Palbociclib Commercial Manufacturing Process Development. Part III

Article pubs.acs.org/OPRD

Palbociclib Commercial Manufacturing Process Development. Part III. Deprotection Followed by Crystallization for API Particle Property Control Brian P. Chekal,*,† Jason Ewers,† Steven M. Guinness,† Nathan D. Ide,† Kyle R. Leeman,† Ronald J. Post,† Anil M. Rane,† Karen Sutherland,† Ke Wang,† Mark Webster,† Gregory J. Withbroe,† John Draper,‡ Denis Lynch,§ Marie McAuliffe,§ and Joseph Keane§ †

Pharmaceutical Sciences, Pfizer Inc., Eastern Point Road, Groton, Connecticut 06340, United States Pfizer Global R&D, Ramsgate Road, Sandwich, Kent, CT13 9NJ, United Kingdom § Pfizer Global Supply, Ringaskiddy, Co. Cork, Ireland ‡

ABSTRACT: A three-step commercial manufacturing route has been developed for palbociclib, a highly selective, reversible inhibitor of CDK 4/6. The third step of the palbociclib process is composed of an acid-catalyzed deprotection reaction telescoped through extractive workup and distillation operations into a controlled crystallization process. The selection of nbutanol and anisole as the cosolvents for this step allowed for the development of a robust process for each unit operation and for tight control of the API particle size.

1. INTRODUCTION Cyclin-dependent kinases 4 and 6 (CDK4/6) are key regulators of the cell cycles that trigger cellular progression from growth phase (G1) into phases associated with DNA replications (S).1 CDK 4/6, whose increased activity is frequent in estrogen receptor-positive (ER+) breast cancer (BC), are key downstream target of ER signaling in ER+ BC.2 Palbociclib, 1 (Figure 1), is a highly selective, reversible oral targeted agent

melting temperature, substantial crystallinity, and low hygroscopicity.6 An initial API process was developed to deliver the isethionate salt for use in early clinical trials. The solid form of the API was reexamined as commercial development activities were initiated for this project. The pharmaceutical sciences team decided to switch the solid form of the API from the isethionate salt to the free base to address specific issues with the isethionate salt form. From an API development prospective, the isethionate salt has a more complex solid form landscape with multiple polymorphs of the monoisethionate salt having been identified in addition to a di-isethionate salt form. In contrast, the free base has a relatively simple solid form landscape with two identified polymorphs.7 From a drug product development perspective, the clinical dosage form with the isethionate salt had a significant API sticking issue that needed to be resolved to allow for commercial-scale manufacturing. A switch to the free base form of the API resulted in a reduction in the propensity of API sticking in the drug product manufacturing process, which facilitated the commercial-scale manufacturing of the drug product. With this switch in the API solid form for commercialization, development activities started on a new final step to deliver the free base form of palbociclib. These activities focused on developing a single step process that incorporates both the two chemical conversions of the penultimate intermediate (5) to palbociclib (1) and the workup and crystallization unit operations to deliver the desired API chemical and physical quality attributes. As the final step in the manufacturing process, multiple chemical and physical quality attributes of the API were evaluated to ensure that the isolated product would meet all quality requirements. The physical quality attributes of palbociclib and their influence on drug product manufactur-

Figure 1. Palbociclib.

that selectively inhibits CDK 4/6 to regain cell cycle control and block tumor cell proliferation.3 The overall chemistry scheme for manufacturing palbociclib is shown in Figure 2. Following up on the previous two parts in this series, which describe the development of the SNAr coupling mediated by the Grignard base in the first step4 and the installation of the side chain by Heck coupling in the second step,5 the development of the final API step is described here. For the initial development of palbociclib, an isethionate salt was selected as the API solid form. This salt was selected primarily due its high aqueous solubility. The isethionate salt also had advantageous solid form properties including high © XXXX American Chemical Society

Received: March 4, 2016

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DOI: 10.1021/acs.oprd.6b00071 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

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Figure 2. Commercial manufacturing process for palbociclib.

ability and bioperformance was an area of particular emphasis in this research. As palbociclib is a BCS Class II drug with low solubility and high permeability, the API particle size impacts the rate of dissolution of API in the gastrointestinal tract, resulting in a desire to minimize the API particle size. For palbociclib, this drive toward smaller API particle size for bioperformance needed to be balanced against API sticking concerns in the drug product manufacturing process. While the switch from the isethionate salt to the free base form reduced the level of API sticking observed during drug product manufacturing, API sticking remained a concern for palbociclib free base. As sticking is a surface area dependent phenomenon, a decrease in the API particle size will further increase the level of sticking. Due to these conflicting constraints from bioperformance needs and drug product manufacturability, particle engineering approaches were utilized to control the API particle size to a tight range that delivered the desired bioperformance without causing a significant sticking issue. Figure 3. SEM image (1500× magnification) of palbociclib isolated from salt break procedure.

