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A method to crystallize substances that oil out
chemical engineering research and design 8 8 ( 2 0 1 0 ) 1174–1181
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A method to crystallize substances that oil out L. Derdour ∗ AstraZeneca Process R&D, Södertälje SE-151 85, Sweden
a b s t r a c t A new approach to crystallize oily substances is described. The tendency for liquid–liquid phase separation (LLPS) is reduced by decreasing the kinetics of self-association via the formation of an intermediate amorphous network. The path to initial crystal formation followed a sequence of first freeze-drying an emulsion of solute in the solvent system followed by suspending the dried solid in water to obtain a hydrated crystalline form. This new procedure was applied successfully to a pharmaceutical organic substance that was previously isolated only as a viscous oil. Once isolated, crystals of the drug were utilized as seeds to allow the successful transformation of an emulsion of the substance into a suspension of crystalline drug solid thus avoiding the freeze-drying step. The isolated crystalline solid retained its physical and chemical purity at room temperature for at least 3 months. © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Oiling out; Liquid–liquid phase separation; Freeze-drying; Pharmaceuticals
1.
Introduction
Oiling out, also referred to as demixing or liquid–liquid phase separation (LLPS), is a common phenomenon that occurs during crystallization development in the pharmaceutical industry. LLPS is usually unwanted because it impedes crystallization of the drug. Nevertheless, LLPS can be advantageous for the separation of fatty acids as reported by Maeda et al. (1997). In the pharmaceutical industry processes involving LLPS are highly undesirable because they lack sufficient engineering process controls upon scale-up and robustness. Crystallization processes that exhibit LLPS can create molten phases that stick to reactor walls, and render the crystallization procedure useless by concentrating impurities in the solute rich phase which leads to high impurity integration in the crystals upon nucleation. In recent years, Serajudin and Pudipeddi (2002) reported that there was an increase in the number of less hydrophilic and less polar drug candidates upon discovery of more potent and improved target specific drug molecules. This likely explains the parallel increase of case studies involving LLPS reported in recent years in the pharmaceutical industry (Bonnett et al., 2003; Deneau and
Steele, 2005; Kim et al., 2003; Lafferrère et al., 2004; Lu et al., 2004; Svärd et al., 2007; Veesler et al., 2003). Less hydrophilic molecules have an inherent low polarity with a lack of anchoring sites, and therefore they do not easily self-assemble in an organized manner. Most often, during the early development of drug candidates, seeds of crystalline forms are not available and one needs to induce nucleation in order to produce a solid form. Thus, generating supersaturation is a prerequisite for obtaining a solid form. In early stage development, limited data are available regarding the solubility of drug candidates in different solvents. Therefore, supersaturation is usually created by screening solvents and using different means such as cooling, evaporation or antisolvent addition or a combination of the stated methods. Trial and error is also utilized at this stage. However, attempts to crystallize drug substances often lead to a phase separation in which the active molecule is either concentrated in one phase or is distributed between many phases. This paper reports a case study of an AstraZeneca developmental drug that exhibits oiling out and demonstrates a relatively original approach to produce a solid crystalline phase of that substance.
Abbreviations: LLPS, liquid–liquid phase separation; PXRD, powder X-ray diffraction; HNMR, proton nuclear magnetic resonance; DSC, diffrential scanning calorimetry; TGA, thermal gravimetric analysis; LC/MS, liquid chromatography/mass spectrometry; KF, Karl Fischer analysis. ∗ Current address: Bristol-Myers Squibb Co., Process Research and Development, 1 Squibb Dr., Bld 50, Rm 232C, New Brunswick, NJ 08903, USA. Tel.: +1 732 325 7704; fax: +1 732 227 3002. E-mail address: lotfi[email protected]. Received 10 December 2008; Received in revised form 27 January 2010; Accepted 2 February 2010 0263-8762/$ – see front matter © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.cherd.2010.02.001
chemical engineering research and design 8 8 ( 2 0 1 0 ) 1174–1181
2.
Background
Supersaturation is mandatory to crystallize a substance. In the pharmaceutical industry, supersaturation is usually achieved in solution by different means, as mentioned above, depending on the solubility of the substance in the solvent system. However, crystallization of molecules with low polarity and low propensity for hydrogen bonding is not easily achieved because of the following reasons: - At low supersaturation, nucleation does not occur because the thermodynamic barrier to overcome is too high, i.e. the molecules do not self-assemble easily. - At high supersaturation, the molecules adopt the closest metastable state, which can be 2 liquids of different compositions. Energetics and kinetics are frequently used to explain the occurrence of LLPS at high supersaturation as reported by Deneau and Steele (2005) and Lafferrère et al. (2004), among others. Vivares and Bonneté (2004), Liu et al. (1996), Thomson et al. (1987), and Galkin and Vekilov (2000), among others also attribute the occurrence of LLPS during the crystallization of polymers to the large difference in molecular weights between solutes and solvents. Theoretically, if the solution concentration is maintained in the region between the solubility curve and the LLPS curve for enough time, crystallization should occur. However, in early stage development, data for solubility and LLPS curves for drug candidates are usually scarce. Besides, when trying to crystallize a molecule with a low polarity, the induction time prior to nucleation can be very high. This can mislead one to believe that the solution is undersaturated and that the supersaturation needs to be increased. This will ultimately bring the system beyond the LLPS curve and provoke liquid–liquid separation. LLPS can also occur in the metastable zone of crystallization because of thermodynamic reasons, i.e. high activation energy for crystallization as mentioned by Lee et al. (1992), for the crystallization of polymers. LLPS can also occur for substances that are relatively straightforward to crystallize as shown by Lee et al. (2008), in which oiling out was demonstrated for the case of sulfathiazole because of rapid evaporation of the solvent. The conclusion drawn from such studies is that crystallization is impossible in such a solvent system, but in reality it could have been achieved if sufficient time was provided for the onset of nucleation in the metastable zone. As an alternative technique, ultrasound has also been used to generate crystals and avoid oiling out as demonstrated successfully by Kim et al. (2003). Nonetheless, this approach is still in early development and usually succeeds only when crystallization is not difficult to achieve. In some cases, oiling out was also found to be a necessary step before nucleation. Using simultaneous measurements of solute concentration and turbidity, Groen and Robert (2001) found that concentrated micro-droplets form first in a continuous phase of lower concentration followed by nucleation at the interface of the droplets-continuous phase. Lastly, it is worth mentioning that modelling was not extensively used to predict the eventual occurrence of LLPS in the case of crystallization until recently as reported by Kiesow et al. (2008), for the crystallization of 4,4 -dihydroxydiphenylsulfone. Therefore, in early development, the unavailability of seeds of the drug and the lack of reliable modelling tools make
