(198g) Continuous Protein Crystallization in a 3D Printed Airlift Crystallizer

Mathew Thomas, K. - Presenter, The Hong Kong University of Science and Technology
Jin, Y., Hong Kong University of Science and Technology
Lakerveld, R., The Hong Kong University of Science and Technology
Continuous crystallization has been receiving growing interest for processes with a small throughput such as those found in pharmaceutical and biotechnology industries. Continuous processing offers several potential advantages compared to conventional batch-wise processing such as easier scale-up, reduction in equipment footprint, and the ability to implement simpler control strategies.1,2 Mixed-suspension, mixed-product removal (MSMPR) is one of the traditional modes of operation for continuous crystallization and is generally preferred for slow-growing crystals that require a long residence time or for crystals that have a high tendency for fouling.3 Protein crystallization typically requires a long residence time and hence calls for the use of an MSMPR mode of operation. An additional advantage of using the MSMPR mode is that an existing batch crystallizer can easily be adopted for continuous crystallization,4 which is important for multi-purpose production facilities. Airlift crystallizers are well suitable for MSMPR crystallization because of the ease of operating for long residence times and rigorous mixing.

An airlift crystallizer5,6 is a pneumatically agitated column generally consisting of a riser and a downcommer. Gas is introduced into the riser through a sparger, which reduces the density of the materials in the riser. The resulting difference in the density between the contents of the riser and the downcommer leads to the liquid circulation in the airlift crystallizer, which keeps the crystals in suspension. Recently, we have developed a 3D-printed airlift crystallizer (ALC) for batch protein crystallization. This 3D printed airlift crystallizer showed a reduction in the induction time for nucleation and generally larger and less agglomerated crystals could be produced in the ALC compared to a conventional stirred tank crystallizer (STC) for the case of lysozyme crystallization.7 The reduction in the induction time was expected and likely caused by heterogeneous nucleation supported by the gas-liquid surface. Generally, it is known that secondary nucleation due to attrition is dominant in an STC,8 as protein crystals are known to be more fragile than crystals of small organic molecules.9 Furthermore, different dominating mechanisms for secondary nucleation exists in both ALC and STC.10 The technical feasibility of the airlift crystallizer for reactive11 and cooling crystallization12 in continuous flow for salts and small organic molecules has been demonstrated. However, airlift crystallizers in continuous flow have not been studied systematically yet. Since different nucleation mechanisms are present in either an ALC or a STC, it is likely that the steady state and transient behaviour of an ALC operated in continuous flow mode will be different compared to an STC. Therefore, in the present work, we have extended protein crystallization in an airlift crystallizer from batch-mode to continuous flow mode.

The objective of this work is to develop and characterise a 3D printed ALC for continuous crystallization of proteins in MSMPR mode and to compare its performance in terms of throughput and product quality with that of an STC operated in MSMPR mode. Lysozyme is used as the model compound with sodium chloride as the precipitant. The ALC is designed and fabricated using 3D printing (stereolithography). Continuous crystallization experiments will be carried out in the ALC and the performance will be compared to that of the STC operated in MSMPR mode. MSMPR operation is achieved by continuously feeding the crystallizer at a relatively low flow rate and removing the slurry from the crystallizer at a higher flow rate in intervals. The crystallizer is operated for a duration of 9 hours (about three residence time). The protein concentration reaches steady-state quickly in an STC, which justifies the relative short operation time and will be compared to the protein concentration as function of time in an MSMPR ALC. The possible classification of the crystals during product removal will be investigated. Finally, seeding will be explored to enhance the start-up of the ALC.

Acknowledgement: The work described in this abstract was supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China, Project No. 16242916.


(1) Lee, S. L.; O’Connor, T. F.; Yang, X.; Cruz, C. N.; Chatterjee, S.; Madurawe, R. D.; Moore, C. M. V; Yu, L. X.; Woodcock, J. Modernizing Pharmaceutical Manufacturing: From Batch to Continuous Production. J. Pharm. Innov. 2015, 10 (3), 191–199.

(2) Lakerveld, R.; Benyahia, B.; Heider, P. L.; Zhang, H.; Wolfe, A.; Testa, C. J.; Ogden, S.; Hersey, D. R.; Mascia, S.; Evans, J. M. B.; et al. The Application of an Automated Control Strategy for an Integrated Continuous Pharmaceutical Pilot Plant. Org. Process Res. Dev. 2015, 19 (9), 1088–1100.

(3) Quon, J. L.; Zhang, H.; Alvarez, A.; Evans, J.; Myerson, A. S.; Trout, B. L. Continuous Crystallization of Aliskiren Hemifumarate. Cryst. Growth Des. 2012, 12 (6), 3036–3044.

(4) Wong, S. Y.; Tatusko, A. P.; Trout, B. L.; Myerson, A. S. Development of Continuous Crystallization Processes Using a Single-Stage Mixed-Suspension, Mixed-Product Removal Crystallizer with Recycle. Cryst. Growth Des. 2012, 12 (11), 5701–5707.

(5) Soare, A.; Lakerveld, R.; Van Royen, J.; Zocchi, G.; Stankiewicz, A. I.; Kramer, H. J. M. Minimization of Attrition and Breakage in an Airlift Crystallizer. Ind. Eng. Chem. Res. 2012, 51 (33), 10895–10909.

(6) Lakerveld, R.; Van Krochten, J. J. H.; Kramer, H. J. M. An Air-Lift Crystallizer Can Suppress Secondary Nucleation at a Higher Supersaturation Compared to a Stirred Crystallizer. Cryst. Growth Des. 2014, 14 (7), 3264–3275.

(7) Mathew Thomas, K.; Lakerveld, R. A 3D-Printed Airlift Crystallizer for Protein Crystallization. Submited 2019.

(8) Tait, S.; White, E. T.; Litster, J. D. A Study on Nucleation for Protein Crystallization in Mixed Vessels. Cryst. Growth Des. 2009, 9 (5), 2198–2206.

(9) Tait, S.; White, E. T.; Litster, J. D. Mechanical Characterization of Protein Crystals. Part. Part. Syst. Charact. 2008, 25 (3), 266–276.

(10) Anisi, F.; Kramer, H. J. M. Crystallization Kinetics in an Airlift and a Stirred Draft Tube Crystallizer; Secondary Nucleation Models Revisited. Chem. Eng. Res. Des. 2018, 138, 200–211.

(11) Gonzalez-Contreras, P.; Weijma, J.; Buisman, C. J. N. Bioscorodite Crystallization in an Airlift Reactor for Arsenic Removal. Cryst. Growth Des. 2012, 12 (5), 2699–2706.

(12) Anisi, F. Control of the Key Phenomena in Continuous and Batch Crystallization Processes: Novel Process and Equipment Design, TU Delft, 2019.