2. DISCUSSION AND RESULTS 2.1. Overview of Process Design. An overall goal for the final step of the palbociclib process is to complete both the chemical conversion of the penultimate intermediate to the API and the controlled crystallization of API to deliver API with the desired particle size and physical properties. Previous development work demonstrated that the conversion of 5 to 1 occurred readily at low pH levels in aqueous solvent mixtures using a variety of strong acids. Initial studies completed as part of the commercial API process development isolated the free base form of the palbociclib by pH adjusting the aqueous reaction mixture using an aqueous base after the reaction was complete. This pH adjustment process resulted in the formation of small particle size API (typical size-scale of 1−5 μm) due to the rapid crystallization caused by the dramatic change in solubility with adjustment of the pH. The API produced by this crystallization method exhibited a high degree of sticking and was highly static prone. The small primary particles also tended to form large, hard agglomerates (typical size scale of 200−1000 μm) that were difficult to disperse by sieving.7 A representative scanning electron microscope (SEM) image of the API produced by this process is shown in Figure 3. Due to these poor API physical properties, palbociclib produced by this method was not considered suitable for further development. To address the poor physical properties of the API produced by the salt break procedure, a recrystallization process was developed. The initial challenge in developing this recrystallization process was identifying a solvent system which yields sufficient API solubility to allow for reasonable solvent volumes.

Preliminary room temperature solubility screening of a diverse set of 38 organic solvents indicated less than 1 mg/mL solubility in almost all of the solvents tested. Only three solvents tested, dichloroethane, 1,4-dioxane, and N-methyl-2pyrrolidone (NMP), resulted in solubility values in the range of 1−3 mg/mL; however, all three solvents are not preferred solvents for the development of a final API crystallization process, due to toxicity issues. To examine the potential of finding a recrystallization solvent system, a pair of follow-up solubility and recrystallization screens was run. In the first high temperature screening study, 50 mg/mL samples of palbociclib were prepared in 14 highboiling solvents and placed into sealed vials. The contents of these vials were then heated to reflux, and visual observation identified the samples that went into solution. For the samples that did completely dissolve, the samples were allowed to cool to room temperature without stirring, and photomicroscopy then was used to characterize the particles produced. The results of this screen study are in listed in Table 1. Based on these small-scale crystallization studies, anisole became the focus of additional crystallization and solubility studies because the particles produced were large. In addition, as anisole is an ICH Class III solvent, control of the residual solvent level is less stringent. Anisole is also ranked as a recommended solvent based on its environmental, health, and safety impacts.8 This screening study also identified pyridine, m-xylene, and mesitylene as potential solvent systems, although B



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While the solubility results from the NMP and NMP/nbutanol systems appeared promising, subsequent UPLC/MS testing of the saturated solution from experiment nos. 3 and 11 in the table showed the presence of a previously unseen impurity peak. These results indicated that degradation occurred in these experiments, suggesting a potential stability issue with the use of NMP as a recrystallization solvent. Although the propylene glycol/n-butanol/anisole mixtures showed improved solubility as compared to the n-butanol/ anisole mixtures, the former solvent system was not pursued because of the potential challenges of working with propylene glycol in a manufacturing environment due to its high viscosity and boiling point. A follow-up solubility study examined nbutanol/anisole mixtures with 30%, 40%, 50%, and 60% nbutanol in order to better define the location of maximum synergetic solubility. This follow-up study showed that the maximum solubility occurred at 40% n-butanol/anisole. As such, the 40% n-butanol/60% anisole solvent mixture was selected as the recrystallization solvent system. Using this solvent mixture, a recrystallization procedure was developed where palbociclib is dissolved with 40 mL/g of solvent (concentration of 25 mg/mL) by heating to 95−100 °C before being crystallized using a controlled cooling profile with seeding to induce nucleation. This recrystallization process resulted in the isolation of palbociclib particles with a larger primary particle size, which also are not static prone and do not form agglomerates. A polarized light microscopy (PLM) image of particles produced by this process is shown in Figure 4.

Table 1. Summary of Results from Preliminary Recrystallization Screening Study solvent

results

cyclopentylmethyl ether n-butyl acetate n-butanol trifluorotoluene toluene chlorobenzene DMF NMP propylene glycol anisole pyridine sulfolane m-xylene mesitylene

did not dissolve did not dissolve did not dissolve did not dissolve did not dissolve recrystallization results in small irregular shaped particles recrystallization results in small needle shaped particles recrystallization results in small irregular shaped particles recrystallization results in small irregular shaped particles recrystallization results in large particles (lathes or tomahawk shape) recrystallization results in small lathe shaped particles recrystallization results in small irregular shaped particles recrystallization results in small/medium tomahawk shaped particles recrystallization results in small needle shaped particles