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obtaining a crystalline form of a molecule of low polarity quite arduous and time consuming. From the literature two types of oiling out are described: 1. Liquid–liquid separation occurs where each phase contains reasonable amounts of solute. According to Veesler et al. (2003), this occurs usually when a mixture of solvents is used. In this case, the solute is generally not evenly distributed between the two phases. Lafferrère et al. (2004), reports that the large difference in affinity of the drug for each solvent can be the driving force for LLPS which can be avoided if the right crystallization conditions, particularly the temperature of nucleation, are selected. Deneau and Steele (2005), gave an explanation of LLPS in terms of energetics and mentioned that it spontaneously occurs if the concentration of solute is located between the binodal points, defined as concentrations with minimal mixing free energy, and the spinodal points at which spontaneous phase separation occurs. In this type of LLPS, the crystallization of the drug is possible by adjusting the crystallization condition in order to avoid the LLPS region. One of the solutions to tackle this problem is seeding which was used successfully by Veesler et al. (2003), Beckmann (2000), and Deneau and Steele (2005), to prevent this kind of LLPS from occurring or to crystallize a solute from an emulsion. 2. Liquid–liquid separation occurs where one phase contains the solvent(s) and the other phase is mainly formed by the solute in the form of a very heavy viscous oil-like phase, hence the name oiling out. This phenomenon occurs usually for high solute concentrations at moderately high temperatures as reported by Svärd et al. (2007), for the vanillin/water system. These authors attribute the LLPS of highly concentrated solutions to the closeness of the temperature of separation and the melting point of the solute. In such cases, lowering the concentration of the solution should prevent oiling out from occurring (cf. Fig. 1a). However, if phase separation occurs at a low temperature and for a dilute solution a crystalline form of the drug may exist but it melts at a temperature lower than the isolation temperature of the solid, which in most cases lies between 0 ◦ C and room temperature. Therefore, the drug separates from the solvent as a melt at room temperature. In this case, attempts to crystallize the drug can prove to be a daunting task requiring a special crystallization procedure and storage conditions. Thus, such molecules are usually discarded from further development unless they provide unique and highly valuable pharmaceutical attributes. As mentioned above, large differences in molecular weighs between solutes and solvents as in the case of crystallization of polymers can also lead to LLPS. In all cases, the determination of the phase diagram can help greatly in selecting crystallization conditions to avoid LLPS phenomenon. Phase diagrams can be very different depending on the system. Fig. 1a, b, c and d are examples of the main types of phase diagrams encountered in the literature. These figures show no general trend regarding the occurrence of LLPS. However, it appears that a critical solute concentration in the metastable region is a prerequisite for causing LLPS. Fig. 1a is a typical depiction of oiling out from a single solvent and shows that high temperature and high concentration are conditions that favour LLPS. Tendency for LLPS was also studied by Maeda et al. (2001), who indicate that the width of the metastable zone for LLPS
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Fig. 1 – (a) Phase diagram, type 1 (Svärd et al., 2007). (b) Phase diagram, type 2 (Bonnett et al., 2003). (c) Phase diagram, type 3 (Veesler et al., 2003). (d) Phase diagram, type 4 (Inspired from Maeda et al., 2001). CLMZ: curve of the limit of the metastable zone. is much narrower that the one for crystallization and that this characteristic can explain the occurrence of LLPS in the metastable zone for crystallization even for low concentrations. This is depicted in Fig. 1d in which cooling down a mixture from point B will cross the solute solubility, but then will cross the liquid–liquid solubility and the curve of the limit of the metastable zone (CLMZ) for LLPS before reaching the CLMZ for crystallization. If the kinetics of LLPS are fast enough, this will result in a metastable LLPS. In this case, the emulsion obtained can in theory be converted into suspension. On the other hand, cooling a highly concentrated mixture (point A in Fig. 1d) will result in a stable LLPS because the phase separation occurs outside the metastable zone for crystallization which excludes the possibility of solid formation. As shown in Fig. 1d, LLPS can be avoided by lowering the solute concentration, i.e. starting from point C. If the phase diagram is available, it is straightforward to select crystallization conditions that should prevent LLPS from occurring. Unfortunately, constructing the phase diagram is time consuming, labour intensive and is impractical to complete during drug development in the pharmaceutical industry. Besides, there is no consensus on conditions that favour LLPS, and the crystallization is system dependent. Therefore, in industry, where time and labour are major factors, scientists usually rely on experience to select screening conditions that will produce crystalline materials.
3.
Materials and methods
Crystallization attempts were carried in a 24-well temperature regulated shaker, Eppendorf AG, Hamburg, Germany.
PXRD was determined using a Philips XC ¸ Pert-MPD diffractometer, PANalytical Inc., Westborough, MA. Thermal analysis was performed using a DSC822e and a TGA/SDTA851e, Mettler Toledo, Columbus OH. Karl Fischer (KF) analyses were performed using a Metrohm 841 Volumetric Karl Fischer Titrando, Westbury, NY. Freeze-drying was carried out in a Heto PowerDry PL9000 Freeze-Dryer, Breda, The Netherlands. AstraZeneca’s developmental drug was supplied with a purity higher than 99.3%. Chemical purity was assessed using a LCMS-2010EV, Duisburg, Germany. Solution HNMR was determined using a Bruker Ultrashield 400 MHz/54 mm, Bremen, Germany. All solvents were obtained from Sigma–Aldrich Sweden AB, Stockholm, Sweden, and were of HPLC grade.
4.
