Crystallization of Cyclosporine in a Multistage Continuous MSMPR

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Crystallization of Cyclosporine in a Multistage Continuous MSMPR Crystallizer Alejandro J. Alvarez,† Aniruddh Singh,‡ and Allan S. Myerson*,‡ †

Department of Chemical Engineering, Tecnologico de Monterrey, Campus Monterrey, Av. Eugenio Garza Sada 2501, Monterrey, N.L. 64849, Mexico ‡ Novartis-MIT Center for Continuous Manufacturing and Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States ABSTRACT: Crystallization processes can be batch or continuous. Potential advantages such as operating at steady state, small equipment size (relative to batch), and ability to recycle are encouraging the pharmaceutical industry to investigate continuous processes. In this work, a continuous cooling crystallization process for the immunosuppressant drug cyclosporine was developed. A multistage mixed suspension mixed product removal (MSMPR) crystallizer was employed which allowed simple analysis of kinetic parameters employing the population balance. Experimentally, the continuous crystallization system was able to operate without any clogging issues for more than four residence times. The experimental yield and purity of the crystals was determined as 71% and 96%, respectively (without recycle) and 87% and 94%, respectively (with recycle). In a batch cooling crystallization experiment, carried out under conditions similar to those of the continuous experiment without recycle, the experimental yield and purity of the crystals were 74% and 95%, respectively. The equilibrium distribution coefficients of cyclosporine impurities were measured experimentally as a function of impurity % of the starting solution. The distribution coefficients increase with a decrease in the purity of the starting solution, indicating a decrease in purification. The MSMPR model was used to estimate the nucleation and crystal growth rate kinetic parameters for cyclosporine crystallization and to evaluate the effect of process conditions on the purity of the crystals and the process yield. Results showed that the temperature of the third stage has a large impact on the final purity of the crystals. As the temperature of the third stage increases, the purity of the crystals also increases while the yield of the process decreases. The effect of recycle ratio on both crystal purity and process yield was also evaluated. A 93% process yield was obtained with a recycle ratio of 0.9. The yield of the process can be significantly improved by increasing the recycle ratio while the crystal purity decreases.

1. INTRODUCTION Crystallization is a separation and purification technique used to produce a wide variety of materials.1 In the pharmaceutical industry, crystallization is an essential operation because the majority of active pharmaceutical ingredients (APIs) are produced in solid form. Control over the crystallization process is desirable, as there is a need to ensure regulatory authorities that APIs of high and reproducible quality and bioavailability can be delivered for formulation and to the patient.2 Crystallization also affects the efficiency of downstream operations such as filtration, drying, and formulation, and the efficacy of the drug can be dependent on the final crystal form.3 Continuous manufacturing has long been well established in the food, dairy, and chemical industries, but manufacturing in batch is still preferred in the pharmaceutical industry.4 Crystallization processes can be batch or continuous. Continuous processing offers the advantages of enhanced reproducibility of results with the material crystallizing under uniform conditions whereas in batch operation conditions change with time, resulting in crystal characteristics difficult to control and inconsistent from batch to batch.5 Also, several factors, such as cost reduction, improved process efficiency, optimal use of equipment, flexibility r 2011 American Chemical Society

in production capacity, etc., are encouraging the pharmaceutical industry to investigate continuous processes.6 Thus, there is a need to develop robust continuous crystallization processes which produce API crystals with high yield and purity. The mixed suspension mixed product removal (MSMPR) crystallizer is a well-mixed continuous crystallizer which is often used in studies of crystallization. It is an idealized vessel which makes theoretical analysis simple. Homogeneous feed solution enters the vessel, supersaturation is generated (by cooling, evaporation, etc.), and nuclei are formed and grow into crystals. Product slurry is continuously withdrawn, and it is assumed that it has exactly the same composition as the vessel contents. This slurry exhibits a crystal size distribution, as the crystals have varying probabilities of residence time in the vessel.7 MSMPR cooling crystallizers are often used, at steady state for the measurement of crystallization kinetics.813 The goal of this work was to carry out continuous crystallization experiments of the immunosuppressant drug cyclosporine and to Received: April 29, 2011 Revised: August 8, 2011 Published: August 10, 2011 4392

