtained with this experimental setup. In our study, experiments have shown that onset time decreases as temperature increases. One possible reason is that the onset time is largely influenced by the nucleation kinetic of the α-form and nucleation kinetic generally increases with temperature. However, further study needs to be carried out to confirm this postulation, which is beyond the scope of this study. Hence, the shortest (worst case) onset time will occur at the highest operating temperature, which is the seeding temperature during seeded crystallization (i.e., 40−45 °C for the current study). If the onset time is too short at the highest temperature, seeded crystallization will not result in products with high δ-form purity, as the seed crystals will progressively transform to the α-form during the process. If this is the case, lower seeding temperature may be required, allowing for a longer onset time. Experiments to study the onset time of polymorphic transformation were conducted in methanol and 1,4-dioxane at 45 °C. To observe the onset of transformation, solid samples were taken every 5 min and observed under a microscope to verify the polymorphic form. In 1,4-dioxane, the δ-form crystals did not transform within 2 h, and a good reproducibility was obtained from a few repeat experiments. On the other hand, the transformation of δ-form crystals in methanol showed less reproducibility, with the δ-form crystals for some experiments showing signs of transformation to the α-form within 2 h. The difference in transformation reproducibility in the two solvents could be due to the difference in volatility of solvents. Consequently, it was observed that experiments done in the more volatile methanol caused nucleation and growth on the wall above the solution phase (i.e., creeping up), which was not observed in experiments done in 1,4-dioxane. This could have been be a source of accidental seeding with the α-form. Further
γ-form
T (°C)
solubility (g/kg)
T (°C)
solubility (g/kg)
14.5 20.5 28.0 30.5 35.0 38.0 43.0
10.9 13.8 18.6 21.0 24.7 29.2 34.8
10.0 17.7 27.5 35.5 37.0 40.0 43.0
9.5 13.1 21.0 26.0 31.0 33.7 36.0
• In 1,4-dioxane, a cooling rate of 0.1 °C/min produced the stable α-form, while cooling at faster rates of 1.0 and 1.7 °C/min produced the δ-form. From the above observation, water may not be a suitable solvent to isolate the metastable forms, since it has a high preference for the stable form. Furthermore, it is noted that the δ-form is more accessible than the γ-form when crystallized from methanol and 1,4-dioxane. Hence, the δ form has a higher chance to be produced consistently and is selected as the desired polymorph. The solvent candidates were also narrowed down further to methanol and 1,4-dioxane. Another important observation was that the cooling rate required to produce the δ-form by homogeneous nucleation was rather high and is impractical for operation on a manufacturing scale. Furthermore, the stochastic nature of primary nucleation is not desirable for batch-to-batch consistency. One way to overcome this issue is to seed the crystallization process with the desired polymorph.30,31 Seeding potentially allows operation at a lower cooling rate and improves process robustness by suppressing nucleation. Since it is desirable to operate seeded crystallization at a lower cooling rate, it is important to study the onset time of polymorphic transformation from the δ- to α-form as this will give us an understanding on the feasibility of seeded crystallization. Note that, though induction times measured from experiments with a magnetic stirrer (as carried out in this study) may not be the same as those measured from experiments with an overhead stirrer (as in scaled-up experiments), this study still gives an indication whether or not seeded crystallization is feasible with the selected seeding D
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onset time studies were also conducted at 25 °C, and no noticeable transformation occurred within 6 h in both solvents. From these results, it was concluded that it was feasible to produce the δ-form from seeded crystallization. Further Solvent Consideration. Based on the polymorphic accessibility study, 1,4-dioxane has a better propensity to produce the desired δ-form as compared to methanol, since it is able to produce δ-form from a wider range of cooling rates and has less tendency for creeping-up. Seeded crystallization experiments also showed that 1,4-dioxane produced the δform more consistently than methanol. However, there are some undesirable effects of using pure 1,4-dioxane as the solvent. First, PZA has lower solubility in 1,4-dioxane and the solubility plot has less curvature, especially at higher temperatures (Figure 4). In addition, 1,4-dioxane freezes at 11.8 °C, which imposes a limiting constraint to the operating temperature. This implies that using 1,4-dioxane as the solvent will result in poorer yield (i.e., cooling from 45 to 15 °C would give 60% theoretical yield for 1,4-dioxane, while cooling from 45 to 5 °C would give 77% yield for methanol) and requires more solvent per product mass (i.e., approximately 73 and 35 kg solvent per kg product for 