In situ and Ex situ Analysis of Salmeterol Xinafoate Microcrystal

May 13, 2008 - ABSTRACT: Salmeterol xinafoate (SX) crystallization was investigated under different conditions of stirring, antisolvent addition,...
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In situ and Ex situ Analysis of Salmeterol Xinafoate Microcrystal Formation from Poly(ethylene glycol) 400-Water Cosolvent Mixtures Darragh Murnane, Christopher Marriott, and Gary P Martin*

CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 6 1855–1862

Drug DeliVery Research Group, Pharmaceutical Sciences DiVision, King’s College London, 150 Stamford Street, London SE1 9NH, United Kingdom ReceiVed October 1, 2007; ReVised Manuscript ReceiVed February 8, 2008

ABSTRACT: Salmeterol xinafoate (SX) crystallization was investigated under different conditions of stirring, antisolvent addition, and supersaturation to identify factors limiting particle growth to enable the production of respirable SX microcrystals from PEG 400. Plastic behavior was observed from rheometry of SX-PEG 400 crystallization media indicating a three-dimensional structure following formation of the crystal phase. Above the yield point, the plastic viscosity of the crystallization medium was identical to PEG solutions. Crystallization was concurrent with mixing regardless of the antisolvent addition method. The crystal size distribution (CSD) depended on the stirring conditions indicating that the CSD depended on a balance of the micro-, meso-, and macromixing steps in the turbulent mixing process arising from the viscous and microviscous properties of PEG. Crystallization from PEG 400 followed nucleation theory with the smallest microcrystals being produced at higher SX supersaturation. The degree of nucleation depended on the initial supersaturation and determined the final crystal median diameter. The latter finding was supported by focused beam reflectance measurement and particle vision and measurement analysis. The nascent microcrystals appeared to be stabilized against agglomeration and extensive particle growth by reversible flocculation. Introduction For optimal therapeutic efficacy in the localized treatment of respiratory disease, the aerosol particles should possess an aerodynamic particle size in the range 2.5–6 µm.1 Particles of active pharmaceutical ingredients (APIs) are generally produced by crude crystallization followed by drying and a size-reduction step involving micronization.2 The particles are then typically formulated as pressurized metered dose inhaler (MDI) or as dry powder inhaler (DPI) formulations. The crystallization process is often developed empirically without optimization or a thorough understanding of the process parameters that affect the physical properties of the crystals.3 The micronization step is, perhaps, the most detrimental process during the production of particles for inhalation. Micronization exerts poor control over the particle size, shape and particle size distribution (PSD) of the milled particles. The latter three factors are crucial for the aerodynamic properties of particles.4 The high energy milling process has been shown to introduce amorphous character into drug particles,5–8 leading also to an alteration in particle surface energy.9–12 The combination of altered crystallinity and surface energy directly affects the suitability of the particles for use in MDI and DPI formulations.13–17 Precipitation crystallization employing conventional and supercritical antisolvent systems has attracted much attention in recent years as a constructive means of particle production.18 Although particles suitable for respiratory delivery can be produced by aggressive antisolvent micronization, particles do not always show desirable levels of crystallinity 19,20 and potentially toxic and/or environmentally harmful organic solvents are required to process hydrophobic APIs. Such a technique is difficult to scale-up and obtain a tight suitable PSD due to inhomogeneity of mixing,21 dispersity in nucleation and growth rates2 and postcrystallization events such as aging and * To whom correspondence should be addressed. E-mail: gary.martin@ kcl.ac.uk.

agglomeration.22 Crystal growth inhibitors have been investigated to prevent aging;19,23,24 however, it is undesirable for the latter compounds (typically polymers) to be included in the final formulation. Poly(ethylene glycol), PEG, has received considerable interest as a replacement for conventional solvents in recent years because appropriate grades of the polymer display good solubility in a variety of polar and nonpolar solvents. PEG is proposed as an ideal environmentally acceptable solvent25 demonstrating low flammability, nonvolatility and low toxicity as well as being biodegradable.26,27 Solid and waxy grades of PEG have been used as solvent systems for chemical reactions28,29 and PEG has been recommended as an alternative solvent for organic transformations.30 Aqueous solutions of PEG have been employed frequently in the purification, crystallization, and recovery of proteins31–33 including the pharmaceutically relevant protein interferon alpha-2a.34 More recently, aqueous solutions of low-molecular-weight PEG have been investigated for use in antisolvent crystallization of low-molecular-weight pharmaceuticals.35 It has been established in certain cases that APIs formulated as solid dispersions in PEG release a microfine suspension of drug crystals into the dissolution medium.36 We have established a system, termed Amphiphilic Crystallization, for the antisolvent micronization of APIs based on the addition of water to solutions of APIs in PEG37 and have shown the utility of high-molecular-weight grades of PEG for antisolvent micronization of APIs.38 To be able to control and optimize the properties of crystals, it is essential to understand the effect of process parameters on nucleation and growth. It is necessary, therefore, to consider the kinetics and thermodynamics of the microcrystallization process for the PEG/water antisolvent system. Since nucleation, crystal growth and solvent mixing occur concurrently during antisolvent micronization, the accepted method of monitoring the mass balance during desupersaturation39 to evaluate the kinetics of crystallization is not fully appropriate. A thorough understanding of microcrystal formation from PEG solvents

