Raman Spectroscopy for Monitoring the Continuous Crystallization of

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Raman Spectroscopy for Monitoring the Continuous Crystallization of Carbamazepine David Acevedo, Xiaochuan Yang, ADIL MOHAMMAD, Naresh Pavurala, Wei-Lee Wu, Thomas F. O'Connor, Zoltan K Nagy, and Celia N. Cruz Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.7b00322 • Publication Date (Web): 29 Dec 2017 Downloaded from http://pubs.acs.org on January 1, 2018

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Organic Process Research & Development

Raman Spectroscopy for Monitoring the Continuous Crystallization of Carbamazepine David Acevedoa, Xiaochuan Yanga*, Adil Mohammada, Naresh Pavuralaa, Wei-Lee Wua, Thomas F. O’Connora, Zoltan K. Nagyb, Celia N. Cruza a

Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland 20993-0002

b Davidson School of Chemical Engineering, Purdue University, West Lafayette, Indiana 47907 *[email protected] **This publication only reflects the views of the authors and should not be construed to represent FDA’s views or policies.

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Abstract Crystallization has a significant impact on the properties of the active pharmaceutical ingredient (API) since it is the final step in the manufacturing of the drug substance and determines particle size distribution, purity, shape, and polymorphs. Many publications have focused on the implementation of Process Analytical Technology (PAT) tools for monitoring batch and continuous operation; however, a comprehensive method development and validation of Raman spectroscopy to monitor continuous crystallization has not been presented. This work demonstrates the development and validation of a method to monitor the solute concentration of Carbamazepine and quantifies the limit of detection for a metastable polymorphic form. The experiments were based on the cooling crystallization of Carbamazepine to produce the most stable form. The method was validated following the principles described in USP general chapter validation procedures for analytical methods. The results demonstrate the model can predict the solute concentration with a root-mean-square-error of prediction of 2.46 mg/ml. The repeatability and intermediate precision was evaluated and the relative standard deviation is below 5 percent. The limit of detection for the metastable form was determined by monitoring the ratio of characteristic peaks when increasing the percentage of the metastable form in the total amount of crystals in the solution. A significant change on the peak ratio is observed when 22.9 percent of the crystals are of the metastable form. In addition, this PAT method was used to monitor a continuous run for 10 residence times, in which the system reached a controlled state of operation after 6 residence times. Keywords: Continuous crystallization, Process Analytical Technology (PAT), Raman microscopy, method development, method validation 1. Introduction Recently, the pharmaceutical industry is experiencing a shift from batch to continuous manufacturing.1 The Food and Drug Administration (FDA) has identified continuous manufacturing as an innovation that could improve the agility, flexibility, and robustness of the pharmaceutical manufacturing sector.2 To support the industrial implementation of continuous manufacturing, the agency has supported throughout the past few years the use of PAT tools to 3



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monitor and control continuous processes, and the development of statistical or modeling approaches to monitor variations in the process and product quality.2b, 3 Crystallization is an important separation and purification unit operation for manufacturing the drug substance. Crystallization determines several quality attributes of drug substances, such as size distribution, shape, polymorphic form, and purity. Variations in these attributes may have significant impacts on the drug dissolution, bioavailability, efficacy, and in some cases safety.3a, 4 The concentration, supersaturation and impurity profile in the crystallizer can affect the crystal attributes.

There have been many PAT tools developed in recent decades that facilitate

monitoring and control of these quality attributes and important process parameters for the pharmaceutical crystallization;3a from the use of laser back scattering techniques for particle size information to online cameras.5 Also, spectroscopic tools such as ultraviolet, mid- and nearinfrared have been shown to be suitable for monitoring the solute concentration during various types of crystallization processes.6 Near-infrared (NIR) spectrophotometry was utilized to monitor concentration in a batch anti-solvent crystallization of naproxen in the NaproxenEudragit L100-alcohol system.6a,

6b

Zhou et al. demonstrated the implementation of an in-situ

attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectrophotometer and partial least-squares (PLS) model for inline concentration measurement and supersaturation control of a batch anti-solvent crystallization process.6c In addition, a NIR method was also developed to track solid phase information such as polymorph transformations in a wet granulation process.7 However, the infrared techniques have several drawbacks: (1) it measures the properties of the materials in direct contact with the probe; (2) Solvents, such as water, may have a very intense absorption band often obscuring the other peaks of interest.8 Therefore, an alternative has been the use of Raman spectroscopy for monitoring solid phase in solution; it offers the advantage where significant information of both the liquid and the solid phase can be extracted from the same spectrum. The use of Raman spectroscopy to monitor and control the polymorphic form during a crystallization process has been widely demonstrated.6d, 9 In-line Raman has been implemented during the crystallization of Carvedilol and used within a feedback control strategy in which the peak ratio characteristic to two polymorphs was controlled throughout the process.9a Simone et 4