dx.doi.org/10.1021/cg200546g | Cryst. Growth Des. 2011, 11, 4392–4400

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Figure 1. (a) Chemical structure of cyclosporine (also known as cyclosporine A) (Abu, L-2-aminobutanoic acid; MeGly, N-methylglycine; MeLeu, N-methyl-L-leucine; Val, valine; Ala, alanine; D-Ala, Dalanine; MeVal, N-methyl-L-valine). (b) X-ray powder diffraction pattern of crystalline (top) and amorphous cyclosporine (bottom).

study the parameters which would influence the overall yield and purity. To accomplish this goal, a multistage continuous MSMPR crystallizer was employed in conjunction with a population balance model and experimentally determined equilibrium distribution coefficients for impurity partitioning between the solution and solid. Although cascades of MSMPR crystallizers have been previously studied and reported in the literature, their analysis was usually focused either on the effect on crystal size14,15 or on the purity of the product.16 This work suggests an integrated approach to evaluate the effect of process parameters both on crystal properties as well as on the process yield.

2. EXPERIMENTAL SECTION 2.1. Materials. Cyclosporine is an immunosuppressant drug used in organ transplant to reduce the activity of the patient’s immune system.17 Cyclosporine was received in both amorphous (90% pure) and crystalline form (95% pure) from Novartis Pharma AG, Basel, Switzerland. Acetone (99.5%) was purchased from SigmaAldrich Corp., St Louis, MO, USA. Amorphous and crystalline cyclosporine were characterized as received, using X-ray powder diffraction (XRPD) and high-performance liquid chromatography (HPLC). The chemical structure (a) and X-ray diffraction patterns (b) of cyclosporine are shown in Figure 1. 2.2. Continuous Crystallization Apparatus and Analytical Instruments. Multistage continuous crystallization experiments were carried out in a three stage continuous crystallizer. Each stage was operated as a mixed suspension mixed product removal (MSMPR) crystallizer. The experimental system consisted of three jacketed glass reactors (50 mL each) with independent temperature control and