10.1021/cg700953k CCC: $40.75  2008 American Chemical Society Published on Web 05/13/2008

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Table 1. Experimental Conditions Employed for the Antisolvent Crystallization of Salmeterol Xinafoate batch

SX concentration (% w/w)

solution: antisolvent ratio

σmax

stirrer speed (rpm)

final weight (g)

end time (min)

antisolvent addition

SX 1 SX 2 SX 3 SX 4 SXA SXB SXC

3.62 3.62 3.62 3.62 0.99 3.50 5.97

17.63 16.24 16.47 16.41 16.98 18.44 16.09

3.16 3.21 3.20 3.20 1.79 3.13 3.63

400 400 1200 2000 400 400 400

140 140 140 140 150 150 150

30 30 30 30 20 20 20

standard reverse reverse reverse standard standard standard

would be gained best by combining knowledge of the kinetics of desupersaturation with the measurement of the evolution of the crystal size distribution (CSD). Probe-based process analytical technologies such as focused beam reflectance measurement (FBRM) and particle vision and measurement (PVM)40 provide a means to detect nucleation events 41,42 and study the dynamics of crystal growth including breakage, aggregration42 and polymorphic transformation.43 The aim of this work was to analyze the microcrystallization of salmeterol xinafoate (SX), a long-acting bronchodilator, from PEG 400. This involved applying in situ and ex situ analytical techniques to investigate the thermodynamic and kinetic factors controlling microparticle formation and to investigate the evolution of the CSD during amphiphilic crystallization. Experimental Section Materials. PEG 400, Analar grade cyclohexane and HiPerSolv grade ammonium acetate were purchased from BDH (VWR International Ltd., Poole, U.K.). Span 80 was purchased from Sigma-Aldrich Company Ltd. (Gillingham, U.K.). High performance liquid chromatography grade methanol was purchased from Fisher Scientific Ltd. (Loughborough, U.K.) or from VWR International Ltd. (Poole, U.K.). An Inertsil ODS 2 column (5 µm, 200 × 4.6 mm i.d.) and a Luna ODS-2 column (3 µm, 150 × 4.6 mm i.d) were obtained from Capital HPLC Ltd. (Broxburn, U.K.) and Phenomenex Ltd. (Macclesfield, U.K.). Nylon filters (0.45 µm pore size, 47 mm diameter), cellulose acetate syringe filters (0.45 µm pore size, Schleicher and Schuell brand), and Anotop small volume syringe filters were obtained from Whatman Intl. Ltd. (Maidstone, U.K.). Plastic syringes were purchased from Becton and Dickinson (Oxford, U.K.). Water was produced by reverse osmosis using an ElgaStat unit (Elga LabWater, Marlow, U.K.). Silica gel was purchase from Prolabo (VWR International, U.K.). Salmeterol xinafoate (SX) was a generous gift from GlaxoSmithKline Pharmaceutical Development (Ware, U.K.). Batch-Stirred Tank Crystallization of Salmeterol Xinafoate. Standard Antisolvent Addition Crystallization. Solutions of SX in PEG 400 were prepared at the desired concentration as reported previously using high shear stirring (Silverson L4RT Mixer, Silverson Machines Ltd., Chesham, U.K.)38 and filtered through a 0.45 µm membrane filter. The correct amount of SX in PEG solution was weighed into a 250 mL glass beaker (diameter ) 7.0 cm). The beaker was placed on a laboratory elevator platform, under a Eurostar digitally controlled overhead stirrer (IKAwerk GmbH and Co. KG) equipped with a stainless steel four blade turbine-propeller stirrer (cross-section 4.0 cm). The platform was raised under the beaker, such that the propeller was placed directly in the center. The vertical position of the stirrer was such that the underside of the stirrer was directly above the base of the beaker, but not in contact with the glass. Table 1 details the experimental conditions for antisolvent crystallization according to this protocol (experiments SX 1 and SX A, B, and C). Supersaturation was defined as maximal supersaturation (σmax) assuming instantaneous homogeneous mixing (eq 1) or as actual supersaturation (σ) (eq 2)