none of these solvents also have the ICH class III listing or EHS ranking similar to anisole. As a follow-up to these initial small-scale crystallization studies, targeted higher temperature solubility studies were conducted. A set of 16 solvent systems were examined at a fixed palbociclib concentration of 25 mg/mL. In this study, the dissolution temperature was measured using a kinetic solubility method up to a maximum temperature of 110 °C. Synergistic solubility behavior as predicted by a COSMOtherm solubility model9 of palbociclib was used to select the binary and ternary solvent systems included in this screening study. The results of these studies are listed in Table 2. For experiments listed as “no solution” in the table, the solids did not dissolve in the solvent upon heating to 110 °C, indicating that the solubility of palbociclib is less than 25 mg/mL at 110 °C in this solvent. Table 2. Kinetic Solubility Measurements of Dissolution Temperature for 25 mg/mL Palbociclib Suspensions experiment no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

solvent n-BuOH DMF NMP DMSO DMAc n-butyl acetate anisole 10% n-BuOH/anisole (v/v) 20% n-BuOH/anisole (v/v) 40% n-BuOH/anisole (v/v) 10% n-BuOH/NMP (v/v) 25% n-BuOH/NMP (v/v) 10% 1,4-butanediol/anisole (v/v) 25% 1,4-butanediol/anisole(v/v) 1:1:8 propylene glycol/n-BuOH/ anisole (v/v) 2:1:7 propylene glycol/n-BuOH/ anisole (v/v)

dissolution temperature (°C) no solution no solution 97.9 no solution no solution no solution no solution no solution 109.7 101.4 103.7 no solution 109.8 104.8 91.2

Figure 4. PLM image (100× magnification) of palbociclib isolated from anisole/n-butanol recrystallization process.

With the development of a recrystallization process, the next objective was the integration of the reaction and recrystallization operations into a single step to allow for an efficient process for producing API. An integrated process was proposed where the reaction to convert 5 to 1 is carried out in an aqueous solvent system under acidic conditions, followed by the addition of the 40% n-butanol/anisole organic solvent mixture. The neutralization of the final reaction mixture is then completed under biphasic conditions at elevated temperature, which allowed for palbociclib free base to remain in solution. Phase break and subsequent water wash of the organic phase removed any byproduct salts formed during neutralization.

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at low pH in aqueous solvent mixtures using a variety of strong acids. Screening studies examined a variety of strong acids and solvent systems to complete this conversion. In addition to the isethionic acid that was previously used for this reaction, these studies identified a variety of strong acids as suitable for this reaction including methanesulfonic acid, hydrochloric acid, and sulfuric acid. Based on the results of these screening studies, hydrochloric acid was selected as the reagent for this step as it is considered the simplest option. Next, a range of aqueous/ organic solvent mixtures were screened and found to be suitable. It is possible to use water for this reaction; however, the mixing behavior of the reaction was not favorable for scaleup due to the poor wetting characteristics of 5 in water. The poor wetting characteristic is improved with the addition of 10% or more of an organic cosolvent. As part of integrating the crystallization operation with reaction portion of the step, additional reaction screening studies examined the use of nbutanol/water mixtures for this reaction. As n-butanol has limited water miscibility in water (approximately 8 wt % nbutanol in water at 20 °C), initial reaction studies examined 10:1 to 12:1 (v/v) ratios of water/n-butanol to keep the solvent system as single phase. In order to further simplify the overall process, additional studies examined adding upfront to the reaction the full amount of n-butanol (15.5 mL/g) required for the crystallization of the free-base. This process change would reduce the number of n-butanol charges, but could potentially slow the reaction due to formation of a biphasic mixture due to the ratio of water to n-butanol. These subsequent reaction studies showed no change to the reaction profile with the charge of additional n-butanol, indicating that the presence of a separate organic phase does not impact the rate of the acid catalyzed BOC deprotection. Process optimization and robustness studies examined a variety of parameters that may impact the rate of this reaction and the purity of the product. These studies examined a range of water volumes, reaction temperature, equivalents of HCl, HCl addition time, and agitation rate during the reaction. The volume of n-butanol charged to the reaction mixture was not examined as part of this study as previous process development studies showed no impact of additional n-butanol on the reaction rate. Since the reaction is conducted as a biphasic mixture (two liquid phases), the potential existed for the intensity of mixing to be factor influencing the reaction performance. In order to ensure the mixing hydrodynamics in the laboratory equipment would be a suitable predictor for equipment that could be used at manufacturing scale, we utilized commercial software that calculates mixing intensity based on the physical system properties as well as vessel geometric parameters. A number of various markers for mixing intensity may be used (maximum energy dissipation rate, tip speed, maximum shear rate, micro or macro mixing times, etc.), the selection of which depends upon the system under study. For this reaction step, the maximum energy dissipation rate was selected as the appropriate parameter for scaling. These factors were translated into agitation speed variations for inclusion in the designed experiments. The results of the process optimization studies showed that reaction temperature was the only process parameter that had an impact on the reaction rate and that no parameter impacted the purity of the reaction mixture at the end of the reaction. The degree of dependence of reaction rate on reaction temperature was unexpectedly strong with observed reaction completion times increasing from approximately 1 h at 80 °C to 4 h at 70 °C to 240 h at 50 °C.