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magnetic stirring. The crystallizers were connected in series using insulated polyethylene tubing. A solution of amorphous cyclosporine in acetone was continuously added to the first crystallizer using low flow peristaltic pumps (flow range 0.038.2 mL/min, VWR International). The slurry of cyclosporine crystals was removed from each stage and transferred to the next stage using medium flow peristaltic pumps (flow range 0.485 mL/min, VWR International). A timer was used to control the intermittent operation of these pumps. The slurry removed from the last crystallizer (stage 3) was transferred to a volumetric flask (500 mL) maintained at a controlled temperature. A sample of slurry from the third stage was withdrawn at steady state. The crystals in the third stage were filtered, dried, and characterized for purity, crystallinity, shape, and size, using high performance liquid chromatography (HPLC), X-ray powder diffraction (XRPD), optical microscopy, and focused beam reflectance measurement (FBRM), respectively. Also, the cyclosporine concentration in the mother liquor was measured using HPLC. XRPD patterns were obtained with a Rigaku Miniflex diffractometer using monochromatic Cu Kα radiation with a nickel filter (λ = 1.54 Å) generated at 30 kV and 15 mA. The data were collected from 2 to 40° with a step size of 0.1° at a scan rate of 1.0°/min. Aluminum sample holders with a zero background silicon plate were used to carry out measurements. The HPLC used was an Agilent 1200 HPLC system with a Zorbax Eclipse XDBC18 column (4.6 mm 250 mm, 5 μm). A Nikon Eclipse ME 600 optical microscope from Nikon Co. was used to observe the crystals. The FBRM device was a Lasentec S400 probe from Mettler Toledo, with a measurement range from 785 nm to 1000 μm. The FBRM probe uses a focused beam of laser light to measure a chord length, which is defined as the distance across a particle as observed by optics collecting backscattered light from a laser crossing the particle. The number of such chords measured in a specific time period yields a chord length distribution. In this work, the chord length distribution (mass-weighted) provided from the FBRM probe is used as a characteristic crystal size distribution (CSD) of the particle. 2.3. Procedure. 2.3.1. Continuous Crystallization Experiment without Recycle. Cooling crystallization experiments were carried out using the continuous crystallization apparatus described above. A 30% (m/m) solution of amorphous cyclosporine in acetone at 53 °C was used as the feed solution. The cyclosporine solution was added to the first reactor at 0.17 mL/min flow rate using a low flow peristaltic pump. The working volume per stage was 30 mL, and the total residence time was 8 h and 50 min. The temperature of stages 1, 2, and 3 was 30, 14, and 14 °C, respectively. The total duration of the experiment was 35 h and 20 min (corresponding to four residence times). 2.3.2. Batch Crystallization Experiment. A batch cooling crystallization experiment was carried out in a 20 mL glass vial. A 30% (m/m) solution of amorphous cyclosporine in acetone at 53 °C was cooled to 30 °C and maintained at that temperature for 3 h. After 3 h the vial was cooled to 14 °C and maintained at that temperature for 6 h. Samples of the crystallized solid and the mother liquor were removed after 3, 6, and 9 h for purity and concentration measurement. 2.3.3. Continuous Crystallization Experiment with Recycle. In order to increase the yield of the process, a cooling crystallization experiment with recycle was carried out. The experiment was designed to simulate the conditions in an experiment where the outlet stream would be filtered and the mother liquor concentrated to the equilibrium concentration of the mother liquor leaving the first reactor. This recycle stream would then be added to the second reactor. To simulate these conditions, a 30% m/m solution of cyclosporine in acetone was crystallized at 30 °C in a batch process. The slurry was filtered, and the mother liquor was saved to be used as a recycle stream. A 30% (m/m) solution of amorphous cyclosporine in acetone at 53 °C was used as the feed solution. The cyclosporine solution was added to the first reactor at 0.17 mL/min flow rate using a low flow peristaltic pump. The working volume per stage was 30 mL, and the total residence time was 8 h and 20 min. The temperature of stages 1, 2, and 3 4393

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coefficient as defined in eq 1. This procedure was repeated in steps of 5 °C (i.e., the recovered mother liquor (ML 1) from the vacuum filter in the first experiment at 35 °C was used in a second experiment at 30 °C, after which that mother liquor (ML 2) was used in a third experiment at 25 °C. A fourth and fifth experiment were carried out at 20 and 15 °C, respectively, as shown in Figure 3.

3. POPULATION BALANCE MODEL Figure 2. Experimental procedure for continuous crystallization experiment with recycle.

Figure 3. Sequence of cooling crystallization experiments to determine the equilibrium distribution coefficients. was 30, 14, and 14 °C, respectively. The recycle stream concentration was 20% m/m, and the recycle stream flow rate was 0.10 mL/min. The total duration of the experiment was 33 h and 30 min. Figure 2 shows the experimental procedure for this experiment. 2.3.4. Experiments To Determine Equilibrium Distribution Coefficients. When impurities incorporate at a small level into the crystal, the incorporation can be characterized by an equilibrium distribution coefficient, DC, numerically equal to the ratio of impurity concentration to the host compound concentration in the solid phase divided by that ratio in the liquid phase. The segregation of an impurity between a liquid phase and crystalline phase at equilibrium has been previously studied.1820 Givand et al. have previously measured impurity distribution coefficients for model amino acid systems and used them to predict purity data in isomorphic systems in a common solvent.21,22 Equation 1 defines the distribution coefficient. Distribution Coeff ¼

ðCimp =CCycA Þsolid ðCimp =CCycA Þliquid

ð1Þ

3.1. Model of Multistage MSMPR without Recycle. A model of the crystallization process based on the simultaneous solution of the population balance equation (PBE) and mass balance was developed. The purpose of the model is to predict the crystal purity, yield, and size distribution of the crystals obtained in the multistage continuous crystallizer. The equilibrium distribution coefficients of the impurities and the kinetic parameters were determined experimentally and used in the model so that yield, purity, and cycle time can be optimized around the desired product quality. The multistage continuous crystallization system can be described as a series of a mixed suspension and mixed product removal (MSMPR) crystallizers, which can be modeled with population balance equations. Assuming a continuous steady state operation with negligible agglomeration or breakage, where the crystal size distribution of the product is the same as that found within the crystallizer and the feed stream is free of suspended solids in the first stage, the general population balance equations for the first three stages can be reduced to dn1 þ n1 ¼ 0 ð2Þ G1 τ 1 dL

