(558cd) A Droplet-Based Evaporative Crystallization System for Protein Crystallization Kinetics Estimation | AIChE

(558cd) A Droplet-Based Evaporative Crystallization System for Protein Crystallization Kinetics Estimation


Hong, M. S. - Presenter, Massachusetts Institute of Technology
Lu, A. E., Massachusetts Institute of Technology
Bae, J., Seoul National University
Lee, J. M., Seoul National University
Braatz, R. D., Massachusetts Institute of Technology
Packed-bed chromatography, which is the currently dominant method for bioseparations, is expensive for high-dose biopharmaceuticals, because its operating costs scale linearly with throughput. On the other hand, crystallization has proved to be an inexpensive industrial separation method for inorganic and organic molecules for satisfying adequate purity and production. Operating costs for crystallization from liquid solution scale sub-linearly with throughput. Although purification of some therapeutic proteins such as insulin use crystallization, crystallization technology effective for large-molecule therapeutic proteins is still lacking and needs to be developed (see [1] for a literature review).

Although numerous micro-batch protein crystallization experiments have been published, few studies consider scale-up to production scale or characterize the crystallization kinetics needed for systematic scale-up. Limited quantity of protein is available during the initial stage of process development, which motivates the development of small-scale experimental systems with the capability of collecting experimental data of sufficient quantity and quality for the estimation of crystallization kinetics. This work describes the design of a droplet-based evaporative crystallization system for the evaluation of candidate crystallization conditions and the estimation of crystallization kinetics using only a minimum quantity of protein. The developed droplet-based evaporative crystallization system is validated for model proteins including lysozyme which has well-characterizing solubility and crystallization kinetics.

In the evaporative crystallization system, a droplet containing aqueous protein solution is hung on the glass cover inside an optically visible chamber for the real-time imaging through a microscope. The chamber was designed in house and manufactured by 3D printing. Air of controlled temperature and humidity is continuously fed to the chamber for controlled evaporation and rehydration of the droplet. The humidity in the chamber is controlled by manipulating the ratio of the flow rates of wet air from a designed humidifier and dry air from a separate supply, and the temperature is controlled using a heat exchanger integrated into the equipment. Evaporation and rehydration are used as handles for dynamically increasing and decreasing the supersaturation, which is the driving force for crystallization. A multi-angle in-situ imaging system is described that enables the measurement of the solute concentration, solubility, and crystal size distribution. The experimental data are then analyzed to estimate the crystallization kinetics.


[1] M. S. Hong, K. A. Severson, M. Jiang, A. E. Lu, J. C. Love, and R. D. Braatz, Challenges and opportunities in biopharmaceutical manufacturing control, Comput. Chem. Eng., vol. 110, pp. 106-114, 2018.