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al. demonstrated the use of in-line Raman coupled with an array of PAT tools to characterize the polymorphic transformation of various materials.9b, 9c Fevotte presented an extensive review on in-line Raman spectroscopy for control of pharmaceutical crystallization processes which demonstrates the feasibility of this tool for monitoring and control of polymorph.9d Also, Raman has been used to monitor the solute concentration of active ingredients such as carbamazepine (CBZ) and flufenamic acid.10 However, the implementation of the PAT tools requires extensive study since it could be affected by temperature, agitation speed, among others. Hence, a good validation procedure should be followed in order to develop an accurate and precise method for monitoring and control purposes.11 This work addresses the development and implementation of PAT methods in the continuous crystallization of the API carbamazepine (CBZ). The development of a two stage MSMPR continuous crystallization system was discussed in a previous paper.3c Before further study on process dynamics and control of the continuous crystallization process, the development and validation of PAT methods should be addressed. Here, the study seeks to investigate the development, validation, and implementation of a PAT method (i.e. Raman spectroscopy) to measure the solute concentration in a continuous crystallization process for the case study of CBZ. A systematic validation approach is presented following the principles described in USP general chapter validation approach for analytical methods. The validated method was implemented in a single stage MSMPR platform in order to monitor the continuous crystallization of a specific polymorphic form of CBZ. It should be noted that in our case we present validation for all the typical analytical performance characteristics, except specificity, mentioned in USP for the solute concentration method. However, the requirement of analytical performance characteristics for a particular PAT method should be based on your intended use of that method, and may be further discussed with FDA through the new emerging technology team12. 2. Materials and Methods 2.1.Materials and equipment

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Carbamazepine (CBZ, Ria International) was chosen as the model compound for this work. CBZ is an anti-epileptic drug and belongs to the biopharmaceutics classification system (BCS) class II.13 CBZ was purchased from Sigma Aldrich. The raw material was tested using X-ray Powder Diffraction (XRPD) and is polymorph III which is the most stable form at room temperature.14 Ethanol 200 proof (Decon Laboratories) was used as solvent for this work. A Raman Rxn2 system from Kaiser Optical Systems, Inc. (785nm, 150-3425cm-1) was used for this study. The experiments performed for the development and implementation of the Raman method were performed on a 400ml glass reactor (ID: 72.6mm). The temperature and stirring speed are controlled via the EasyMax 402 system (Mettler Toledo). A four-upward impeller pitched-blade element (outer diameter: 38mm) was used as an agitator throughout all the studies performed in this work. The temperature was measured using a Pt100 resistance temperature detector. The system was also used for the various continuous cooling crystallization studies by introducing two peristaltic pumps (Ismatec ISM829).3c Tubing with an inner diameter of 2.49 mm was used throughout the continuous cooling crystallization experiments. A level control system which maintains the desired working volume (e.g. 400ml) by adjusting feed pump (Feedback) was developed in LabView (National Instruments). The output flow rate was kept fixed at the desired flow rate. An alternating filtration system was used where the direction of the output flow could be sent to different Buchner funnels using a pneumatic valve. 2.2.Seed preparation Seed crystals of form III were prepared by cooling 400ml of a saturated solution of CBZ (40.0mg/ml Ethanol) from 40°C to 20°C. A linear cooling profile was implemented at a fixed cooling rate of 0.25°C per min. The temperature was held constant for 1 hour to allow the system to equilibrate. A similar procedure was implemented in order to obtain form II, however, 25 ml of deionized water were added during the cooling process and the final temperature was set to 5°C. The crystals were filtered and washed using deionized water (MilliQ, Millipore System) maintained at room temperature; afterwards, the crystals were dried at room temperature for 48 hours. The dried crystals were sieved using an Advantech Sonic Sifter; various sieve trays were used in order to create a set of seed crystals with different crystal size distribution (CSD) ranging from 63 to 425µm. 6