σmax ) ln σ ) ln

csws wcrysseq

(1)

ccrys seq

(2)

where cs is the concentration of SX in the solution (g g-1) prior to the addition of the antisolvent, ws is the mass of the solution taken prior to

the addition of the antisolvent, wcrys is the final weight of the crystallization medium following addition of the antisolvent, and seq is the equilibrium solubility of SX (g g-1) in the fully mixed cosolvent mixture, ccrys is the experimentally determined concentration of SX in the crystallization medium, and seq is the equilibrium solubility38 in the fully mixed cosolvent mixture. Crystallization was initiated by the addition of reverse osmosis water (filtered through a 0.45 µm nylon membrane) at an addition rate of 200 g min-1 using a peristaltic pump (Watson Marlow Bredel Pumps Ltd., Falmouth, U.K.) equipped with 6 mm internal diameter silicon tubing, which had a 2 mm thick wall. A 1 mL plastic Gilson pipet tip fixed to the end of the silicon tubing was used to direct the water flow. The antisolvent was directed to flow down the side wall of the glass beaker. Samples (∼1 mL; n ) 3) were removed from the approximate midpoint of the crystallization medium by syringe 60–90 s after addition of the antisolvent was terminated, and upon termination (∼4 mL, n ) 3) of the crystallization process. The liquor was filtered through a 0.45 µm syringe filter, weighed into a volumetric flask (20 mL), and made up to volume for HPLC analysis of SX.44 The crystals were harvested by vacuum filtration using a 0.45 µm nylon membrane filter (47 mm diameter) housed in a glass filter unit (Millipore (UK) Ltd., Watford, U.K.). The wet cake was washed with 2 × 100 mL volumes of water; the latter having been filtered through a 0.45 µm nylon filter and precooled to 4 °C. The washed filter cakes were transferred to Petri dishes, covered with perforated aluminum foil, and dried in vacuo at 50 °C overnight (Vacutherm, Heraeus GmbH, Hanau, Germany). The dry cakes were transferred to sealed glass vials and stored at room temperature over dried silica gel in a glass desiccator. Reverse Antisolvent Addition Crystallization. Three batches of crystals (SX 2, 3, and 4) were prepared by reverse antisolvent addition of a 3.62% w/w solution of SX in PEG 400. The experimental configuration was identical to that detailed for standard antisolvent addition crystallization above; with the exception that water (140.00 g) was initially weighed into the beaker in place of the SX/PEG solution. All other experimental conditions are detailed in Table 1. Drug solutions were weighed (to the nearest 0.001 g) into plastic syringes, and were introduced into the center of the crystallization liquor at a rate of 60 g min-1 to initiate crystallization. Samples were removed for assay and crystallization was terminated as described above for the standard antisolvent addition method. Kinetic Analysis of Supersaturation during Antisolvent Crystallization of SX. A further three batches of crystals were crystallized by standard antisolvent addition using a solution containing 3.49% w/w SX in PEG 400 (batch size varied from 140–170 g). The σmax was 3.06 and stirring was maintained at 400 rpm. Samples (1 mL; n ) 3) were withdrawn using a 5 mL plastic syringe at 30, 60, 5 and 20 min (4 mL; n ) 3) after complete antisolvent addition as described. Each sample was taken simultaneously by three individuals and filtered through either a 0.02, 0.1, or 0.2 µm Anotop inorganic syringe filter into preweighed 20 mL volumetric flasks. The solutions were made up to volume and assayed for SX content by HPLC using a Luna ODS-2 column in place of the Inertsil ODS-2 column to allow a more rapid analysis time. All other HPLC conditions were identical. Comparison of In situ and Ex situ Monitoring of SX Crystallization. A 3.49% w/w solution of SX in PEG 400 (21 g) was crystallized (n ) 3) by reverse antisolvent addition to water (241 g) in a 600 mL glass beaker (diameter 8.5 cm) in the same configuration as above. The σmax was 3.29 and stirring was initiated using the overhead stirrer at a stirring rate of 510 rpm. Samples were removed and filtered for HPLC analysis of SX content at 60 s, 5 min (1 mL; n ) 1) and 20 min (4 mL; n ) 1) after addition of the drug solution. The progress of crystallization was monitored in situ using FBRM and PVM. The Lasentec FBRM S400 probe and Lasentec PVM 700 system (both