Controlled cooling of the organic solution resulted in the crystallization of palbociclib with the desired particle properties. Early in the development of this integrated process, it was noted that the organic phase retained approximately 5 wt % water and that this level of water had a significant impact on the solubility of palbociclib. Figure 5 illustrates the difference in the

Figure 5. Temperature-dependent solubility of palbociclib in nbutanol/anisole mixtures.

temperature-dependent solubility of palbociclib in n-butanol/ anisole solvent mixtures. The temperature-dependent solubility of palbociclib is essentially unchanged if the ratio of n-butanol to anisole is adjusted between 30% and 40% (v/v) n-butanol, while the solubility greatly increases if the 40% n-butanol/ anisole mixture is saturated with water at room temperature. Therefore, a final addition to the process was made with the introduction of a distillation operation prior to crystallization in order to reduce the water content of the solvent mixture and thereby reduce the solubility of palbociclib. With the design phase of developing the final step of the API process completed, individual units of work were completed to examine the robustness and to optimize the individual unit operations in this step. 2.2. Reaction. Figure 6 depicts the reaction of penultimate intermediate (5) to palbociclib (1) in detail. Early API process

Figure 6. Detailed reaction scheme.

development studies indicated that the hydrolysis of the enol ether occurred extremely rapidly under acid conditions as compared to the removal of the tert-butyloxycarbonyl (BOC) protecting group. Therefore, analytical techniques developed for following this reaction looked for the consumption of the in situ intermediate, 6. Early development work on the API synthetic route demonstrated that the conversion of 5 to 1 occurred readily D



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Figure 7. Measured off-gas rates for CO2 and isobutylene during reaction.

understanding of the rate and amount of its formation was a critical part of the development of this reaction. Detailed studies utilized a mass spectrometer to quantify the rate of gas flow from this BOC removal reaction.11 Reaction conditions that produced the fastest overall rate of reaction were examined in order to provide a worst-case scenario for gas generation. With these conditions that allowed for complete conversion of 6 to 1 in approximately 1 h, the maximum rate of isobutylene release was measured as 0.05 L/min/mol of 6. The total amount of isobutylene released was measured as 5 L/mol of 6. It is hypothesized that the substoichiometric amount of isobutylene released is due to a portion of tert-butyl cation

The observed independence of reaction rate on equivalents of HCl is in contrast to the second-order dependence noted previously for a BOC removal reaction.10 Additional process development studies examined the rate of off-gassing during the reaction. The removal of the BOC protecting group may liberate an equivalent of carbon dioxide and an equivalent of a highly reactive tert-butyl cation. For this reaction, the tert-butyl cation can decompose to form isobutylene, or it can react with water or n-butanol solvents to form tert-butyl alcohol or n-butyl t-butyl ether, respectively.10 As the generation and release of highly flammable isobutylene is a concern from a process safety and environmental perspective, E



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being trapped as t-butanol or t-butyl n-butyl ether. The same reaction conditions released carbon dioxide at a maximum rate of 1.2 L/min/mol of 6, with a total amount of 17 L/mol of 6. Figure 7 depicts the measured off-gas rate for isobutylene and carbon dioxide from this experiment. (Note that the rate of isobutylene off-gas does not return to the baseline level at the end of the experiment due to some interference in the mass spectrometer method from the n-butanol solvent vapor.) The measured peak flow and total release of isobutylene were within the safe operating limits for the large-scale manufacturing equipment based on the process venting system design. Therefore, the process was considered suitable for scale-up. 2.3. Neutralization and Extractive Workup. A neutralization and aqueous extractive workup process was developed to neutralize the in situ HCl salt formed during the reaction and to remove the inorganic byproducts of neutralization. Early in development of the free base form of palbociclib, sodium hydroxide was selected as the base for the neutralization of the in situ salt. The neutralization and workup operations are completed at elevated temperatures so that the liberated palbociclib free base remains in solution. The phase separation rate and quality of the phase split from this extractive workup was an initial concern during the design of the process as anisole has a room temperature density (0.995 g/mL) that is quite close to water. As part of solubility studies of the solvent mixture used for this extractive workup, liquid−liquid equilibrium (LLE) data was measured for the anisole/nbutanol/water system. As anisole is not a common solvent used for process chemistry, this type of data was not available in the literature. For this LLE study, the water and anisole composition of the organic and aqueous phase of samples was measured via Karl Fischer titration (KF) and UPLC after the sample was stirred overnight. The initial sample was prepared with a 50% anisole, 25% n-butanol, and 25% water (w/w) to mimic solvent volumes used for the process. The measurements indicate that organic phase contained 66% anisole, 30% n-butanol, and 4% water (w/w), while analysis of the aqueous phase indicates this phase contains approximately 0.5% anisole, 3% n-butanol, and 96.5% water. As the less dense n-butanol partitions primarily to the organic phase, this leads to a significant difference in the density of the organic and aqueous phases which facilitates rapid phase splits. Subsequent measurement of the density of each phase indicated that the density of the organic phase is 0.888 g/mL, while the density of the aqueous phase is 0.989 g/mL. Optimization studies examined the impact of the neutralization and extractive workup process parameters on the processing performance and quality of palbociclib produced. Parameters examined included the stoichiometry of sodium hydroxide, the volumes of water, and n-butanol used for the reaction, the volume of anisole added prior to the neutralization, and the phase separation temperature. An examination of the impact of these process parameters on the rate of phase separation and the quality of phase separation showed that poor phase separations could occur at 70 °C if the stoichiometry of sodium hydroxide to hydrochloric acid was 1:1 leading to a pH value below 9 after the neutralization. For experiments where an excess of sodium hydroxide was used, resulting in pH values above 9, a fast and clear phase separation was noted across the range of temperatures examined. Examples of poor and clean phase separations are shown in Figure 8. Analytical testing of the organic phase at the end of the neutralization and workup procedure for each of the DoE