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2.3.Methodology for the study of solid concentration and temperature effect on Raman spectra The solid concentration effect was investigated by performing a series of experiments in saturated solution of CBZ in ethanol at room temperature (25.5 mg/ml, 300 ml). Form III was added progressively in the solution and monitored over a period of 1-2 hours before addition of more CBZ. The solid concentrations tested were + 25 %, + 50 %, and + 75 % solid. The “+ 0% solid” baseline was assumed as 7.65 g, which is the amount of CBZ in the 300mL clear solution before any solid addition. The exposure time and number of scans were set to 15s and 5, respectively. The temperature effect on the Raman spectra was evaluated by monitoring the solute peak when an undersaturated solution is cooled down to the saturation temperature. A 300ml solution of CBZ in ethanol was cooled down from 75 to 55 °C in a stepwise manner. The temperature was changed after collecting five spectra. The exposure time and number of scans were set to 15s and 5, respectively. The total batch time was set to a maximum of 60 minutes to avoid any significant evaporation of ethanol which could affect the online measurement. 2.4.Methodology for the development and validation of solute concentration model The model for the prediction of solute concentration using Raman spectra was developed using clear solutions at different solute concentrations. Figure 1 shows the concentrations and temperature considered for the model development. The clear solutions were added to the 400mL crystallizer and heated to a minimum of 15 °C above the saturation temperature. The temperature was decreased step wise up to the point in which nucleation started.9b The lowest temperature considered was the previous point before nucleation was observed. Due to the existence of induction time, measurement can be taken in a supersaturated solution before nucleation happens. Five spectra were collected for each concentration and temperature. The time exposure and number of scans were set to 15 s and 5, respectively.

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100 Calibr. Solubility

80 60 40 20 0 -20

0

20

40

60

80

100

Temperature ( °C) Figure 1. Illustration of concentration points used for model calibration on a solubility curve plot.

The solute concentration method was validated following the same principles as described in USP general chapter validation procedures for analytical methods. The accuracy was validated by measuring the concentration of five clear solutions (15, 25, 40, 55, and 75 mg/ml) in the crystallizer at constant agitation speed and temperature. Samples were obtained for off-line analysis using HPLC method which was previously developed and validated; refer to section 2.6 for detailed explanation of HPLC method. The repeatability was evaluated by monitoring three undersaturated solutions (i.e. 15, 35, and 65 mg/ml). The clear undersaturated solutions were prepared every day and were monitored for periods of one hour during the same day. After each monitoring period, the equipment was turned off for 30 min. Then, the system was initiated and the same sample was monitored for another hour. This was performed for three monitoring periods on the same day. The intermediate precision was assessed by measuring the solute concentration of clear CBZ solutions over a 3-day period; in order to avoid difference due to evaporation of ethanol, a 1.2L solution (20mg/ml) was prepared on the first day and stored under refrigeration. Then, 300ml aliquots were used every day for the intermediate precision studies. The limit of detection (LOD) and limit of quantitation (LOQ) was determined based on the standard deviation and mean of the blank.15 The blank response was determined from the spectra of 200 proof ethanol over 1 hour period. 2.5.Methodology for the determination of LOD for form II 8


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The ratio of doublet near 1050 and 1027cm-1 were used to distinguish form II and III. A specific amount of form III (i.e. 9 g of form III, equals to 0.6 g of form II / g of CBZ in solution) was added initially to a saturated solution (300 mL). The solution was saturated at 20 °C (i.e. 18.44 mg/ml) and the agitation speed was fixed to 300 rpm. The temperature was measured using a Pt100 and controlled at 20 °C. The slurry was monitored for two to three hours to obtain fifty spectra (N=50). Then, specific amounts of form II seed crystals were added to the slurry to achieve the desired solid concentration; increments of 0.5-5% on the form II solid concentration were performed. Five spectra were collected immediately after the addition to avoid transformation to form III. This addition process was completed within 10 minutes. Previously we have stirred form II crystals in ethanol for at least 30 minutes, no form III was found in offline XRD analysis. The form II solid loading evaluated ranged between 0.05% to 80% (g of form II / g of CBZ in solution). The time exposure and number of scans were set to 15 s and 5, respectively. The total batch time after starting the addition of form II was two hours. 2.6.HPLC Method The HPLC method used in this work was developed following a method described in literature.16 A Zorbax Eclipse XBD HPLC Column C18 (150 x 4.6mm, 3.5µm) (Agilent Technologies USA) was used for separation. The column temperature was set at 30°C and the UV wavelength was set to 285nm; an injection volume of 10µL was used for all the samples analyzed. A flowrate of 0.8 ml per min was set for the mobile phase which consisted of acetonitrile (37% v/v) and phosphate buffer (63% v/v). The phosphate buffer was prepared by mixing 6.66g KH2PO4 in 1000ml DI water (pH 3 with H3PO4). A run time of 9 minutes was set for all the different studies. Methanol was used as the diluent for the standard solutions and off-line samples analyzed through this work. A calibration curve was developed based on measured peak area of five standard solutions (5 to 500 µg/ml) and linear least-square regression analysis with a correlation coefficient of 0.9999. A relative standard deviation (RSD) of 0.78% was achieved during the validation procedure of the method for six consecutives injections. The highest RSD achieved over a period of three days was 0.70% for three concentrations analyzed. 2.7.Data processing in Raman method 9