Microcrystal Formation from PEG 400-Water Cosolvent Mixtures Mettler-Toledo AutoChem, Leicester, U.K.) were immersed into the crystallization vessel. Particle count statistics of chord length (range 1–1000 µm) as a function of time and the video stream were collected using the Lasentec FBRM control and image control software, on an interfaced personal computer. The crystals were harvested by filtration and dried as above. Measurement of the Crystal Size Distribution. Particle sizing was carried out by laser diffraction using the Malvern Mastersizer X (Malvern Instruments Ltd., Malvern, U.K.) equipped with a 100 mm focal length lens and an MS7 magnetically stirred cell using the 2 NHE software presentation. The dispersant medium was an SX-saturated solution of 0.5% w/v Span 80 in cyclohexane. The saturated dispersant solution was filtered through 0.45 µm cellulose acetate syringe filters into the sample cell. The crystals of SX (approximately 1 mg) were added to an 8 mL glass vial and 2 mL of filtered dispersant was added. The suspension was sonicated in a water bath for 5 min (Sonicleaner, Dawe Ultrasonics Ltd., USA) to allow dispersion of the particles and aliquots were added successively to the sample cell by means of a Gilson pipet to achieve a satisfactory obscuration level (20% obscuration) and allowed to equilibrate for 60 s. Each individual sample was measured five times using 2500 measurement sweeps at a stirrer speed of 3 scale units. Rheology of PEG Crystallization Media. A series of PEG 400 aqueous solutions was prepared in the concentration range 0–100% w/w. Additionally, a solution of SX in PEG 400 (3.5% w/w) was prepared. Portions of the solution (between 0.5 and 4.0 g) were weighed accurately into glass vials, and set volumes of water (varied from 63.2 µg to 9.15 g) were weighed by difference into the solution to generate a series of crystallization media with varying ratios of drug solution to antisolvent. A magnetic follower was added to the mixture, and the mixture was stirred at room temperature for 5 min on a magnetic stirrer (Stuart Scientific, U.K.). The rheological properties of PEG 400 solutions and the SX crystallization suspensions were studied using a Carri-Med CSL 100 Rheometer (TA Instruments Ltd., Crawley, U.K.). Rheometry was performed at 25 °C using the Peltier temperature controlled lower plate. Samples (∼1 mL) were applied to the plate. The operational conditions were the following: measurement system type, parallel plate; plate diameter, 4.0 cm; measurement system gap, 250 µm; measurement system inertia, 1.510 µN m s2; equilibration time, 60 s; equilibration mode, shear stress sweep; start stress, 0.010 Pa; end stress, 5.000 Pa; stress mode, linear; ascent time, 60 s; peak hold time, 0 s; and descent time, 60 s. Aqueous solutions of PEG 400 and suspensions of SX in aqueous PEG 400 crystallization media were measured in triplicate. Dynamic viscosity was determined as the slope of a plot of shear stress as a function of the shear strain using TA Rheology Advantage software (version 5.1.42, TA Instruments, U.K.).

Results and Discussion Rheology of PEG Crystallization Media. A plot of the logarithm of dynamic viscosity as a function of PEG 400 concentration measured by rotational rheometry demonstrated linearity (Figure 1), although there was some suggestion of a plateau being attained at high concentrations of PEG (>90% w/w). It was desirable to employ rotational rheometry in the current study to allow comparison with systems where solids were suspended in the PEG solutions during crystallization. Employing rotational rheometry for Newtonian systems such as aqueous PEG solutions was shown to be valid in a previous study.38 The viscosity plot shows the presence of PEG confers a marked viscosity upon the crystallization medium and this has the potential to affect the mixing of the drug solution and antisolvent.45 It was especially evident that the highest viscosity was exhibited by PEG media at high concentrations of PEG. Such high PEG concentrations represent the equivalent to the initial stages of solution-antisolvent mixing and crystal formation, and would be expected to provide a barrier to solute diffusion once micromixing of the polymeric solvent and water antisolvent has occurred. Arising from the slow diffusion of

Crystal Growth & Design, Vol. 8, No. 6, 2008 1857

Figure 1. Plot of the logarithm of dynamic viscosity as a function of weight concentration of PEG 400 in water of aqueous PEG solutions (×) and salmeterol xinafoate-PEG 400 crystallization media (b) determined at 25 °C (mean ( SD, n ) 3).

solute in the viscous medium, nucleation could display an equivalent time scale to the generation of maximum supersaturation.21 Upon further addition of antisolvent (equivalent to the dilution of PEG), the viscosity of the crystallization medium decreased. This ensures an improvement in solute diffusivity capable of maintaining nucleation rates. Plots of shear stress against shear rate for the crystallization media of SX in PEG 400 demonstrated plastic behavior when the concentration was decreased below 60% w/w PEG and a low yield stress value was apparent. The appearance of the yield stress corresponded to the generation of a crystalline phase, as seen by the clear solution suddenly becoming cloudy. Solutions above 60% w/w PEG 400 were therefore analyzed according to the Newtonian viscosity model (eq 3):

σ ) ηγ

(3)

where σ is the shear stress, η is the dynamic viscosity and γ is the shear rate; and those below according to the Bingham model (eq 4)

σ ) a + η/γ

(4)

where a is the yield stress, and η* is the plastic dynamic viscosity. The dynamic viscosity of crystallization media as a function of PEG 400 concentration is also presented in Figure 1. It is clear from the plot of (plastic) dynamic viscosity of SX-PEG 400 suspensions (crystallization media) as a function of PEG 400 concentration is very similar to the equivalent plot of PEG 400 alone in water. This is clear also from a comparison of the regression equations for PEG 400 (eq 5) and SX-PEG 400 crystallization media (eq 6)

η ) 0.0188((0.0002)PEGconc - 2.938((0.019) (r2 ) 0.992) (5) η * ) 0.0190((0.0004)PEGconc - 2.895((0.025) (r2 ) 0.986) (6) Table 2 presents the yield values calculated according to the Bingham model as a function of PEG concentration. The plot of viscosity as a function of concentration shows that a decrease in crystallization medium viscosity occurs upon addition of water, but this is accompanied by an increase in yield stress. The increase in yield stress was also in accordance with the

1858 Crystal Growth & Design, Vol. 8, No. 6, 2008 Table 2. Yield Stress of SX/PEG 400 Crystallization Media at Varying PEG Weight Concentrations in Water concentration of PEG (% w/w)

yield stress (Pa)