G2 τ 2

dn2 þ n2 ¼ n1 dL

ð3Þ

G3 τ 3

dn3 þ n3 ¼ n2 dL

ð4Þ

where L is the characteristic size of the crystal, n(L) is the number density of crystals per unit volume of size between L and L + dL, G is the size-independent crystal growth rate, and τ is the mean residence time. These ordinary differential equations can be solved analytically, using the boundary condition n(0) = n0, where n0 is the crystal density of zero size nuclei. Randolph and Larson have analytically solved these ODEs.23 The analytical solution is

where Cimp is the concentration of impurity and CCycA is the concentration of the host. A sequence of cooling crystallization experiments of cyclosporine (with acetone as the solvent) were carried out to determine the equilibrium distribution coefficients of the impurities. A saturated solution of cyclosporine in acetone at 40 °C was prepared (an excess of cyclosporine was equilibrated in acetone at 40 °C for 24 h, and the suspension was filtered to obtain a saturated solution of cyclosporine. The saturated solution was cooled down to 35 °C, and once crystals of cyclosporine could be visually observed, the temperature was held constant at 35 °C for at least 24 h to ensure equilibrium. The contents of the vessel were poured through a 0.45 μm filter under vacuum. The filtrate was labeled ML 1. The wet crystals on the filter paper were washed with approximately 100 mL of cold water in order to remove any adhering mother liquor. The washed crystals were dried overnight in a vacuum oven at 35 °C and were labeled Solid 1. The relative amounts of cyclosporine A and impurities in the solid phase (Solid 1) and in the mother liquor (ML 1) were determined by HPLC analysis. These results were used to calculate the distribution

L G1 τ1 L n2 ¼ n02 exp G2 τ2 G1 τ 1 L L 0 þ n1 exp exp G1 τ1 G2 τ2 G1 τ1 G2 τ2 n1 ¼ n01 exp

ð5Þ

ð6Þ

L G2 τ 2 L L þ n02 exp exp n3 ¼ n03 exp G3 τ 3 G2 τ 2 G3 τ3 G2 τ 2 G3 τ 3 ( ) n01 G21 τ21 L L exp þ exp G1 τ 1 G3 τ3 ðG1 τ1 G2 τ2 ÞðG2 τ2 G3 τ3 Þ ( ) 0 n 1 G1 G2 τ 1 τ 2 L L exp exp G2 τ 2 G3 τ 3 ðG1 τ1 G2 τ2 ÞðG2 τ2 G3 τ3 Þ

ð7Þ 4394



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The rate at which solute is lost from the solution phase is equal to the rate at which mass is gained by the solid phase. Thus, a mass balance on the solute crystallized in each stage, assuming steady state, gives C0 = Ci + MTi, where C0 is the solute concentration at feed, Ci is the solute concentration at stage i, and MTi is the suspension density at stage i, which is calculated from the third moment of the population density Z MTi ¼ kv F L3 ni dL ð8Þ where F is the solid density, kv is a volume shape factor, and ni is the population density at stage i. The crystal growth rate and nucleation rate can be expressed as a function of supersaturation in the form of empirical power-law eqs 9, 10, 11, and 12, where Gi and Bi are the crystal growth rate and nucleation rate at stage i, respectively, Ci is the steady state concentration at stage i, Cisat is the equilibrium concentration at stage i, T is the temperature, and R is the gas constant [8.314 J/(mol K)], while kg, kg0, kg1, g, kb, and b are model parameters. g Ci Csat i Gi ¼ kg ð9Þ Csat i kg ¼ kg0 exp

kg1 RðT þ 273Þ

ð10Þ

n0i ¼

kb1 RðT þ 273Þ

ð11Þ

Bi Gi

ð12Þ ð13Þ

The volume based distribution of the steady state crystal size distribution (CSD) experimental data can be converted to population density data using the relationship nexp ¼