Crystallization procedure development
Numerous attempts were done to crystallize the substance or at least obtain a solid form. The first approach was to induce solidification by solvent evaporation. A multitude of solvents of varying polarities were used. Using this approach, the drug substance was isolated as a highly viscous molten phase that was difficult to process. This first series of experiments were beneficial in identifying potential solvents and anti-solvents for the drug substance. From this information, anti-solvent crystallization schemes were designed in an attempt to crystallize the substance. Several solvent systems were also tested but liquid–liquid separation occurred in all cases as shown in the photographs in Fig. 2 which shows the formation of an emulsion upon addition of the anti-solvent. Upon observation under the microscope, the solvent evaporated and a
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Fig. 2 – Emulsion observed under the microscope. molten phase of the drug was obtained. This observation indicated that a crystalline form of the drug substance could have a lower melting point than normal conditions, making attempts to crystallize the substance at room temperature impossible. Therefore crystallization of the substance as a solvate/hydrate or a co-crystal was investigated. Attempts to crystallize a salt form of the drug were ruled out because the molecule lacked basic or acidic sites. Trials to isolate a cocrystal of the drug substance were also abandoned because the periphery of the drug molecule lacked reliable H-bond donors and/or acceptors. The low propensity for H-bond formation makes self-assembly quite difficult especially at high molecular mobility, i.e. high temperature. Therefore, this explains the difficulty of the drug molecules to create a crystal lattice at room temperature. The drug molecule does not have reliable H-bond donors and acceptors on its periphery but it does contain a reliable Hbond acceptor. However, it is believed to be embedded deep within the molecule and hence cannot create bonds with neighbouring drug molecules.
4.1.
Approach to crystallize the drug substance
The approach that we adopted in attempts to obtain a crystalline form for the developmental drug was based on two fundamental ideas:
1. The presence of an embedded reliable H-bond acceptor prompted us to investigate the possibilities of isolating a solvated form of the drug. More particularly, to crystallize the drug molecule as a hydrate. This choice was based on the thought that water molecules are small enough to reach the H-bond acceptor that is embedded in the drug molecule and could therefore link 2 drug molecules by creating 2 Hbonds with the H-bond acceptors of each drug molecule. From this rationale, water can link drug molecules through H-bonding and, consequently, a monohydrate-drug solid would be possible to isolate. In the subsequent sections, the drug molecule will be represented by R1 –A–R2 where
R1 and R2 are 2 bulky parts of the molecule and A is the reliable H-bond acceptor. 2. Next, we chose to use the strategy of producing an amorphous form of the substance followed by providing a small amount of energy to slowly relax the substance to a crystalline form (Hancock and Zografi, 1997). One of the main drivers to LLPS is the self-association of drug molecules via hydrophobic interactions and self-association of solvent molecules via dipoles/H-bonding as depicted in Fig. 3 for the case where the solvent is water. The rationale in forming a crystalline solid through an amorphous structure is to reduce the molecular mobility of the drug substance, i.e. to decrease the equilibrium constant for the drug association: ka1 . Freezing the drug molecules in an amorphous structure should substantially reduce the molecular mobility within the drug substance and decrease the kinetics of self-association. Fig. 3 shows the proposed mechanism that occurs in the liquid state which compares high kinetics for LLPS and crystallization.
The first attempt to isolate a solid hydrate form of the drug involved a simple procedure in which the drug is dissolved in an organic solvent and then water is added as an anti-solvent. This attempt failed as liquid–liquid separation occurred as mentioned above. The second approach is depicted in Fig. 4 using the creation of an amorphous network. Essentially, the drug was dissolved in a low boiling point organic solvent such as methanol, stage A, then add water to oil out the drug substance, stage B. The organic solvent was then removed by vacuum distillation to obtain an emulsion of the drug and the water, stage C. This emulsion was then mixed vigorously to create a dispersion of micro-droplets of the drug in water, stage D. Then, while being mixed, the emulsion was frozen to −50 ◦ C, stage E. Finally the frozen mixture was lyophilized. The dried material, stage F, was porous and agglomerated with no distinct faces, and as shown by microscope images in Fig. 5, it did not exhibit birefringence.
Fig. 3 – Molecular association and competition between LLPS and crystallization.
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Fig. 4 – First attempt to crystallize R1 –A–R2 . However, microscope images also revealed that the lyophilized solid material melted rapidly and a viscous oil was obtained when warmed to room temperature as depicted in Fig. 5, stage G. As expected, water content analysis revealed that all water had sublimed, which meant that the compound did not form a hydrate. Further solid state analysis could not be performed on the freeze-dried solid because, as shown in Fig. 5, it melted quickly. HNMR analysis confirmed that the drug substance is free of residual solvent. This observation confirmed that the substance is in a molten phase under normal conditions of temperature and pressure as a neat form.
4.2. Use of the lyophilized solid to obtain a crystalline form of a monohydrate-drug Our next approach was to mix the solid obtained by freeze-drying with liquid water again to promote favourable conditions for isolating a hydrated solid form. The amorphous solid obtained by freeze-drying was highly porous and should have a lower molecular mobility compared to the molten phase which would reduce the kinetics of self-association. It is expected that upon mixing with liquid water, the later will diffuse into the pores and come in close contact with the substance for a sufficient time that permits the nucle-
ation of a hydrated form before drug molecules and water self-association prevent that from happening. Conversely, the hydrate-drug solid is thermodynamically possible, but it is blocked by the drug self-association when it is in the liquid state and by the high molecular mobility encountered in the molten phase. The solid amorphous material has a lower molecular mobility and a higher relaxation time than the molten phase and can provide enough time for the molecules to assemble in an ordered fashion, i.e. crystalline form. Therefore, the solid obtained by freeze-drying was rapidly suspended in water at a low temperature in order to slow down the transformation from the supercooled liquid, i.e. amorphous solid, to the molten phase. The mixture water/drug was mixed in a multi-well mixer and the solid was monitored periodically for possible form changes. Approximately 1 h after the start of the mixing some peaks started to appear in the PXRD pattern. The solid was isolated after 2 h of mixing and after filtration and drying under vacuum it was analyzed by PXRD which revealed that it was highly crystalline. Thermal analysis was also determined and DSC revealed an endothermic event at the same temperature interval where the TGA displays a weight loss. Hot-stage microscopy was then used to ascertain the cause of the endotherm and revealed a melting event at that temperature interval indicating that the endothermic
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Fig. 5 – Melting of solid obtained by freeze-drying observed under microscope. event corresponds to melting combined with dehydration. The heat involved during the endotherm displayed by the DSC event suggests that the lattice energy is low, probably due to the few H-bonds per unit cell. Crystals initially obtained with the procedure involving lyophillization were used as seeds in subsequent crystallization experiments carried out at room temperature. This seeded crystallization afforded crystalline material with the same characteristics as the one initially obtained. The water content of the solid was also determined by KF analysis and TGA and it was found to correspond to the level of the monohydrate which confirmed our initial hypothesis that a water molecule can act as a link between two drug molecules. The chemical purity was determined by LC/MS and HNMR and was found to be identical to the starting material. The overall procedure to isolate the crystalline form of the drug is summarized in Fig. 6.