5.1 10.1 20.5 30.5 40.8 51.1 61.8

0.516 ( 0.011 0.501 ( 0.009 0.463 ( 0.012 0.305 ( 0.022 0.222 ( 0.007 0.155 ( 0.005 0.022 ( 0.005

concomitant rise in the phase volume of crystals present in the medium. The presence of a yield stress suggests the presence of three-dimensional structure in the crystallizing suspensions. Effects of Mixing on Crystal Size Distribution. Previous investigations into crystallization of low-molecular-weight compounds from PEG solvents has suggested that the crystallization process depends on the hydrodynamic conditions in the crystallizer.37,46 It has been suggested that segregated quasiemulsions form depending on the stirring rate when aqueous PEG 300 and water are mixed in a 1:1 ratio.46 In the current study, it was found that precipitation occurred during the mixing phase for crystallization of SX from PEG 400 drug solutions by reverse and standard antisolvent addition covering a range of supersaturation values (σmax ≈ 1.8 (SX A) to σmax ≈ 3.6 (SX C)). Reverse addition crystallization is a recognized method to produce the smallest possible crystals during precipitation,47 but this was not observed in the current study (Table 3). Particles produced using the standard addition method were statistically smaller than those produced by reverse antisolvent addition (p < 0.03, Student’s t-test), however there was relatively little difference between the PSD of crystals produced by the standard addition (SX 1) and reverse antisolvent addition method at the same level of supersaturation and stirring (SX 2). An example of a typical PSD is presented for Batch SX 2 in Figure 2. No significant difference was observed between the supersaturation levels in the latter two batches following complete mixing of the drug solution and antisolvent (σ at 1.5 and 30 min, Table 3). The equivalence of the supersaturation values listed in Table 3 indicate that mixing was homogeneous in all cases and contradicts the observations of nonhomogenous mixing and longer induction times for reverse compared to normal antisolvent addition for viscous PEG-water mixtures.46 The data in Table 3 indicated that the particle size distribution (PSD) became broader when the stirring rate was increased. For example, the D(v, 0.1) values were observed to decrease (ANOVA, p < 0.05) in the following order: 2000 rpm 0.05) between crystals manufactured at 1200 and 2000 rpm, which were both smaller than those produced at 400 rpm. The homogeneity of mixing of conventional nonviscous solvents increases as the stirring speed is increased during reverse antisolvent addition, thus leading to a lower rate of nucleation,48 and accordingly, the resultant crystals possess a larger particle size. In antisolvent crystallization, particle formation takes place at the same time as mixing.21 This arises because the nucleation rate is generally higher than the rate of molecular mixing (micromixing), which must occur for solute molecules to nucleate. An instantaneous interfacial tension exists between miscible solvents of different viscosity such as PEG and water.49 As a result, localized segregation of PEG solutions and the antisolvent may exist and the presence of an interfacial tension will ensure that droplets of PEG require mixing above the

Murnane et al.

Kolmogorov microscale (mesomixing).50 The interfacial tension must be overcome to enable micromixing and subsequent nucleation. Reverse addition antisolvent crystallization with PEG is equivalent to the dissolution of PEG. Dissolving PEG displays a microviscosity,51,52 which provides the resistance to molecular motion as well as a macroviscosity (the resistance of fluid to flow). Thus diffusive micromixing will be slow during reverse addition crystallization. An increased microviscosity results in the retarded interdiffusion of SX solute and water solvent molecules prior to nucleation. PEG microviscosity can, however, be reduced by increasing the intensity of stirring.51 The subsequent improvement in the diffusivities of PEG, water, and SX molecules allow improved rates of nucleation, providing the opportunity for the decrease in the D(v, 0.1) values. For standard antisolvent addition crystallization, viscous PEG represents the inital continuous phase during mixing. A viscous continuous phase promotes poorer macromixing, and this results in the localized peaking of supersaturation ensuring high nucleation rates as the dominant method of desupersaturation. The high viscosity of a PEG continuous phase retards solute diffusion, which is necessary for nucleation. Thus nucleation and mixing rates may be of comparable timescales. Upon further addition of water, the diffusion and micromixing processes are facilitated by the decreased viscosity of the crystallization medium, facilitating the maintenance of nucleation rates. Nucleation is facilitated by improved micromixing rates when stirring is increased,53 but macromixing is also improved,54 leading to a reduction in the localization of nucleation. This was exemplified by the fact that only the supersaturation at 90 s of the crystallization medium SX 4 was higher than that produced by standard addition and that the median diameter appeared to plateau beyond a speed of 1200 rpm (Table 2). That is, to say, improved homogeneity of mixing (and the resultant decreased rate of nucleation) was achieved by increasing the stirring rate to 2000 rpm. The increased homogeneity of supersaturation due to concurrently improved macromixing can facilitate crystal growth leading to the broadening of the particle size distribution observed with Batches SX 2, 3, and 4. The accelerated diffusional growth of crystals with improved stirring may dominate any effects of improved mixing to increase nucleation rates.55 Samples of SX 1, SX 2, and SX 3 were stored for a week to study the potential for aging of the precipitate and particle growth was evident from the PSD data of the samples (Table 3). ANOVA revealed significant differences (p < 0.001) in the particle sizes after 30 min and aging both for batches produced by reverse antisolvent addition and the standard control method. It was shown using Tukey’s test that the median diameters and cumulative 10 and 90% undersize diameters of crystals produced at 1200 rpm were larger than those produced at 400 rpm or by standard crystallization (p < 0.05). In the case of reverse antisolvent addition crystallization, significant increases upon storage were observed in D(v, 0.1), D(v, 0.5), and D(v, 0.9)values (p < 0.03). For the standard crystallization batch, no significant increase was observed in the D(v, 0.1) (p ) 0.112), although the median and 90% cumulative were shown to increase (p < 0.035). Crystal aging was greatest in the case of crystals produced by reverse antisolvent addition at 1200 rpm. The changes in the particle size distributions were consistent with Ostwald ripening (isothermal recrystallization) and consolidation of the CSD. The limited increase in crystal size when SX was crystallized at lower stirring rates was due to the lower number of unstable small particles, which subsequently dissolve and