ΔMMT ΔLkv FL3

ð14Þ

where nexp is the experimental population density, ΔM is the mass based CSD (%), MT is the slurry density, ΔL is the size range, kv is the volume shape factor, L is the characteristic size of the crystal, and F is the crystal density. If the error is defined as the difference between the measured (experimental) and calculated values of the crystal size distribution, then a least-squares optimization procedure to estimate the kinetic parameters consists of finding the values of θ that minimize the objective function Φ(θ), defined as the sum of squares of the error, where θ is the vector of model parameters, θ = [kg0 kg1 g kb b]. The optimization problem is expressed as24,25 min ΦðθÞ ¼ θ

#stages Lmax

0

Distribution of Impurities and Crystal Purity. The distribution of the impurity between the solid and liquid phases at each stage of crystallization is determined by the equilibrium distribution coefficient (eq 1), which can be expressed as DCj ¼

ðMi, j =MA, j Þ ðCi, j =CA:j Þ

∑1 ∑0 ½ðnexp nðLÞÞ2

ð15Þ

ð17Þ

Assuming steady state, a mass balance for the impurity over a stage j has terms for the impurity coming in (Ci,j1 and Mi,j1), a term for the impurity going out in solution (Ci,j), and a term for the impurity accumulated in the crystals (Mi,j) Ci, j1 þ Mi, j1 ¼ Ci, j þ Mi, j

b Ci Csat i Bi ¼ kb Csat i kb ¼ kb0 exp

the experimental data. The value of the parameters θ is then recalculated and the procedure is repeated until a minimum value for the sum of squares of the error is found. Once the optimal values for kinetics parameters are found, the crystal growth rates and nucleation rate at steady state are calculated for each stage, using eqs 9, 10, 11 and 12. The mass based mean size of the crystals can be estimated for each stage i from the third and fourth moments of the size distribution. Z ∞ L4 ni dL 0 ð16Þ ðL4, 3 Þi ¼ Z ∞ 3 L ni dL

ð18Þ

where Ci,j1 and Mi,j1 are the concentrations of impurity in the liquid and solid phases entering stage j and Ci,j and Mi,j are the concentrations of impurity in the liquid and solid phases leaving stage j. From the experimental data shown in Figure 4, the distribution coefficient at each stage is a function of the concentration of impurity, which can be expressed as follows ! Ci, j DCj ¼ a þ b ð19Þ Ci, j þ CA, j where a and b are experimental parameters. Equations 17, 18, and 19 can be solved simultaneously to yield the concentration of impurity at stage j. Finally, the purity of the host compound is calculated as the mass fraction of the host in the solid phase. 3.2. Model of Multistage MSMPR with Recycle. The multistage MSMPR model was modified to include a recycle stream. The outlet stream from the third crystallizer is filtered and the mother liquor concentrated to the equilibrium concentration of the mother liquor leaving the first reactor. This recycle stream is then added to the second crystallizer as shown in Figure 5. F0, F1, F2, F3, F4, and F8 are the flow rates of the feed, stage 1, stage 2, stage 3, filtrate mother liquor stream, and recycle stream, respectively. Mass balance expressions described in section 3.1 were modified to represent the recycle-based arrangement shown in Figure 5. The new mass balance expressions for the impurity at each stage are

subject to eqs 512. Thus, the population balance equations for each stage, as well as the mass balances and expressions for kinetics of crystallization are solved with initial values for the parameters θ, and the obtained crystal size distribution for each stage is compared to 4395

Ci0 ¼ Ci1 þ Mi1

ð20Þ

Ci1 F1 þ Mi1 F1 þ Ci8 F8 ¼ Ci2 F2 þ Mi2 F2

ð21Þ

Ci2 F2 þ Mi2 F2 ¼ Ci3 F3 þ Mi3 F3

ð22Þ



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Figure 4. Equilibrium distribution coefficients as a function of the purity of the starting solution.