Figure 8. Phase split quality at minimum NaOH:HCl ratio. Left: Result from phase separation conducted at 70 °C (after 21 min, poor phase separation). Right: Result from phase separation conducted at 75 °C (after 30 s, clean separation).

studies showed no dependence of the purity of this phase on any of the process parameters examined, indicating a robust operating space for this process. No negative impact on the purity of the organic phase was noted, even for the conditions that lead to a poor phase split. 2.4. Distillation. The role of the distillation process in the final step for the manufacture of palbociclib is to reduce the water content of the organic phase in order to decrease the solubility of API. Due to the high normal boiling points of anisole and n-butanol (154 and 118 °C, respectively), initial distillation studies examined partial vacuum distillation. In separate experiments, distilling at reduced pressures of 450 mbar and 500 mbar was undertaken with boiling points at 75 and 77 °C observed, respectively. Each experiment was distilled to a final pot temperature of 100 °C. The product isolated from these experiments showed the typical purity profile and typical yield. Additional experiments used an atmospheric distillation process to avoid any processing complexity caused by needing to control the level of vacuum during the distillation process. These studies identified that the system first began to reflux at 92 °C, which is close to the binary azeotrope noted for nbutanol and water (93 °C) and anisole and water (96 °C).12 Continuing to a distillation end temperature of 120 °C resulted in removal of approximately 6 L/kg of distillate, which formed two phases in the receiver. Analysis of the mother liquor by KF showed the water content was reduced to approximately 0.5 wt % by atmospheric distillation. UPLC analysis of the mother liquor also did not show the presence of any new impurities indicating that the system is thermally stable even at an elevated temperature of 120 °C. Additional stressing experiments were run holding the system for extended time at the distillation end point to look for the formation of any impurities. Analysis of samples pulled from this stressing experiment showed that the atmospheric distillation process is chemically stable. A subsequent robustness DoE study examined a range of distillation end temperatures of 110 to 130 °C. Statistical analysis of the results from this DoE study indicated that the distillation end temperature had an impact on the volume of organic distillate removed. The analysis also showed that the water content of the mother liquor depended on the distillation end temperature. Increasing the distillation end temperature from 110 to 130 °C corresponds with a decrease in mother F



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and anisole solvents charged, as well as the phase separation and distillation temperatures, which all may impact the crystallization solvent composition and solvent volume. A Resolution III fractional factorial design (212−8) was executed to efficiently examine this large number of process parameters. This screening design allowed for the evaluation of 12 parameters in 20 experiments, which included four replicate experiments to examine the reproducibility. Analysis of the results from this screening DoE study allowed for process parameters that showed no impact on the measured responses to be excluded from follow-up DoE studies. The DoE analysis indicated that the phase separation temperature had no impact on the solvent composition of the organic phase prior to distillation. Consistent with this observation, analysis of the measured API quality attributes showed no dependence on the phase separation temperature. The distillation end temperature was shown to impact the mother liquor solvent composition but had no impact on the measured quality attributes of the API (particle size, purity, or assay). The screening DoE study indicated that the particle size averages of the API are impacted by the seed amount, seed size, seeding temperature, hold time after seeding, and isolation temperature. All of the observed dependencies of particle size on crystallization process parameters were consistent with expected crystallization behavior, with larger particle size averages observed with larger seed, lower seed charges, and longer hold time after seeding. A follow-up DoE study was designed to examine more closely the impact of six process parameters identified as having an impact on quality attributes in the initial screening DoE study. Table 3 outlines the parameters examined and the ranges

liquor water content from 0.9 wt % to 0.2 wt %. The distillation end temperature also impacts the anisole/n-butanol ratio in the mother liquor, with higher anisole content noted as the distillation end temperature is increased. (illustrated in Figure 9). Although the solvent composition and total volume of