Microcrystal Formation from PEG 400-Water Cosolvent Mixtures

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Table 3. Changes in Supersaturation (σ) (mean ( SD, of 3 filtered samples from each crystallization batch) and Particle Size Distribution (mean ( SD, n ) 5) with Time Following the Antisolvent Crystallization of Salmeterol Xinafoate from PEG 400 Solutions experiment

time

σ

standard 400 rpm (SX 1)

1.5 min 30 min 1 week 1.5 min 30 min 1 week 1.5 min 30 min 1 week 1.5 min 30 min

1.93 ( 0.01 0.10 ( 0.03

reverse 400 rpm (SX 2) reverse 1200 rpm (SX 3) reverse 2000 rpm (SX 4)

2.15 ( 0.13 0.00 ( 0.17 2.15 ( 0.17 0.11 ( 0.21 2.31 ( 0.02 0.10 ( 0.01

recrystallize onto larger particles. The relative stability of some suspensions upon storage for as long as week demonstrates the utility of the PEG crystallization media because most antisolvent precipitation systems routinely demonstrate a dramatic evolution of CSD over time.56 Thermodynamic Factors Controlling Crystallization from PEG 400. Table 4 presents the PSDs of SX crystals isolated 20 min after addition of the water antisolvent at 400 rpm, employing increasing levels of supersaturation (SX A, SX B, and SX C). The quantity of SX-PEG 400 solution was maintained at a constant level to avoid any change in shear conditions. It was found that the D(v, 0.1) was lower for SX crystallized at higher supersaturation levels (one-way ANOVA; p < 0.05), though no significant difference was observed between SX B and SX C (p > 0.05). D(v, 0.5) and the D(v, 0.9) also decreased when the supersaturation was increased (p < 0.05), although there was no difference between SX B and SX C batches (p > 0.05).

Figure 2. Sample particle size distribution obtained by laser diffraction analysis of Batch SX 2 microcrystals of salmeterol xinafoate.

D(v, 0.1) (µm)

D(v, 0.5) (µm)

D(v, 0.9) (µm)

0.70 ( 0.01 1.26 ( 0.54

3.59 ( 0.10 5.70 ( 1.30

9.33 ( 0.21 12.97 ( 2.16

0.77 ( 0.02 0.98 ( 0.11

4.04 ( 0.18 5.34 ( 0.76

9.88 ( 0.41 12.46 ( 1.70

0.70 ( 0.02 2.21 ( 0.41

3.26 ( 0.21 10.05 ( 1.03

10.25 ( 1.96 22.04 ( 2.53

0.66 ( 0.02

2.97 ( 0.37

12.61 ( 2.40

Table 4. Particle Size Distribution (mean ( SD, n ) 5) of Salmeterol Xinafoate Crystallized by Standard Antisolvent Addition to PEG 400 Solutions at Different Levels of Drug Supersaturation (σ), and the Supersaturation of the Crystallization Media as a Function of Time (mean ( SD of 3 filtered samples from each crystallization medium) experiment

σ

D(v, 0.1) (µm)

D(v, 0.5) (µm)

D(v, 0.9) (µm)

SX A SX B SX C

1.79 3.13 3.63

0.87 ( 0.05 0.77 ( 0.05 0.70 ( 0.02

5.41 ( 0.47 4.40 ( 0.36 3.67 ( 0.31

12.45 ( 0.79 10.74 ( 0.51 9.55 ( 0.65

SX A SX B SX C

σmax

σ1 min

σtermination

∆σa

1.79 3.13 3.63

1.69 ( 0.04 1.81 ( 0.01 1.75 ( 0.24

0.74 ( 0.01 0.54 ( 0.05 0.22 ( 0.07

0.95 ( 0.04 1.28 ( 0.05 1.52 ( 0.25

a ∆σ corresponds to the change in supersaturation between 1 min and the termination of crystallization.

