(624l) Improving Purity and Size Distribution in Biopharmaceutical Crystallization Using PAT-Based Feedback Control Strategies

Simone, E. - Presenter, Loughborough University
Zhang, W. - Presenter, North China Pharmaceutical group Corporation
Nagy, Z. K. - Presenter, Purdue University


Purity is a critical quality attribute for both pharmaceutical and biopharmaceutical products. The presence of impurities (solvents, salts or byproducts of the synthetic path) in drugs can cause a reduction of their effectiveness or can even be toxic for the patients. Biopharmaceuticals are produced by biological processes which are difficult to control. Therefore the amount of impurities that has to be removed can be significantly higher than in the case of synthetic pharmaceuticals. In this work process analytical technology (PAT) tools and different feedback control strategies (T-control, direct nucleation control and supersaturation control) were tested for the crystallization of a biopharmaceutical product. UV/Vis spectroscopy, focused beam reflectance measurement (FBRM) combined with CryPRINS were used to improve the crystal size distribution and purity of crystallized vitamin B12. The different feedback control strategies were compared to classical crystallization techniques in terms of purity of the final crystal and quality of the crystal size distribution and it is shown that using suitable crystallization feedback control strategies, the purity and quality of crystals can be improved. The fragility of the vitamin B12 molecule and the presence of impurity, together with its needle-like morphology made the implementation of PAT based control strategies particularly challenging. A direct nucleation control based on FBRM data and a calibration-free supersaturation control using ATR-UV/Vis were implemented on the system and compared to specific T-control strategies (for example temperature cycling). In the direct nucleation feedback control strategy the number of counts obtained from the FBRM is kept constant by heating and cooling steps. If the counts are higher than the desired setpoint range, the controller reacts by increasing the temperature in the crystallizer and dissolving the smaller particles. If the number of counts is too low instead, the system is cooled down promoting growth or maybe nucleation. This strategy does not require any calibration and was found to be effective in obtaining large crystals of paracetamol (Saleemi et al. 2012b, Saleemi et al. 2012a, Saleemi 2011), glycine (Abu Bakar et al. 2009) as well as in enhancing the crystalline properties of a cardiovascular API (Saleemi et al. 2012). Temperature cycling (with no feedback control) was also implemented in order to check its efficacy. This technique was successfully used to control both size and polymorphic form of sulfathiazole (Abu Bakar et al. 2010, Abu Bakar 2010, Abu Bakar et al. 2010) and to reduce liquid inclusion in the crystallization of cyclotrimethylene trinitramine (RDX) (Kim et al. 2011). Calibration based supersaturation control is a now common technique widely tested for different APIs; however, only few examples of calibration-free strategies are present in literature (Duffy et al. 2013, Barrett et al. 2010, Kee et al. 2009, Chew et al. 2007). A calibration-free supersaturation control approach is also implemented for the crystallization of vitamin B12 and the results are compared among all feedback control approaches.


Materials and Methodology

Crude Vitamin B12 extracted from biofermentation was donated by Hebei Welcome Pharmaceutical Co., LTD (China). Pure water or solutions of water and ethanol were used as solvents for the experiments. Experiments were conducted at both Loughborough University and Purdue University with similar, but not exactly identical instruments. A jacketed stirred vessel (500ml) was used for the crystallization experiments. The temperature in the vessel was controlled using a Huber thermostat connected to a thermocouple and the CryPRINS software. An ATR-UV/Vis immersion probe was used for the monitoring of solute concentration and for the supersaturation control experiments while a focused beam reflectance measurement (FBRM) probe was used for the direct nucleation control experiments. The data from the FBRM, ATR-UV/Vis and the Huber could be transmitted in real-time to the CryPRINS software (Crystallisation Process Informatics System). This allows real-time monitoring and control of the FBRM count/s, ATR-UV/Vis signal and the temperature, as well as setting a temperature profile and performing supersaturation control.

Slow linear cooling experiments: simple cooling experiments (-0.075 and -0.1 °C/min) were performed with raw and recrystallized vitamin B12. The quality of the crystals obtained was compared to the one from feedback controlled experiments.

Supersaturation control experiments: a calibration-free approach was used to perform the seeded supersaturation control experiments. In this approach an inferential solubility curve expressed as UV signal versus temperature is used instead of the classical concentration versus temperature relationship. Both crude material and purified (vitamin B12 crystallized and washed with acetone once) were used for the experiments, and because of their difference in purity two solubility equations were measured. Those equations were inserted in CryPRINS which automatically changed the temperature in the crystallizer in order to keep the supersaturation at the desired set-point level after seeding. The supersaturation setpoint is calculated as the difference between the desired UV signal at a given temperature and the value of the inferential solubility of vitamin B12 at that temperature.

Direct nucleation control experiments: the direct nucleation control technique is based on the use of both FBRM and CryPRINS to keep the number of crystal counts/s in the vessel constant during the whole batch. One of the statistics (usually total counts/s) measured by the FBRM is sent to CryPRINS and temperature is decreased if the measured total counts/s is lower than the set point range, or increased if it is higher (crystals in excess are then dissolved). The parameters that have to be inserted in CryPRINS are the chosen statistics setpoint as well as the desired cooling and heating rate.

Temperature cycling experiments: several experiments using different cooling/heating rates and number of cycles were performed. Both crude and recrystallized material was used to prepare the solution, while pure seeds were added (about 3 % of the total solute). The amplitude of every cycle was 7-9 °C and cycles converged towards a final temperature of 6 °C.

HPLC analysis: the HPLC method was developed by the company who provided the material and then slightly changed for the available instrument. The purity of the crystals obtained during the experiments was measured using this technique.

Results and discussion

Simple cooling crystallization experiments resulted in a product of poor size distribution characterized by very small crystals together with big ones. However, the crystal size distribution could be considerably improved using feedback control strategies. Calibration-free supersaturation control is the simplest approach that gave a narrow distribution compared to simple linear cooling of material crystallized the same number of times.  Temperature cycling was also found to improve the crystal size distribution, although determining the optimal operating conditions is more difficult compared to supersaturation control because of the higher number of parameters to set (number and amplitude of the cycles, heating and cooling rates). Experiments showed that direct nucleation control is not suitable for vitamin B12 because of the specific morphology of the compound: the growth of needle crystals is accompanied by an increase in the total counts/measurement, therefore, trying to keep the counts constant prevent the particles from growing. In general, using material which was crystallized once to prepare the solution, helped in improving the final crystal size distribution regardless of the type of crystallization strategy used. Among the experiments with material recrystallized once, temperature cycling with fast heating/cooling rate gave the highest purification efficiency.


Three feedback control strategies were tested on a biopharmaceutical and two of them were proved to increase the quality of the CSD and purity of the final product compared to traditional slow cooling. Direct nucleation control could not be used for the studied molecule because of its needle-like morphology. Temperature cycling gave a highest increase in purity compared to calibration-free supersaturation control.


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