Figure 9. DoE model predictions for anisole content of the mother liquor.

solvent are impacted by the distillation end temperature, analysis of the API particle size results from this DoE study showed no dependence on the distillation end temperature. 2.5. Crystallization. For the final step of the palbociclib API process, development of the crystallization unit operation was considered most important, as control of the particle size distribution was critical for assuring both performance and manufacturability of the drug product. A narrow range for API particle size was targeted to balance the need to ensure rapid API dissolution in vivo with the need to minimize API sticking during the drug product manufacturing process. Biopharmaceutics studies indicated that the API particle size needed to be below an upper limit to ensure bioavailability of the drug. Drug product manufacturing studies for the selected capsule formulation of palbociclib indicated that API sticking becomes a more significant issue as the API particle size is reduced. Based on these overlapping constraints, a range of 10−45 μm was targeted for the D(50) particle size average. In addition to setting a narrow target range for the D(50) particle size average, the span of the particle size distribution, which is defined in eq 1, was targeted to a value below 3.0 in order to constrain the breadth of particle size distribution. Additional quality attributes analyzed during the development of the crystallization process included the assay, and total purity of palbociclib and the level of specific impurities. span =

D(90) − D(10) D(50)

Table 3. Crystallization Process Parameters Examined in DOptimal DoE Study crystallization process parameter

range explored

seed amount seed D[4,3] particle size seeding temperature hold time after seeding cooling rate isolation temperature

0.1−5.0 wt % 3−30 μm 80−100 °C 1−4 h 0.1−0.5 °C/min 0−20 °C

explored in this second DoE study. For this study, a D-optimal design was selected to most efficiently examine the desired experimental space. A D-optimal design is a computergenerated design with an objective function minimizing the variance of parameter estimates for a prespecified model, and is often used as an alternative to traditional factorial designs when the experiment space is subject to constraints.13 In this study, a constrained experimental space was selected for the seed amount and seed particle size process parameters in order to focus on experimental regions where the expected particle size results are close to the desired targeted range. The impact of specific impurities or the total purity of the organic phase on the particle size and other API quality attributes was not examined in this study as previous development experience and robustness studies had shown that the impurity levels have been extremely consistent after the aqueous washes. A total of 27 experiments based on D-optimal design were generated to evaluate all main effects and all two-factor interactions for the six parameters. Figure 10 depicts the design points over the constrained space of seed amount and seed particle size.

(1)

An initial assessment of the crystallization process identified 12 process parameters that could impact the quality attributes of palbociclib. In addition to standard crystallization related process parameters such as seeding temperature and cooling rate, the assessment process identified the volume of n-butanol G



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Analysis of the measured span of the particle size distribution for the experiments run as part of the D-Optimal DoE study indicated that span has a strong dependence on the seed size and hold time postseeding and only a weak dependence on the seeding temperature. Figure 12 depicts the dependence of the

Figure 10. Summary of experimental space investigated in D-optimal DoE study for seed amount and seed size parameters. The number next to markers indicates the number of replicates run with that combination of parameter values.

Analysis of the results from the D-optimal DoE study indicated that the API particle size averages showed a dependence on three of the parameters examined: seed amount, seed size, and seeding temperature. Similar to the results from the previous DoE study, particle size averages increased with larger seed particle size and with a decrease in the amount of seed added. The D-optimal DoE study indicated a strong dependence of the API particle size average with the seeding temperature. The analysis of the DoE data showed an increase in the particle size averages with an increase in the seeding temperature as shown for the D[4,3] average in Figure 11. This dependence of the particle size on seeding temperature suggests that secondary nucleation at the seeding temperature plays an important role in determining the final API particle size distribution. As such, seeding at an elevated temperature would reduce the population of secondary nuclei formed, thereby allowing each particle to grow to a larger size. Unfortunately, direct observation of the particle size distributions over the course of this crystallization was not possible due to the prolonged exposure to temperatures above 90 °C, which is the maximum safe operating temperature rating for the FBRM equipment. Comparing the impact of the various process parameters on the API particle size, it is interesting to note that an increase in seeding temperature from 80 to 100 °C has a comparable impact as an increase in the seed particle size from 3 to 30 μm.