Although mixing effects are crucial for determining the rate of generation of supersaturation, it is the degree of supersaturation that provides the thermodynamic driving force for crystallization.57 For crystal batches A-C, the hydrodynamic conditions were kept constant and it was appropriate to compare the maximal supersaturations (σmax; eq 1). Calculation of σmax is limited by the fact that at high supersaturation particle formation takes place concurrent with mixing and the actual degree of supersaturation is not possible to specify.21 Nevertheless, the CSD data (Table 4) show that antisolvent crystallization from PEG 400 obeys classical nucleation theory, which states that the lowest particle size is achieved with the highest possible supersaturation. The rate of nucleation increases with supersaturation,22 and thus the degree of supersaturation generated following mixing is reduced more by nucleation than by crystal growth.58 Regardless of the starting σmax, there was no difference between the levels of supersaturation 1 min after terminating the addition of the antisolvent (ANOVA, p ) 0.588) (Table 4). The greater decrease in supersaturation during the initial 1.5 min produced a higher nucleation rate, as shown by the smaller crystal diameters of Batch SX C than SX A. Kinetic Factors Controlling Crystallization of SX from PEG 400. Three solutions of 3.49% w/w SX in PEG 400 were crystallized using a stirrer speed of 400 rpm and an addition rate of antisolvent of 200 g min-1. Figure 3 shows a bar chart plot of the concentration of drug in the crystallization media (filtered through inorganic membrane filters of 0.2, 0.1 or 0.02 µm) as a function of time after addition of the antisolvent. There was no significant difference between the concentration of SX in the filtrates passing through the different pore sizes at an individual time point (ANOVA, p > 0.32). Precipitation at high degrees of supersaturation leads to rapid nucleation rates59 capable of producing nanoparticles,22 and crystal growth may occur by means of nanoaggregation. The above filtration

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Figure 4. Plot of supersaturation as determined by HPLC of salmeterol xinafoate as a function of time following addition of a drug in PEG 400 solution to water (mean ( SD of a single sample taken from each of three crystallization batches). Figure 3. Bar chart showing the concentration of salmeterol xinafoate in filtrates of the crystallization medium, sampled simultaneously using filters of varying exclusion size (0.02, 0.10, and 0.20 µm) as a function of time (one sample was filtered at each time point through each filter; the bars represent the mean ( SD of n ) 3 batches unless otherwise indicated); grey, white, and hatched boxes correspond to the concentration of SX in filtrates through 0.2, 0.1, and 0.02 µm filters, respectively. Table 5. Particle Size Analysis by Laser Diffraction (mean ( SD, n ) 5) and In situ Determined Chord Lengths and Count Statistics of Salmeterol Xinafoate Crystallized from PEG 400 experiment

D(v, 0.1) (µm)

D(v, 0.5) (µm)

D(v, 0.9) (µm)

Batch 1 Batch 2 Batch 3 mean

0.66 ( 0.02 1.06 ( 0.09 0.84 ( 0.08 0.85 ( 0.07

3.70 ( 0.47 5.72 ( 0.34 4.77 ( 0.75 4.73 ( 0.55

11.01 ( 0.82 12.66 ( 0.75 11.17 ( 1.40 11.61 ( 1.03

mean unweighted mean square-weighted chord length chord length experiment (1.5 min) (µm) (20 min) (µm) Batch 1 Batch 2 Batch 3 mean

4.73 4.67 4.86 4.75 ( 0.10

103.37 119.07 116.69 113.04 ( 8.46

counts s-1 in range 1–5 µm (1.5 min) 31603.5 33010.7 30480.4 31698.2 ( 1267.8

experiments indicated that the SX detected by HPLC analysis is present as molecular solute rather than filterable nanoparticles, because if the latter had been produced, differences would have existed in the concentrations detected following differential filtration. Comparison of Ex situ and In situ Crystallization Analysis. Three batches of SX crystals were produced by reverse antisolvent crystallization, with FBRM and PVM monitoring probes positioned in the crystallization media. The particle size distribution of these SX crystals, when isolated upon termination of the crystallization at 20 min was determined by laser diffraction (Table 5). A plot of supersaturation of the above crystallization media, as determined by HPLC, as a function of time (Figure 4) demonstrated a rapid decrease in the concentration of SX during the first minute after addition of the antisolvent. The majority of supersaturation was dissipated during the initial mixing period, suggesting that nucleation determined the CSD of the SX crystals. The majority of crystals were confirmed to be formed in the initial stages of crystallization by in situ particle size analysis using FBRM (by studying the chord length distribution (CLD) at different time points) and by studying particle behavior using PVM. FBRM revealed the immediate crystallization of a large number (high counts) of particles in the 1–5 and 1–10 µm chord length range (Table 5 and Figure 5). Observations using the PVM probe showed that these particles were highly adhesive,