Figure 5. Schematic diagram of the multistage MSMPR system with recycle.

where Ci0, Ci1, Ci2, Ci3, and Ci8 are the impurity concentration at feed, stages 1, 2, 3, and recycle stream, respectively, and Mi1, Mi2, Mi3, and Mi8 are the mass of impurity in the solid phase per unit volume in stages 1, 2, 3 and the recycle stream, which are calculated from the equilibrium distribution coefficient at each stage. Similar mass balance expressions are used for the host. These equations are solved simultaneously with the population balances and kinetic expressions to obtain crystal purity, crystal size, and process yield.

4. RESULTS AND DISCUSSION 4.1. Continuous Crystallization Experiment without Recycle. The continuous crystallization system was able to operate

without any clogging issues for more than four residence times. The experimental yield was determined as 40%, 62%, and 71% in the first, second, and third stages, respectively. The yield in the first stage was close to the theoretical value, indicating that the steady state in vessel 1 had a low supersaturation while the values in stages 2 and 3 were much further from the theoretical value, indicating a higher supersaturation value in these vessels. The supersaturation (relative supersaturation = (c c*)/c*, where c is solute concentration in solution and c* is the equilibrium solute concentration at that temperature) in each stage, based on yield and temperature, was 0.01, 0.64, and 0.25 for stages 1, 2, and 3, respectively. The purity of the crystals obtained in the experiment was measured as 96% using HPLC. The solids obtained in the experiment were confirmed as being crystalline by XRPD. The diffraction pattern and a microimage of the crystals obtained in the experiment without recycle are shown in Figure 6. The CSD of the crystals obtained in the experiment was collected

Figure 6. X-ray diffraction pattern and a microimage of cyclosporine crystals obtained in a continuous crystallization experiment without recycle.

and used to estimate nucleation and crystal growth kinetics parameters as described in section 4.5. 4.2. Batch Crystallization Experiment. The crystallization yield after 3, 6, and 9 h was 25%, 73%, and 74%, respectively. Mother liquor samples were removed at these times to make the experiment comparable to the continuous experiment without recycle, where the residence time for each of the three stages was 2 h and 56 min. The purity of the crystals obtained in the experiment was measured as 95% using HPLC. It is interesting to compare these results with the steady state experimental yield and purity values obtained in the continuous experiment without 4396



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Table 1. Purity Values of the Feed Solution and the Crystal and Mother Liquor Samples Taken during the Distribution Coefficient Experiment

Table 2. Estimated Nucleation and Crystal Growth Kinetic Parameters parameter

value

units

sample id

cyclosporine A

impurities

kg @ 30 °C

1.19 10

m/min

feed solution

89.9%

10.1%

solid 1

96.7%

3.3%

kg @ 14 °C kg0

2.26 107 1.13 107

m/min m/min

solid 2

95.4%

4.6%

kg1

9057

J/mol

solid 3

95.0%

5.0%

kb @ 30 °C

4.08 1010

#/(m3 min)

solid 4 solid 5

93.5% 93.4%

6.5% 6.6%

kb @ 14 °C

1.12 1010

#/(m3 min)

kb0

4.80 10

#/(m3 min)