4.3.
Conversion of an emulsion into a suspension
The strategy followed used freeze-drying as a path to produce seeds of crystalline monohydrate. This result indicates that mixing the drug with water results in a metastable LLPS. Hence, the possibilities of using the seeds in order drive the crystallization from an emulsion of the drug substance in water or a mixture of water and an organic solvent without performing freeze-drying were investigated. We decided to introduce the seeds of the hydrate-drug in an emulsion of the drug in water and to stir the mixture for few hours. Temperature of the mixture was kept in the range 0–5 ◦ C in order to limit stress to the system. It was observed that after 2 h, the emulsion turned into a slurry suspension. After filtration and drying, the solid was analyzed by PXRD and had the same drug monohydrate form as the seeds. This process proved reliable as it was repeated several times and produced the crystalline R1 –A–R2 ·H2 O form consistently. Thus, the freeze-drying step
was not needed furthermore and a simple seeded crystallization from an emulsion sufficed to produce the highly crystalline pharmaceutically acceptable crystalline solid drug. The transformation from the emulsion to a suspension of crystalline solid indicates that the LLPS obtained is indeed metastable even though the emulsion remained stable for up to 3 months without seeding. Next, the mixture was subjected to temperature fluctuations to test the robustness of the procedure. Agitation rate was also varied. The outcome of the study was always a crystalline R1 –A–R2 ·H2 O with the same PXRD, DSC and water content. The method thus proved to be robust and reliable. Also, no issues related to the rheology were observed which suggest that a priori no potential problems should be expected upon scale-up. The stability at room temperature of the solid was also tested by monitoring the chemical and physical purity for 3 months after isolation using PXRD, KF, LC/MS and HNMR. The analyses did not show any detectable change proving that the crystalline R1 –A–R2 ·H2 O was stable at room temperature for up to 3 months.
4.4. Possible obstacles to nucleation in the liquid homogeneous phase It is believed that in the liquid state, nucleation of the crystalline hydrate form is prevented because of the following reasons: 1. High molecular mobility in molten phase: H-bonds are continuously formed and disrupted and the duration of Hbonding is short because of the kinetic mobility inherent to the liquid state. This prevents clusters of hydrate substance from reaching the critical size for growth. 2. Self-association: water molecules self-associate by Hbonding. Water is known to have a high self-affinity and
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Fig. 6 – Procedure followed to isolate a solid monohydrate crystalline form of the drug. tends to form H-bond linked clusters. Hydrophobic drug molecules also self-associate by hydrophobic interaction as described by Scheraga (1998). The strategy used in this study to obtain initial crystals of R1 –A–R2 ·H2 O was to lower the kinetics of self-association of the drug molecules by freezing the substance in an amorphous structure and adding water to form the monohydrate.
4.4.1. phase
Thermodynamic barrier to nucleation in liquid
Self-association also suggests that the energy required to form a nuclei of R1 –A–R2 ·H2 O of a reasonable size is significantly higher than the one needed for the formation by LLPS. In order to create embryos of R1 –A–R2 ·H2 O, water molecules have to self-disrupt their clusters and disrupt the drug molecules hydrophobic clusters. This task is very energy demanding and as observed experimentally is not possible without an external aid, i.e. seeding. Crystalline R1 –A–R2 ·H2 O has lower energy than the liquid R1 –A–R2 because creating H-bonds lowers the energy much more than hydrophobic interactions do. Consequently, transformation from LLPS to a suspension is possible once crystals of R1 –A–R2 ·H2 O are added to the suspension.
4.4.2.
Kinetic barrier to nucleation in liquid phase
Once clusters of water and drug are disrupted, water has to form H-bonds with the drug that last long enough to permit self-assembly with other hydrated drug molecules. Because of high molecular mobility in the liquid phase, the life time of Hbonds is expected to be short. Thus the size of the embryos of R1 –A–R2 ·H2 O formed in the liquid phase cannot reach the critical size for nucleation making the latter impossible in the molten phase. On the other hand, the amorphous matrix lowers the molecular mobility of the drug which can increase the life time of H-bonds created between water and the drug. As a result, embryos of R1 –A–R2 ·H2 O created can grow larger than in the liquid phase. According to the concept of sigma cooperativity, as mentioned by Morris (1999), these clusters grow stronger and larger as more molecules are added to them via
H-bonds. This will ultimately lead to self-sustaining clusters that will attain the critical size for nucleation and, consequently, nucleate making the crystallization of R1 –A–R2 ·H2 O possible.
5.
Conclusions
A case study of a LLPS encountered during trials to crystallize a developmental drug and a novel method to generate a crystalline form of that substance were presented. Because of a lack of reliable hydrogen bond donors and acceptors on its periphery and sterical hindrance, the substance did not crystallize at room temperature. A crystalline form was achieved by utilizing water molecules as bridging links between drug molecules to form a solid hydrate of the substance. Lyophillization was involved in the path to the first crystals of a monohydrate crystalline form of the drug. These crystals were then used as seeds to drive the crystallization from an emulsion of the drug in water. Drug molecules selfassociation combined with high molecular mobility in the liquid phase are assumed to block nucleation of the monohydrate in solution. Low molecular mobility in the amorphous solid created by lyophillization is considered to be the facilitator of nucleation. The solid obtained proved to be stable for up to 3 months at room temperature.
Acknowledgements The author is thankful to AstraZeneca’s senior management at Södertälje, Sweden, for assistance and follow-up during the preparation of this paper. Dr. Sebhathu T. is also sincerely thanked for determining PXRDs.