Figure 5. Unweighted chord length distribution (counts s-1(y-axis)) as a function of chord length (x-axis)) of salmeterol xinafoate crystallized from PEG 400 at the indicated time points.

which necessitated removal of the FBRM probe after approximately 3 min from the crystallization vessel to clean the laser window. Nuclei precipitated rapidly in this fashion frequently agglomerate in order to overcome the interfacial energy resulting from their large surface area.60 The degree of supersaturation decreased more slowly between 1 and 5 min (Figure 4) in agreement with the observations from Figure 3. There was a small but statistically significant difference (Student’s t-test, p ) 0.050) between the supersaturation at 5 and 20 min. A marked level of supersaturation, therefore, persisted following initial nucleation. Persistent supersaturation following nucleation has been implicated previously in unwanted CSD changes in crystallizers.61 Figure 5 shows a decrease in the count of particles with a CLD of 1–10 µm after initial rapid precipitation concomitant with a rise in the counts of coarser particles (20–102 and 102–500 µm). The square-weighted CLD (Figure 6) shows that an initial chord length distribution of small volume particles was replaced by a population with low numbers of very large particles. The excess supersaturation could potentially fuse together particles that agglomerate by cementation. Such agglomeration and crystalline bridge formation is a process that is typical during precipitation.48 Increasing the speed of stirring of the crystallization medium resulted in an increase in the counts of smaller sized particles (Figure 7). The larger sized particles were replaced by a larger

Microcrystal Formation from PEG 400-Water Cosolvent Mixtures

Crystal Growth & Design, Vol. 8, No. 6, 2008 1861

Figure 8. Videomicrograph of salmeterol xinafoate crystallized from PEG 400, during the late stages of the batch crystallization. The yand x-axes represent the scale of particle size (µm). Figure 6. Square-weighted chord length distribution (counts s-1(yaxis)) as a function of chord length (x-axis) of salmeterol xinafoate crystallized from PEG 400 at the indicated time points.

was not significantly different to the mean of the D(v, 0.5) values determined by laser diffraction (Table 5) after filtration and drying of each of the three crystallization batches (student’s t-test, p ) 0.972). This observation confirmed that the crystals’ diameter was determined by the initial nucleation period rather than postcrystallization events. Conclusions

Figure 7. Effect of stirrer speed on the unweighted chord length distribution of salmeterol xinafoate crystallized from PEG 400 20 min after mixing drug solution and the water antisolvent.

number of smaller particles at a higher stirrer rate (e.g., 510 rpm versus 100 rpm) (Figure 7). An image obtained from the PVM toward the later stages of the Batch 2 crystallization run is presented in Figure 8. Images obtained with PVM demonstrated that the development of the population of large size crystals was due to the aggregation of the smaller microcrystals, to form large spherical aggregates. It was observed that as the stirrer speed was sequentially increased, the large aggregates broke down to form aggregates of a smaller size. Thus, later desupersaturation and the changes in the CLD over the period of crystallization do not appear to be related to the irreversible cementation of multiple particles into agglomerates under conditions of residual supersaturation. Alternatively, particle aggregration was attributed to reversible floc formation that stabilizes the microcrystals. The median chord length (Table 5) measured 1.5 min after the start of mixing (4.75 ( 0.10 µm)

The crystallization of SX from PEG 400 was shown to follow classical nucleation theory with the crystal diameters determined by the thermodynamic driving force of supersaturation. However, as is typical of antisolvent crystallization, the CSD was also affected by the prevailing mixing conditions, which are responsible for the rate of generation and localization of supersaturation. Interestingly, it was shown that reverse antisolvent crystallization could also be employed under controlled conditions of mixing to achieve an identical CSD. It was hypothesized that the viscous and microviscous properties of PEG solutions determine a balance of mixing timescales leading to rapid generation of supersaturation. This is in agreement with the finding that mesomixing becomes increasingly important as the viscosity of the medium increases.45 Further studies into the altered diffusivities and the balance of micro- and mesomixing scales could be achieved in a jet-reactor device in which the turbulent mixing conditions are more easily characterized and scalable than a stirred beaker. Crystallization of SX was associated with two periods of desupersaturation with the median crystal diameter determined by the extent of initial nucleation. Persisting supersaturation is frequently associated with agglomeration and cementation serving to alter the CSD after initial crystal formation (aging). SX microcrystals appeared to be stabilized against precipitate aging by the formation of shear-reversible aggregates (flocs), as shown by in situ particle analysis and the yield point in the rheograms. Flocs can be ruptured by high shear stirring and upon filtration. Flocculation provides a reasonable explanation for the resistance of the crystallization batches to Ostwald ripening by stabilizing the surface energy of the hydrophobic particles. Acknowledgment. The authors acknowledge King’s College London and MedPharm Ltd. for financial support. We thank Mr. Ian Haley and Mettler-Toledo AutoChem for the provision

1862 Crystal Growth & Design, Vol. 8, No. 6, 2008

of the Lasentec FBRM and PVM analysis units and are very grateful for Mr. Haley’s advice and expertise, which contributed to this study.

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CG700953K