6

20

ML1

88.0%

12.0%

kb1

7.03 10

J/mol

ML2

87.2%

12.8%

g

1.33

dimensionless

ML3

86.0%

14.0%

b

1.5

dimensionless

ML4

85.6%

14.4%

ML5

84.4%

15.6%

recycle. Initially the yield in the batch experiment is lower than the steady state yield obtained in stage 1 in the continuous experiment (25% compared to 40%). The higher steady state yield could be explained due to the presence of crystals in the first stage when the feed solution enters it whereas in the batch experiment, at t = 0 min, the feed solution is not seeded. However, once crystallization has started, the yield in the batch experiment after 6 h is 73% compared to 62% at steady state in stage 2 in the continuous experiment and 74% after 9 h in the batch experiment compared to 71% at steady state in stage 3 in the continuous experiment. The equilibrium yield at 14 °C is 77%. Thus, after 6 and 9 h, the batch yield is closer to the equilibrium value while the steady state in stages 2 and 3 in the continuous experiment has a higher supersaturation. The lower purity of the crystals obtained in the batch experiment (95% compared to 96% in the continuous experiment) could be explained on the basis of the higher yield (74% compared to 71% in the continuous experiment). 4.3. Continuous Crystallization Experiment with Recycle. A mass balance was first completed to obtain the recycle ratio needed for a high theoretical yield of cyclosporine. It was determined that a recycle ratio of 0.75 would give a theoretical yield of 97%. The experimental yield in the continuous crystallization experiment with recycle was 87%. The results of this experiment confirmed that a higher yield can be reached with recycle, when compared with a nonrecycle process. The purity of the crystals obtained in the experiment was measured using HPLC as 94%. 4.4. Experiments To Determine Equilibrium Distribution Coefficients. Table 1 shows the purity values of the feed solution and the crystal and mother liquor samples taken at various stages of the experiment (see Figure 3). The purity of the crystalline solid decreases, as expected, due to the increase in concentration of impurities in the mother liquor as the sequence of cooling crystallization experiments goes on. Equilibrium distribution coefficients for the impurities were calculated by applying eq 1 and using the fractions of cyclosporine and impurities obtained experimentally. A plot of the distribution coefficient as a function of the purity of the initial solution is shown in Figure 4. The distribution coefficient increased as the purity of the initial solution decreased. As per its definition, higher values of the distribution coefficient represent the segregation of the impurity in the crystalline phase rather than the liquid phase. Thus, the experimentally obtained

3

Figure 7. Comparison of the calculated and experimental (FBRM) size distribution (mass-weighted) of cyclosporine crystals in the third stage.

distribution coefficients show that impurity uptake is proportional to the concentration of impurities in the crystallizer. These distribution coefficients can be used to predict the purity of the crystalline phase for any given concentration of impurity in solution. 4.5. Model of Multistage MSMPR without Recycle. As described in section 3.1, the MSMPR model can be used to estimate the nucleation and crystal growth rate kinetic parameters by solving the optimization problem defined in eq 15. This approach was used to obtain kinetics from the continuous crystallization experiment of cyclosporine in acetone. The results of the parameter estimation are shown in Table 2. These parameters were used in the model to predict yield values of 21%, 58%, and 68% for the first, second, and third stages, respectively, under the conditions used in the experiment. The corresponding experimental yield values are 40%, 62%, and 71%. The parameters were estimated from the experiment without recycle using the steady state crystal size distribution. The calculated size distribution using the model agrees reasonably well with the experimental size distribution, as shown in Figure 7. The nucleation and crystal growth kinetics obtained were then used to describe the continuous crystallization process and to evaluate the effect of process conditions on the crystal size. Effect of Process Conditions on Crystal Purity, Crystal Size, and Yield. The multistaged MSMPR model was used to evaluate the effect of process conditions on the purity of the crystals, the size of the crystals, and the process yield for the continuous 4397



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Figure 8. Effect of temperature of stages 1 and 3 on crystal purity (%).

Figure 9. Effect of temperature of stages 1 and 3 on process yield (%).

cooling crystallization of cyclosporine in acetone. The effects of the temperature of stages 1 and 3 were evaluated. The results are presented in Figures 8 and 9. Results show that the temperature of the third stage has a large impact on the final purity of the crystals. As the temperature of the third stage increases, the purity of the crystals also increases. The temperature of the first stage also has some influence on the purity, but the effect is less significant. In batch experiments at 35, 30, and 25 °C, the purity of the crystals was 96.7%, 95.4%, and 95.0%, respectively. The purity obtained with the continuous multistage model was 97.1%, 96.0%, and 94.3% for steady state operation, with the temperature of stages 1, 2, and 3 equal to 35, 30, and 25 °C, respectively. A comparison of Figures 8 and 9 shows that the temperature of stage 3 has an opposite effect on crystal purity and process yield. A decrease in temperature of the third stage reduces the crystal purity but improves the yield of the process at the same time. This behavior is shown in Figure 10. Also, the model predicts a decrease in mean product crystal size with increasing yield. To increase the yield predicted by the model, the temperature of stages 2 and 3 was decreased. This behavior (decreasing crystal size) suggests that the crystal growth kinetics is more sensitive to