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Chemical Engineering Research and Design journal homepage: www.elsevier.com/locate/cherd
A method to crystallize substances that oil out L. Derdour ∗ AstraZeneca Process R&D, Södertälje SE-151 85, Sweden
a b s t r a c t A new approach to crystallize oily substances is described. The tendency for liquid–liquid phase separation (LLPS) is reduced by decreasing the kinetics of self-association via the formation of an intermediate amorphous network. The path to initial crystal formation followed a sequence of first freeze-drying an emulsion of solute in the solvent system followed by suspending the dried solid in water to obtain a hydrated crystalline form. This new procedure was applied successfully to a pharmaceutical organic substance that was previously isolated only as a viscous oil. Once isolated, crystals of the drug were utilized as seeds to allow the successful transformation of an emulsion of the substance into a suspension of crystalline drug solid thus avoiding the freeze-drying step. The isolated crystalline solid retained its physical and chemical purity at room temperature for at least 3 months. © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Oiling out; Liquid–liquid phase separation; Freeze-drying; Pharmaceuticals
1.
Introduction
Oiling out, also referred to as demixing or liquid–liquid phase separation (LLPS), is a common phenomenon that occurs during crystallization development in the pharmaceutical industry. LLPS is usually unwanted because it impedes crystallization of the drug. Nevertheless, LLPS can be advantageous for the separation of fatty acids as reported by Maeda et al. (1997). In the pharmaceutical industry processes involving LLPS are highly undesirable because they lack sufficient engineering process controls upon scale-up and robustness. Crystallization processes that exhibit LLPS can create molten phases that stick to reactor walls, and render the crystallization procedure useless by concentrating impurities in the solute rich phase which leads to high impurity integration in the crystals upon nucleation. In recent years, Serajudin and Pudipeddi (2002) reported that there was an increase in the number of less hydrophilic and less polar drug candidates upon discovery of more potent and improved target specific drug molecules. This likely explains the parallel increase of case studies involving LLPS reported in recent years in the pharmaceutical industry (Bonnett et al., 2003; Deneau and
Steele, 2005; Kim et al., 2003; Lafferrère et al., 2004; Lu et al., 2004; Svärd et al., 2007; Veesler et al., 2003). Less hydrophilic molecules have an inherent low polarity with a lack of anchoring sites, and therefore they do not easily self-assemble in an organized manner. Most often, during the early development of drug candidates, seeds of crystalline forms are not available and one needs to induce nucleation in order to produce a solid form. Thus, generating supersaturation is a prerequisite for obtaining a solid form. In early stage development, limited data are available regarding the solubility of drug candidates in different solvents. Therefore, supersaturation is usually created by screening solvents and using different means such as cooling, evaporation or antisolvent addition or a combination of the stated methods. Trial and error is also utilized at this stage. However, attempts to crystallize drug substances often lead to a phase separation in which the active molecule is either concentrated in one phase or is distributed between many phases. This paper reports a case study of an AstraZeneca developmental drug that exhibits oiling out and demonstrates a relatively original approach to produce a solid crystalline phase of that substance.
Abbreviations: LLPS, liquid–liquid phase separation; PXRD, powder X-ray diffraction; HNMR, proton nuclear magnetic resonance; DSC, diffrential scanning calorimetry; TGA, thermal gravimetric analysis; LC/MS, liquid chromatography/mass spectrometry; KF, Karl Fischer analysis. ∗ Current address: Bristol-Myers Squibb Co., Process Research and Development, 1 Squibb Dr., Bld 50, Rm 232C, New Brunswick, NJ 08903, USA. Tel.: +1 732 325 7704; fax: +1 732 227 3002. E-mail address: lotfi[email protected]. Received 10 December 2008; Received in revised form 27 January 2010; Accepted 2 February 2010 0263-8762/$ – see front matter © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.cherd.2010.02.001
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2.
Background
Supersaturation is mandatory to crystallize a substance. In the pharmaceutical industry, supersaturation is usually achieved in solution by different means, as mentioned above, depending on the solubility of the substance in the solvent system. However, crystallization of molecules with low polarity and low propensity for hydrogen bonding is not easily achieved because of the following reasons: - At low supersaturation, nucleation does not occur because the thermodynamic barrier to overcome is too high, i.e. the molecules do not self-assemble easily. - At high supersaturation, the molecules adopt the closest metastable state, which can be 2 liquids of different compositions. Energetics and kinetics are frequently used to explain the occurrence of LLPS at high supersaturation as reported by Deneau and Steele (2005) and Lafferrère et al. (2004), among others. Vivares and Bonneté (2004), Liu et al. (1996), Thomson et al. (1987), and Galkin and Vekilov (2000), among others also attribute the occurrence of LLPS during the crystallization of polymers to the large difference in molecular weights between solutes and solvents. Theoretically, if the solution concentration is maintained in the region between the solubility curve and the LLPS curve for enough time, crystallization should occur. However, in early stage development, data for solubility and LLPS curves for drug candidates are usually scarce. Besides, when trying to crystallize a molecule with a low polarity, the induction time prior to nucleation can be very high. This can mislead one to believe that the solution is undersaturated and that the supersaturation needs to be increased. This will ultimately bring the system beyond the LLPS curve and provoke liquid–liquid separation. LLPS can also occur in the metastable zone of crystallization because of thermodynamic reasons, i.e. high activation energy for crystallization as mentioned by Lee et al. (1992), for the crystallization of polymers. LLPS can also occur for substances that are relatively straightforward to crystallize as shown by Lee et al. (2008), in which oiling out was demonstrated for the case of sulfathiazole because of rapid evaporation of the solvent. The conclusion drawn from such studies is that crystallization is impossible in such a solvent system, but in reality it could have been achieved if sufficient time was provided for the onset of nucleation in the metastable zone. As an alternative technique, ultrasound has also been used to generate crystals and avoid oiling out as demonstrated successfully by Kim et al. (2003). Nonetheless, this approach is still in early development and usually succeeds only when crystallization is not difficult to achieve. In some cases, oiling out was also found to be a necessary step before nucleation. Using simultaneous measurements of solute concentration and turbidity, Groen and Robert (2001) found that concentrated micro-droplets form first in a continuous phase of lower concentration followed by nucleation at the interface of the droplets-continuous phase. Lastly, it is worth mentioning that modelling was not extensively used to predict the eventual occurrence of LLPS in the case of crystallization until recently as reported by Kiesow et al. (2008), for the crystallization of 4,4 -dihydroxydiphenylsulfone. Therefore, in early development, the unavailability of seeds of the drug and the lack of reliable modelling tools make