1,4-dioxane and methanol, respectively). Second, the cost of 1,4-dioxane per kg is about double to that of methanol (i.e., the price quoted by Merck Millipore is SGD60 and SGD25 per kg for 1.4-dioxane and methanol, respectively). Finally, from the ICH guideline for residual solvents,29 the residuals for 1,4-dioxane and methanol are limited to 380 and 3000 ppm, respectively. Hence, 1,4dioxane is less desirable from the toxicity point of view. To balance the pros and cons of methanol and 1,4-dioxane, a solvent mixture of 1,4-dioxane-methanol was considered. Due to the aforementioned reasons, it is desirable to use the least amount of 1,4-dioxane in the solvent mixture while maintaining consistent production of the δ-form. Hence, a few seeded experiments were carried out in different solvent mixtures, and results showed that 20% 1,4-dioxane−80% methanol resulted in the δ-form production with a good consistency. As an additional benefit, the solubility of PZA in a 20% 1,4dioxane−80% methanol solvent mixture (Table 4) is higher
solubility (g/kg)
5.0 13.3 19.3 27.5 31.5 35.6 43.1 47.0
11.1 15.0 20.0 25.0 30.0 35.0 45.0 50.0
DESIGN SPACE DEVELOPMENT
After selecting the desired polymorph and solvent, the subsequent step is to establish the design space for polymorphic purity, or the region of operation which assures consistent production of the δ-form with polymorphic impurity below a specified limit. Several factors may affect the product polymorphic purity, including cooling rate, seeding temperature, seed loading, solvent mixture composition, and seed preparation technique (Table 5). The seed preparation technique is a qualitative parameter and deserves further explanation. Previous studies26 show that solid-state grinding may cause solid state transformation from the metastable δ-form to the stable α-form. Due to possible inadvertent seeding with the stable α-form through ballmilling seed preparation, an alternative way of seed preparation was sought. Power ultrasound (defined as frequencies from 20 to 100 kHz) has long been applied to heterogeneous reactions to enhance reaction rates,32 and its application to crystallization (sonocrystallization) has gained interest recently.33−37 Sonocrystallization for particle size reduction is particularly relevant to the current study.34,37 Power ultrasound exerts alternate cycles of compression and rarefaction within a liquid, creating bubbles during the rarefaction stage.34 The bubbles survive repeated cycles of compression and rarefaction until a critical size is reached and collapse occurs (i.e., cavitation phenomena). Such cavitation creates intense temperature and pressure pulses in the vicinity of the collapse, causing particle breakage. Using power ultrasound for size reduction generally does not affect crystalline structure.37 In contrast, mechanical force applied during grinding/ballmilling may produce dislocations in the crystal lattice.38 This causes regions of amorphicity on the crystal surface, which may subsequently recrystallize as a different polymorph. Mechanical size reduction, for example, was shown to have negative impact on the stability of albuterol sulfate.39 In this study, δ-form PZA was generated through unseeded crystallization in 1,4-dioxane by cooling saturated solutions from 45 to 15 °C. Subsequently, either seed preparation technique (i.e., ultrasonic or ballmilling) was performed to generate seeds for the design space development (Table 6). After each seed preparation, PXRD measurement was performed to verify that no stable α-form was observed in the seeds. CSD measurements (Figure 7) showed that seeds prepared by both methods had a similar mean size (65−75 μm), though the ballmilled seeds had a slightly wider size distribution than the ultrasonic seeds. To understand the effects of the selected five factors on the product polymorphic purity, Design of Experiments (DOE) was performed in this study. Central Composite Design (CCD) was used for each seed preparation technique, where 28 experiments were carried out in random order (i.e., 16 factorial experiments + 2 × 4 axial experiments at face values + 4 repeat central experiments). For each crystallization experiment, the initial solution was saturated with PZA with respect to the α-form at 44 °C. The initial dissolution of PZA was carried out by heating the system to 48 °C, and maintaining at this temperature for about 20 min before cooling it to the seeding temperature. The final temperature for each experiment was set to 5 °C. The process
Table 4. Solubility Values of α-PZA in 20% 1,4-Dioxane− 80% Methanol Mixture T (°C)
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than in the individual solvents over the whole temperature range, and the plot has a similar curvature to that of methanol (Figure 6). As a result, crystallization from this mixture solvent will result in a similar theoretical yield to that from methanol (i.e., 76%) and require less solvent per product mass (i.e., about 28 kg solvent per kg product) compared to that from individual solvents. E
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Figure 6. Solubility of α-PZA in 20% 1,4-dioxane−80%methanol mixture, methanol, and 1,4-dioxane.