changes (decrease) in temperature than the nucleation kinetics. In other words, the crystal growth effect decreases more than the nucleation effect when the temperature is decreased and, thus, the overall effect is a decrease in crystal size. The results of the model could be used to find out the optimal operating conditions to maximize both crystal purity and yield. The effect of increasing the residence time of the last stage (by increasing the volume) was also studied using the model. The experimental parameters were chosen such that a theoretical yield of 91% is obtained. Increasing the volume of the third stage from 30 mL (the actual working volume in the experiments) to 480 mL increased the process yield from 82% to 89%. This behavior is shown in Figure 11. 4.6. Model of Multistage MSMPR with Recycle. The effect of recycle ratio on both crystal purity and process yield was evaluated. The recycle ratio (R) is defined as the ratio of the recycled stream to the mother liquor stream. The results are presented in Figure 12. The yield of the process can be significantly improved by increasing the recycle ratio. As the recycle rate increases, the amount of solute available for crystallization is increased, which improves the overall yield. A 93% process yield value was obtained with a recycle ratio of 0.9. 4398



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Figure 10. Purity and mean size of the crystals as a function of process yield for a continuous crystallization system without recycle.

Figure 11. Effect of increasing the last stage volume (i.e., increasing residence time) on process yield (%).

Figure 12. Effect of recycle ratio on crystal purity and process yield (%).

However, as the recycle rate increases, the concentration of impurity in stage 2 also increases, which decreases the level of purification of the crystalline phase. A slight decrease in crystal purity was observed with an increasing recycle ratio, as shown in Figure 11. Thus, a compromise between yield and purity has to be made. The mathematical model becomes an excellent tool to select the desired levels of crystal purity and yield for the process. Also, the model can be used to determine the process conditions required to obtain crystals of any size.

5. CONCLUSIONS A continuous cooling crystallization process to produce the immunosuppressant drug cyclosporine with high yield and purity was developed. The multistage crystallization system can be used to perform continuous crystallization studies of pharmaceutical compounds. The continuous crystallization system was able to operate without any clogging issues for more than four residence times. The results obtained were compared with a batch experiment carried out under similar conditions, and similar values of crystal yield and purity were obtained. By using a recycle stream, the experimental yield in the continuous crystallization experiment was increased while the purity of the crystals decreased. The equilibrium distribution coefficients of cyclosporine impurities were measured experimentally as a function of impurity % of the starting solution. The distribution coefficients increase with a decrease in the purity of the starting solution, indicating that the purification gets worse. A population balance model which describes the experimental system was also developed. This model can be used to optimize crystal purity, yield, and crystal size of the product, as well as to obtain kinetic parameters for crystal growth and nucleation. The model was used to evaluate the effect of process parameters on the purity of the crystals and the process yield. Results showed that the temperature in the third stage has a large impact on the final purity of the crystals. As the temperature in the third stage 4399


Crystal Growth & Design increases, the purity of the crystals also increases. The temperature of the first stage also has some influence on the purity, but the effect is less significant. Also, an increase in temperature of the third stage improves the crystal purity but reduces the yield of the process. The effect of recycle ratio on both crystal purity and process yield was evaluated. While the yield of the process can be significantly improved by increasing the recycle ratio, the crystal purity decreased as the recycle ratio increased. Due to the increased interest in the development of continuous crystallization processes in the pharmaceutical industry, there is a need to develop robust continuous crystallization processes which produce API crystals with high yield and purity. While manufacturing in batch is still preferred in the pharmaceutical industry, the development of systems such as the one described in this work will help the industry in moving from batch to continuous manufacturing.

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’ AUTHOR INFORMATION Corresponding Author

*Mail: Professor of the Practice of Chemical Engineering, Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Building 66-568, Cambridge MA, 02139. Phone: 617-452-3790. Fax: 617-253-2072. E-mail: [email protected].

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