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obtaining a crystalline form of a molecule of low polarity quite arduous and time consuming. From the literature two types of oiling out are described: 1. Liquid–liquid separation occurs where each phase contains reasonable amounts of solute. According to Veesler et al. (2003), this occurs usually when a mixture of solvents is used. In this case, the solute is generally not evenly distributed between the two phases. Lafferrère et al. (2004), reports that the large difference in affinity of the drug for each solvent can be the driving force for LLPS which can be avoided if the right crystallization conditions, particularly the temperature of nucleation, are selected. Deneau and Steele (2005), gave an explanation of LLPS in terms of energetics and mentioned that it spontaneously occurs if the concentration of solute is located between the binodal points, defined as concentrations with minimal mixing free energy, and the spinodal points at which spontaneous phase separation occurs. In this type of LLPS, the crystallization of the drug is possible by adjusting the crystallization condition in order to avoid the LLPS region. One of the solutions to tackle this problem is seeding which was used successfully by Veesler et al. (2003), Beckmann (2000), and Deneau and Steele (2005), to prevent this kind of LLPS from occurring or to crystallize a solute from an emulsion. 2. Liquid–liquid separation occurs where one phase contains the solvent(s) and the other phase is mainly formed by the solute in the form of a very heavy viscous oil-like phase, hence the name oiling out. This phenomenon occurs usually for high solute concentrations at moderately high temperatures as reported by Svärd et al. (2007), for the vanillin/water system. These authors attribute the LLPS of highly concentrated solutions to the closeness of the temperature of separation and the melting point of the solute. In such cases, lowering the concentration of the solution should prevent oiling out from occurring (cf. Fig. 1a). However, if phase separation occurs at a low temperature and for a dilute solution a crystalline form of the drug may exist but it melts at a temperature lower than the isolation temperature of the solid, which in most cases lies between 0 ◦ C and room temperature. Therefore, the drug separates from the solvent as a melt at room temperature. In this case, attempts to crystallize the drug can prove to be a daunting task requiring a special crystallization procedure and storage conditions. Thus, such molecules are usually discarded from further development unless they provide unique and highly valuable pharmaceutical attributes. As mentioned above, large differences in molecular weighs between solutes and solvents as in the case of crystallization of polymers can also lead to LLPS. In all cases, the determination of the phase diagram can help greatly in selecting crystallization conditions to avoid LLPS phenomenon. Phase diagrams can be very different depending on the system. Fig. 1a, b, c and d are examples of the main types of phase diagrams encountered in the literature. These figures show no general trend regarding the occurrence of LLPS. However, it appears that a critical solute concentration in the metastable region is a prerequisite for causing LLPS. Fig. 1a is a typical depiction of oiling out from a single solvent and shows that high temperature and high concentration are conditions that favour LLPS. Tendency for LLPS was also studied by Maeda et al. (2001), who indicate that the width of the metastable zone for LLPS
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Fig. 1 – (a) Phase diagram, type 1 (Svärd et al., 2007). (b) Phase diagram, type 2 (Bonnett et al., 2003). (c) Phase diagram, type 3 (Veesler et al., 2003). (d) Phase diagram, type 4 (Inspired from Maeda et al., 2001). CLMZ: curve of the limit of the metastable zone. is much narrower that the one for crystallization and that this characteristic can explain the occurrence of LLPS in the metastable zone for crystallization even for low concentrations. This is depicted in Fig. 1d in which cooling down a mixture from point B will cross the solute solubility, but then will cross the liquid–liquid solubility and the curve of the limit of the metastable zone (CLMZ) for LLPS before reaching the CLMZ for crystallization. If the kinetics of LLPS are fast enough, this will result in a metastable LLPS. In this case, the emulsion obtained can in theory be converted into suspension. On the other hand, cooling a highly concentrated mixture (point A in Fig. 1d) will result in a stable LLPS because the phase separation occurs outside the metastable zone for crystallization which excludes the possibility of solid formation. As shown in Fig. 1d, LLPS can be avoided by lowering the solute concentration, i.e. starting from point C. If the phase diagram is available, it is straightforward to select crystallization conditions that should prevent LLPS from occurring. Unfortunately, constructing the phase diagram is time consuming, labour intensive and is impractical to complete during drug development in the pharmaceutical industry. Besides, there is no consensus on conditions that favour LLPS, and the crystallization is system dependent. Therefore, in industry, where time and labour are major factors, scientists usually rely on experience to select screening conditions that will produce crystalline materials.
3.
Materials and methods
Crystallization attempts were carried in a 24-well temperature regulated shaker, Eppendorf AG, Hamburg, Germany.
PXRD was determined using a Philips XC ¸ Pert-MPD diffractometer, PANalytical Inc., Westborough, MA. Thermal analysis was performed using a DSC822e and a TGA/SDTA851e, Mettler Toledo, Columbus OH. Karl Fischer (KF) analyses were performed using a Metrohm 841 Volumetric Karl Fischer Titrando, Westbury, NY. Freeze-drying was carried out in a Heto PowerDry PL9000 Freeze-Dryer, Breda, The Netherlands. AstraZeneca’s developmental drug was supplied with a purity higher than 99.3%. Chemical purity was assessed using a LCMS-2010EV, Duisburg, Germany. Solution HNMR was determined using a Bruker Ultrashield 400 MHz/54 mm, Bremen, Germany. All solvents were obtained from Sigma–Aldrich Sweden AB, Stockholm, Sweden, and were of HPLC grade.
4.