Preprocessing of the spectra was performed by applying second derivative to distinguish peaks and eliminate baseline shift. Also, smoothing between 13 points and a window of 12 cm-1 was applied using iC-Raman. A time exposure of 15 s and the number of scans was set to 5. The number of scans and the exposure time were fixed throughout this study. 3. Results & Discussion 3.1.Development of Raman Method for Solute Concentration The solute concentration model was calibrated and validated through a series of experiments in which the Raman spectra was collected for clear solutions at different concentrations and temperatures. The detailed experimental procedure is described in section 2. The spectra of clear solutions were compared to off-line spectra obtained for CBZ form III and II, to identify characteristic peak for model development. Figure 2a shows the spectra obtained for a clear solution of CBZ, solid form III, and II. A specific peak for the solute was identified for this system in the region between 357 and 370 cm-1 as observed in Figure 2b, and the peak area with respect to the baseline between these two points was correlated with the solute concentration. The peak for form III around 370 and 380cm-1 appears when solid precipitates in the system. Therefore, the impact of solids in the solute characteristic peak should be evaluated to understand if the univariate dependence holds or a multivariate model should be developed.

(a)

Normalized Raman Intensity

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(b)

Figure 2. Normalized (a) raw and (b) preprocessed spectra for undersaturated CBZ in ethanol solution, solid form III, and form II.

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The effect of solid on the solute peak was evaluated by adding CBZ crystals to a saturated solution (25.5 mg/ml) operated at 25°C. Figure 3 shows the dynamic profile of the solute peak intensity after the addition of 25, 50, and 75% solid to the clear solution. The first addition (25% loading) occurred after monitoring the clear solution for 30 minutes. The clear solution average peak area is 401.8±6.6. The variability (i.e. standard deviation) of the solute peak increases although no reduction of the average peak area is observed. However, a slight reduction is observed after the second addition of raw material where the solid reaches 50% loading. The average peak area decreased from 415.3±13.5 to 403.5±16.4 (i.e. ~ 0.5 mg/mL decrease), when the solid loading was increased from 25 to 50% g solid/ g CBZ solute. At last, the solute peak area decreased to 399.2±20.8 after the solid loading was increased to 75% g solid/ g CBZ solute. The increase in the noise and decrease of the Raman intensity after the third addition may be associated to saturation of the Raman signal. Therefore, the results suggest that constant and low/medium solid loading (e.g. less than 50%) during the continuous crystallization is preferred to avoid the solid effect on the Raman method for solute concentration measurement.

500 Raman Intensity

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400

300

0%

25%

50%

75%

200 0

100

200 300 time (min)

400

Figure 3. (a) Raman intensity for solute peak obtained for a saturated solution of CBZ (25.5 mg/mL, 25 °C) at various solid concentrations (25, 50, and 75% g CBZ solid/g CBZ solute). Three red marks represent the three time points for the three additions of solids. For each solid concentration, a point representing the average value and standard deviation bar of data in that range is added in the figure.

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The effect of the temperature was evaluated by varying the operating temperature of undersaturated solutions of CBZ in ethanol. The solute peak was monitored at various temperatures for a period of 1 hour. Figure 4 shows the impact of temperature on the solute peak intensity where the average intensity increased as the operating temperature raised. The temperature has a linear effect on the peak intensity as shown by the linear correlation. A slight increase of 3% on the solute peak intensity was observed for a span of 15°C. Therefore, multilinear models considering temperature and Raman intensity were developed to capture the independent contribution of temperature on the solute concentration models. Table 1 summarizes the results obtained for the various models developed from the Raman spectra measured, as described in section 2.4. The range considered for all models developed is between 15 to 65 mg/ml.

Raman intensity

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Figure 4. Temperature effect on Raman intensity obtained from undersaturated solution (Tsat 40°C, 40.7 mg/mL) of CBZ in ethanol. Table 1. Model results obtained from least square fit on Raman calibration samples for solute concentration prediction (concentration: 15-65mg/ml, temperature: 0-75°C). Maximum p-values are for the fitted coefficients obtained are shown (red variable refers to p-value above 0.05).

Model

Variables

1

(A)

2

2

(A,A )

2

RMSE (%) 0.16

p-value

R

Raman Spectroscopy for Monitoring the Continuous Crystallization of

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