the response variable (α-form impurity), Box-Cox transformation40 was performed on the response variable (refer to Supporting Information for the detailed model development). From the resulting models, polymorphic impurity contour plots were drawn for different pairs of cooling rate and solvent composition (Figures 8 and 9 for ultrasonic and ballmilled seed preparation techniques, respectively). Note that, in the contour plots, the cooling rates are limited to 0.1−0.5 °C/min to account for the cooling rate limitation in larger vessels (e.g., 60 L pilot plant vessel). Suppose the specification for maximum allowable polymorphic impurity level is 5%. From the contour plots for ultrasonic seeds (Figure 8), operating the process at 0.1 °C/ min and 1,4-dioxane composition less than 10% is undesirable because the product polymorphic impurity will always be >15% regardless of the seed loading and seeding temperature. Increasing the cooling rate or increasing 1,4-dioxane composition in the solvent mixture has a favorable impact (i.e., decrease in polymorphic impurity level), as can be seen from the enlargement of the allowable operating region as either factor increases, with increasing 1,4-dioxane composition
Table 5. Process Parameters Affecting Product Polymorphic Purity and the Ranges Considered in This Study process parameters
ranges
cooling rate (°C/min) seeding temperature (°C) seed loading (wt % of theoretical yield) solvent mixture composition (wt % 1,4-dioxane) seed preparation technique
0.1−1 40−45 0−10 0−20 ultrasonic and ballmilling
Table 6. Description of Seed Preparation Techniques seed preparation technique ultrasonic ballmilling
approach 0.25 g of δ-form in saturated 5 mL solution, sonicated at 40% power for 30 s 0.5 g of δ-form ballmilled at 1/20 Hz for 35 s
parameter values and resulting α-form impurity levels obtained from both seed preparation techniques are given in Table 7. Response surface curves for polymorphic impurity level were then developed from the experimental data. In order to alleviate the nonconstant variance problem and to improve model fit to
Figure 7. CSD of ballmilled and ultrasonic seeds. All seeds have a mean size of 65−75 μm, but the ballmilled seeds have a wider CSD. F
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Table 7. Process Parameter Values and the Resulting α-Form Impurity Percentage no.
cooling rate (°C/min)
seeding temperature (°C)
seed loading (%)
solvent composition (wt % 1,4-dioxane)
α-form impurity level (ultrasonic) (wt %)
α-form impurity level (ballmilling) (wt %)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.1 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55
40.0 40.0 45.0 45.0 40.0 40.0 45.0 45.0 40.0 40.0 45.0 45.0 40.0 40.0 45.0 45.0 42.5 42.5 45.0 40.0 42.5 42.5 42.5 42.5 42.5 42.5 42.5 42.5
1.0 10.0 1.0 10.0 1.0 10.0 1.0 10.0 10.0 10.0 1.0 10.0 1.0 10.0 1.0 10.0 5.5 5.5 5.5 5.5 10.0 1.0 5.5 5.5 5.5 5.5 5.5 5.5
0.0 20.0 20.0 0.0 20.0 0.0 0.0 20.0 20.0 0.0 0.0 20.0 0.0 20.0 20.0 0.0 10.0 10.0 10.0 10.0 10.0 10.0 20.0 0.0 10.0 10.0 10.0 10.0
100.0 0.0 2.8 100.0 81.7 84.1 98.6 100.0 0.0 0.0 46.6 0.0 28.1 0.0 0.0 3.7 0.0 38.7 100.0 0.0 0.0 14.2 0.0 6.1 0.0 0.0 3.2 3.4
85.9 0.0 100.0 100.0 70.5 24.9 100.0 100.0 0.0 0.0 0.0 0.0 58.0 0.0 0.0 75.2 0.9 64.9 13.9 28.5 0.0 61.3 0.0 0.0 0.0 0.0 0.0 0.1
Figure 8. Polymorphic impurity contour plots for the ultrasonic seeds for different pairs of cooling rate and solvent mixture composition. (Red: impurity >15%; yellow: 10% < impurity < 15%; light blue: 5% < impurity < 10%; dark blue: impurity 15%; yellow: 10% < impurity < 15%; light blue: 5% < impurity < 10%; dark blue: impurity