Crystallization procedure development
Numerous attempts were done to crystallize the substance or at least obtain a solid form. The first approach was to induce solidification by solvent evaporation. A multitude of solvents of varying polarities were used. Using this approach, the drug substance was isolated as a highly viscous molten phase that was difficult to process. This first series of experiments were beneficial in identifying potential solvents and anti-solvents for the drug substance. From this information, anti-solvent crystallization schemes were designed in an attempt to crystallize the substance. Several solvent systems were also tested but liquid–liquid separation occurred in all cases as shown in the photographs in Fig. 2 which shows the formation of an emulsion upon addition of the anti-solvent. Upon observation under the microscope, the solvent evaporated and a
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Fig. 2 – Emulsion observed under the microscope. molten phase of the drug was obtained. This observation indicated that a crystalline form of the drug substance could have a lower melting point than normal conditions, making attempts to crystallize the substance at room temperature impossible. Therefore crystallization of the substance as a solvate/hydrate or a co-crystal was investigated. Attempts to crystallize a salt form of the drug were ruled out because the molecule lacked basic or acidic sites. Trials to isolate a cocrystal of the drug substance were also abandoned because the periphery of the drug molecule lacked reliable H-bond donors and/or acceptors. The low propensity for H-bond formation makes self-assembly quite difficult especially at high molecular mobility, i.e. high temperature. Therefore, this explains the difficulty of the drug molecules to create a crystal lattice at room temperature. The drug molecule does not have reliable H-bond donors and acceptors on its periphery but it does contain a reliable Hbond acceptor. However, it is believed to be embedded deep within the molecule and hence cannot create bonds with neighbouring drug molecules.
4.1.
Approach to crystallize the drug substance
The approach that we adopted in attempts to obtain a crystalline form for the developmental drug was based on two fundamental ideas:
1. The presence of an embedded reliable H-bond acceptor prompted us to investigate the possibilities of isolating a solvated form of the drug. More particularly, to crystallize the drug molecule as a hydrate. This choice was based on the thought that water molecules are small enough to reach the H-bond acceptor that is embedded in the drug molecule and could therefore link 2 drug molecules by creating 2 Hbonds with the H-bond acceptors of each drug molecule. From this rationale, water can link drug molecules through H-bonding and, consequently, a monohydrate-drug solid would be possible to isolate. In the subsequent sections, the drug molecule will be represented by R1 –A–R2 where
R1 and R2 are 2 bulky parts of the molecule and A is the reliable H-bond acceptor. 2. Next, we chose to use the strategy of producing an amorphous form of the substance followed by providing a small amount of energy to slowly relax the substance to a crystalline form (Hancock and Zografi, 1997). One of the main drivers to LLPS is the self-association of drug molecules via hydrophobic interactions and self-association of solvent molecules via dipoles/H-bonding as depicted in Fig. 3 for the case where the solvent is water. The rationale in forming a crystalline solid through an amorphous structure is to reduce the molecular mobility of the drug substance, i.e. to decrease the equilibrium constant for the drug association: ka1 . Freezing the drug molecules in an amorphous structure should substantially reduce the molecular mobility within the drug substance and decrease the kinetics of self-association. Fig. 3 shows the proposed mechanism that occurs in the liquid state which compares high kinetics for LLPS and crystallization.
The first attempt to isolate a solid hydrate form of the drug involved a simple procedure in which the drug is dissolved in an organic solvent and then water is added as an anti-solvent. This attempt failed as liquid–liquid separation occurred as mentioned above. The second approach is depicted in Fig. 4 using the creation of an amorphous network. Essentially, the drug was dissolved in a low boiling point organic solvent such as methanol, stage A, then add water to oil out the drug substance, stage B. The organic solvent was then removed by vacuum distillation to obtain an emulsion of the drug and the water, stage C. This emulsion was then mixed vigorously to create a dispersion of micro-droplets of the drug in water, stage D. Then, while being mixed, the emulsion was frozen to −50 ◦ C, stage E. Finally the frozen mixture was lyophilized. The dried material, stage F, was porous and agglomerated with no distinct faces, and as shown by microscope images in Fig. 5, it did not exhibit birefringence.
Fig. 3 – Molecular association and competition between LLPS and crystallization.
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Fig. 4 – First attempt to crystallize R1 –A–R2 . However, microscope images also revealed that the lyophilized solid material melted rapidly and a viscous oil was obtained when warmed to room temperature as depicted in Fig. 5, stage G. As expected, water content analysis revealed that all water had sublimed, which meant that the compound did not form a hydrate. Further solid state analysis could not be performed on the freeze-dried solid because, as shown in Fig. 5, it melted quickly. HNMR analysis confirmed that the drug substance is free of residual solvent. This observation confirmed that the substance is in a molten phase under normal conditions of temperature and pressure as a neat form.
4.2. Use of the lyophilized solid to obtain a crystalline form of a monohydrate-drug Our next approach was to mix the solid obtained by freeze-drying with liquid water again to promote favourable conditions for isolating a hydrated solid form. The amorphous solid obtained by freeze-drying was highly porous and should have a lower molecular mobility compared to the molten phase which would reduce the kinetics of self-association. It is expected that upon mixing with liquid water, the later will diffuse into the pores and come in close contact with the substance for a sufficient time that permits the nucle-
ation of a hydrated form before drug molecules and water self-association prevent that from happening. Conversely, the hydrate-drug solid is thermodynamically possible, but it is blocked by the drug self-association when it is in the liquid state and by the high molecular mobility encountered in the molten phase. The solid amorphous material has a lower molecular mobility and a higher relaxation time than the molten phase and can provide enough time for the molecules to assemble in an ordered fashion, i.e. crystalline form. Therefore, the solid obtained by freeze-drying was rapidly suspended in water at a low temperature in order to slow down the transformation from the supercooled liquid, i.e. amorphous solid, to the molten phase. The mixture water/drug was mixed in a multi-well mixer and the solid was monitored periodically for possible form changes. Approximately 1 h after the start of the mixing some peaks started to appear in the PXRD pattern. The solid was isolated after 2 h of mixing and after filtration and drying under vacuum it was analyzed by PXRD which revealed that it was highly crystalline. Thermal analysis was also determined and DSC revealed an endothermic event at the same temperature interval where the TGA displays a weight loss. Hot-stage microscopy was then used to ascertain the cause of the endotherm and revealed a melting event at that temperature interval indicating that the endothermic
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Fig. 5 – Melting of solid obtained by freeze-drying observed under microscope. event corresponds to melting combined with dehydration. The heat involved during the endotherm displayed by the DSC event suggests that the lattice energy is low, probably due to the few H-bonds per unit cell. Crystals initially obtained with the procedure involving lyophillization were used as seeds in subsequent crystallization experiments carried out at room temperature. This seeded crystallization afforded crystalline material with the same characteristics as the one initially obtained. The water content of the solid was also determined by KF analysis and TGA and it was found to correspond to the level of the monohydrate which confirmed our initial hypothesis that a water molecule can act as a link between two drug molecules. The chemical purity was determined by LC/MS and HNMR and was found to be identical to the starting material. The overall procedure to isolate the crystalline form of the drug is summarized in Fig. 6.