Figure 12. DoE model predictions for span of the particle size distribution at a seeding temperature of 90 °C.

span values on the hold time postseeding and on the seed particle size. The observed dependence of the span value on the hold time postseeding is logical with lower span values observed with increase hold time at an elevated temperature. It is interesting to note that the hold time postseeding was not found to be a statically significant process parameter for any of the individual particle size averages used to calculate the span. Similar statistical analysis of the assay, total purity, and individual impurities tracked through the final step of the API process showed that these measured quality attributes have no dependence on any of the process parameters examined in this D-optimal DoE study. A set of five confirmatory experiments were run to verify the particle size predictions from the D-Optimal DoE statistical model. Comparison of the measured particle size averages with the predicted values shows close agreement with absolute differences ranging between 1 and 6 μm for the D[4,3] average. The results of the confirmatory experiments demonstrated that the statistical model describes the particle size output of the palbociclib crystallization process adequately. Based on models

Figure 11. DoE model predictions for D[4,3] particle size at (A) seeding temperature of 80 °C and (B) seeding temperature of 100 °C. H



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potential for attrition during agitated drying, so continuous agitation could be used during the drying operation if needed.

for the particle size averages and the span of the particle size distribution, a desired operating space for the crystallization process was identified. More than 30 pilot-plant and commercial manufacturing scale batches of palbociclib have been manufactured with a D(50) particle size in the range of 12−14 μm by controlling the appropriate process parameters. 2.6. Final IsolationFiltration, Washing, and Drying. A final unit of work was completed to examine the impact of the filtration, washing, and drying process parameters on the quality of palbociclib. Specifically, these studies examined the impact of the process parameters from these three unit operations on the level of residual solvent in the final API. As both anisole and n-butanol are high boiling solvents, the removal of residual solvent via a standard vacuum drying operation is challenging. Early in the development of the anisole/n-butanol recrystallization process for palbociclib, nheptane was identified as a good wash solvent for reducing the level of residual solvents. The selection of n-heptane as wash solvent was based on several factors including low API solubility, miscibility with anisole and n-butanol, and ICH Q3C Class III status. Early crystallization studies that used a copious n-heptane wash showed low levels of residual solvent after drying for less than 2 h in a 50 °C vacuum oven. These results suggested that n-heptane is a good wash solvent for removal of residual anisole and n-butanol. Process robustness studies examined the impact of filtration, washing, and drying process parameters on the residual anisole, n-butanol, and n-heptane levels. The parameters investigated in these studies, along with the ranges explored are listed in Table 4. In these series of studies, no impact on the level of residual

3. CONCLUSIONS A robust process for producing palbociclib has been developed as part of the commercial manufacturing route for this API. The process combines an efficient acid catalyzed reaction for converting the final isolated intermediate to palbociclib with a crystallization process for controlling the physical properties of the API. After solubility screening studies identified anisole/nbutanol mixture as a potential crystallization solvent system, a cooling crystallization process was developed that delivered palbociclib with the desired particle size to facilitate drug product development. Process robustness studies identified broad parameter ranges for the reaction, neutralization, distillation, and final isolation operations. Control of appropriate process parameters allows for the manufacture of palbociclib within the narrow targeted range of particle size. Using the developed process, more than 30 pilot-plant and commercial manufacturing scale batches of palbociclib have been manufactured with a D(50) particle size in the range of 12−14 μm. 4. EXPERIMENTAL SECTION Reactions were monitored by reverse phase UPLC. UPLC conditions: BEH Shield RP18, 2.1 × 100 mm, 1.7 μm, 45 °C, flow 0.4 mL/min; λ = 295 nm, 2 μL injection volume; A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile. Gradienthold at 5% B for 1 min, then to 95% B in 7 min, hold at 95% B for 1 min, re-equilibrate to 5% B in 0.1 min, with a total run time of 12 min. Sample diluent: 60:40:0.05 DI water−acetonitrile−perchloric acid. 4.1. Mass Spec/Off Gas Conditions. For experiments where the off gas monitoring was conducted, the experimental conditions provided above were followed at approximately 150 mL scale in an automated Systag FlexyCube reactor equipped with a flat glass baffle and vent condenser. For these experiments, reaction conditions were selected that would generate the most off gas (80 °C and 6 equiv of HCl) to represent the worst case set of conditions. A pressurized argon cylinder was used to supply the inert carrier gas at the desired flow rate to ensure the headspace was swept into the connection line for the mass spectrometer. The argon was supplied to the reactor headspace via a needle and septa from the cylinder via a mass flow controller (Matheson model 8270) and 1/8″ I.D. PTFE tubing. The vent line consisted of a vapor condenser connected to a 1/8″ I.D. PTFE tubing connection to a tee connection. The mass spectrometer (Pfeiffer Vacuum OmniStar GSD300) capillary was secured to the side port of the tee via a septa, while the other side of the tee was vented into the lab hood via a sufficiently long piece of tubing to ensure atmospheric air would not be drawn into the mass spec line and compromise the ability to sample from the reactor headspace. This set up created a short delay from reaction initiation and sensing at the mass spec due to the volumes created via reactor headspace and the holdup of the tubing used. The NIST solvent webbook (http://webbook.nist.gov/) or other references can be used to determine the most appropriate ion channel for evaluation of the off gas. For our studies, 41 m/z was used for isobutylene, and 44 m/z was used for carbon dioxide.