4.3.
Conversion of an emulsion into a suspension
The strategy followed used freeze-drying as a path to produce seeds of crystalline monohydrate. This result indicates that mixing the drug with water results in a metastable LLPS. Hence, the possibilities of using the seeds in order drive the crystallization from an emulsion of the drug substance in water or a mixture of water and an organic solvent without performing freeze-drying were investigated. We decided to introduce the seeds of the hydrate-drug in an emulsion of the drug in water and to stir the mixture for few hours. Temperature of the mixture was kept in the range 0–5 ◦ C in order to limit stress to the system. It was observed that after 2 h, the emulsion turned into a slurry suspension. After filtration and drying, the solid was analyzed by PXRD and had the same drug monohydrate form as the seeds. This process proved reliable as it was repeated several times and produced the crystalline R1 –A–R2 ·H2 O form consistently. Thus, the freeze-drying step
was not needed furthermore and a simple seeded crystallization from an emulsion sufficed to produce the highly crystalline pharmaceutically acceptable crystalline solid drug. The transformation from the emulsion to a suspension of crystalline solid indicates that the LLPS obtained is indeed metastable even though the emulsion remained stable for up to 3 months without seeding. Next, the mixture was subjected to temperature fluctuations to test the robustness of the procedure. Agitation rate was also varied. The outcome of the study was always a crystalline R1 –A–R2 ·H2 O with the same PXRD, DSC and water content. The method thus proved to be robust and reliable. Also, no issues related to the rheology were observed which suggest that a priori no potential problems should be expected upon scale-up. The stability at room temperature of the solid was also tested by monitoring the chemical and physical purity for 3 months after isolation using PXRD, KF, LC/MS and HNMR. The analyses did not show any detectable change proving that the crystalline R1 –A–R2 ·H2 O was stable at room temperature for up to 3 months.
4.4. Possible obstacles to nucleation in the liquid homogeneous phase It is believed that in the liquid state, nucleation of the crystalline hydrate form is prevented because of the following reasons: 1. High molecular mobility in molten phase: H-bonds are continuously formed and disrupted and the duration of Hbonding is short because of the kinetic mobility inherent to the liquid state. This prevents clusters of hydrate substance from reaching the critical size for growth. 2. Self-association: water molecules self-associate by Hbonding. Water is known to have a high self-affinity and
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Fig. 6 – Procedure followed to isolate a solid monohydrate crystalline form of the drug. tends to form H-bond linked clusters. Hydrophobic drug molecules also self-associate by hydrophobic interaction as described by Scheraga (1998). The strategy used in this study to obtain initial crystals of R1 –A–R2 ·H2 O was to lower the kinetics of self-association of the drug molecules by freezing the substance in an amorphous structure and adding water to form the monohydrate.
4.4.1. phase
Thermodynamic barrier to nucleation in liquid
Self-association also suggests that the energy required to form a nuclei of R1 –A–R2 ·H2 O of a reasonable size is significantly higher than the one needed for the formation by LLPS. In order to create embryos of R1 –A–R2 ·H2 O, water molecules have to self-disrupt their clusters and disrupt the drug molecules hydrophobic clusters. This task is very energy demanding and as observed experimentally is not possible without an external aid, i.e. seeding. Crystalline R1 –A–R2 ·H2 O has lower energy than the liquid R1 –A–R2 because creating H-bonds lowers the energy much more than hydrophobic interactions do. Consequently, transformation from LLPS to a suspension is possible once crystals of R1 –A–R2 ·H2 O are added to the suspension.
4.4.2.
Kinetic barrier to nucleation in liquid phase
Once clusters of water and drug are disrupted, water has to form H-bonds with the drug that last long enough to permit self-assembly with other hydrated drug molecules. Because of high molecular mobility in the liquid phase, the life time of Hbonds is expected to be short. Thus the size of the embryos of R1 –A–R2 ·H2 O formed in the liquid phase cannot reach the critical size for nucleation making the latter impossible in the molten phase. On the other hand, the amorphous matrix lowers the molecular mobility of the drug which can increase the life time of H-bonds created between water and the drug. As a result, embryos of R1 –A–R2 ·H2 O created can grow larger than in the liquid phase. According to the concept of sigma cooperativity, as mentioned by Morris (1999), these clusters grow stronger and larger as more molecules are added to them via
H-bonds. This will ultimately lead to self-sustaining clusters that will attain the critical size for nucleation and, consequently, nucleate making the crystallization of R1 –A–R2 ·H2 O possible.
5.
Conclusions
A case study of a LLPS encountered during trials to crystallize a developmental drug and a novel method to generate a crystalline form of that substance were presented. Because of a lack of reliable hydrogen bond donors and acceptors on its periphery and sterical hindrance, the substance did not crystallize at room temperature. A crystalline form was achieved by utilizing water molecules as bridging links between drug molecules to form a solid hydrate of the substance. Lyophillization was involved in the path to the first crystals of a monohydrate crystalline form of the drug. These crystals were then used as seeds to drive the crystallization from an emulsion of the drug in water. Drug molecules selfassociation combined with high molecular mobility in the liquid phase are assumed to block nucleation of the monohydrate in solution. Low molecular mobility in the amorphous solid created by lyophillization is considered to be the facilitator of nucleation. The solid obtained proved to be stable for up to 3 months at room temperature.
Acknowledgements The author is thankful to AstraZeneca’s senior management at Södertälje, Sweden, for assistance and follow-up during the preparation of this paper. Dr. Sebhathu T. is also sincerely thanked for determining PXRDs.
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