Table 4. Filtration, Wash, and Drying Parameters Examined crystallization process parameter

range explored

filtration pressure n-heptane wash volume number of n-heptane washes drying temperature drying time

0.4−1.2 bar 1−7 L/kg 1−3 50−90 °C 2−24 h

solvents was observed for any of the parameters examined across the explored ranges. These results indicated that the removal of residual solvents from this crystallization system is facile, even with a small volume of n-heptane wash. Results from the study showed that the level of residual anisole and nbutanol reached a final level after the n-heptane washes, and the level of these solvents was unchanged by vacuum drying even after 24 h at 90 °C. These same results demonstrated that at least 2 h of vacuum drying operation is necessary to reduce the level of residual n-heptane. Additional studies of the vacuum drying operations assessed the potential physical impact of agitation during drying, as large-scale clinical and commercial batches of palbociclib were manufactured using an agitated filter-dryer. These studies used lab-scale tools that were developed to assess the impact of agitated filter drying on the particle properties of APIs and intermediates.14 An examination of the impact of agitation during the initial deliquoring of the n-heptane wet cake showed a low potential for the agglomerates at residual n-heptane levels up to 50 wt %. A second study focused on the potential for attrition of palbociclib particles with prolonged agitation during drying. This lab-scale study showed that palbociclib has a low I



Article

4.1.1. Palbociclib (1). To a reactor was added water (200 mL, 10 vol), 5 (20 g, 33.1 mmol, 1.0 equiv), and n-butanol (230 mL, 11.5 vol). The mixture was heated to 70 °C, and hydrochloric acid solution (36 wt %, 16.2 g, 5.0 equiv) was added. The mixture was stirred for 4 h. Upon reaction completion, anisole (360 mL, 18 vol) was added followed by sodium hydroxide solution (50 wt %, 14.3 g, 5.4 equiv), and the mixture was stirred for 30 min. The aqueous phase was removed, and the organic phase was washed twice with water (200 mL, 10 vol) before being distilled atmospherically to an end temperature of 120 °C. The solution was cooled to 80 °C and then seeded with 1 (0.10 g, 0.007 equiv). The slurry was held at 80 °C and then cooled to 10 °C. The solids were filtered and washed twice with n-heptane (80 mL, 4 vol), then dried at 50 °C to give 14.8 g (84.3% yield) of 1. Solid state characterization data of 1 is available in ref 7.

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(6) Beylin, B. G.; Blackburn, A. C.; Erdman, D. T.; Toogood, P. L. A preparation of isethionate salts of pyridopyrimidine derivative, useful as CDK4 inhibitors. U.S. Patent 7,345,171, Mar. 18, 2008. (7) Chekal, B. P.; Ide, N. D. Solid Forms of a Selective CDK4/6 Inhibitor. U.S. Pat. Appl. 61767761, 2013. (8) Prat, D.; Wells, A.; Hayler, J.; Sneddon, H.; McElroy, R.; AbouShehada, S.; Dunn, P. Green Chem. 2016, 18, 288−296. (9) Loschen, C.; Klamt, A. J. Pharm. Pharmacol. 2015, 67, 803−811. (10) Ashworth, I. W.; Cox, B. G.; Meyrick, B. J. Org. Chem. 2010, 75, 8117−8125. (11) Dias, E. L.; Hettenbach, K. W.; am Ende, D. J. Org. Process Res. Dev. 2005, 9, 39−44. (12) McConville, F. X. The Pilot-Plant Real Book; FXM Engineering and Design: Worcester, MA, 2004; pp 6−31. (13) Myers, R. H.; Montgomery, D. C. Response Surface Methodology: process and product optimization using designed experiments; Wiley Series in Probability and Statistics; Wiley: New York, 1995; pp 364−394. (14) am Ende, D. J.; Birch, M.; Brenek, S. J.; Maloney, M. T. Org. Process Res. Dev. 2013, 17, 1345−1358.

AUTHOR INFORMATION

Corresponding Author

*E-mail: brian.p.chekal@pfizer.com. Present Address

N.D.I.: AbbVie, 1400 Sheridan Road, North Chicago, IL 60064. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the contributions of Jason Mustakis and Yuriy Abramov for the computational solubility predictions of palbociclib in binary solvent systems that facilitated selection of solvents systems for screening studies. We also acknowledge the contributions from Dave am Ende for assistance with the analysis of the reaction off-gas analysis and from Rich Barnhart for the process safety support. The authors also thank our drug product development colleagues, Matt Mullarney, John Kresevic, and Mary am Ende, for their contributions and collaboration on this project. The dedicated efforts of the manufacturing staff at Sandwich Process Development Facility and at the Ringaskiddy Small Equipment Group is also appreciated.

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REFERENCES

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Palbociclib Commercial Manufacturing Process Development. Part III

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