Crystallization is an alternative separation and purification technique to conventional chromatography for proteins due to specific characteristics, such as good scalability, low cost, and the attainable stability of the crystalline products. However, protein crystallization often suffers from long batch time due to the slow crystallization kinetics. Furthermore, protein crystallization is conventionally conducted in the batch mode of operation, which has potential disadvantages compared to continuous operation. These challenges of protein crystallization are addressed in this thesis by employing process intensification principles. The work presented in this thesis will focus on two typical process intensification domains, i.e., the space and time domains involving equipment structuriza...[ Read more ]

Crystallization is an alternative separation and purification technique to conventional chromatography for proteins due to specific characteristics, such as good scalability, low cost, and the attainable stability of the crystalline products. However, protein crystallization often suffers from long batch time due to the slow crystallization kinetics. Furthermore, protein crystallization is conventionally conducted in the batch mode of operation, which has potential disadvantages compared to continuous operation. These challenges of protein crystallization are addressed in this thesis by employing process intensification principles. The work presented in this thesis will focus on two typical process intensification domains, i.e., the space and time domains involving equipment structurization and continuous operation. 3D printing is used as a tool to achieve process intensification through structurization, because of its ability for rapid fabrication of customized process equipment with complex geometries. Novel crystallizer types and process configurations are investigated with the aid of 3D printing as the fabrication technique.

First, a workflow for the fabrication of crystallizers with 3D printing is presented. The workflow aids in the selection of appropriate 3D printing techniques and suitable materials for the fabrication of custom-designed crystallizer. Furthermore, the designs for the crystallizers used in this thesis are developed based on models or heuristics. These designs can be a platform or template for other crystallizers with geometric similarities and can also be the start of a library of crystallizers that can be customized and fabricated as required. 3D printing has found limited applications in the fabrication of equipment for separation processes, including crystallization, despite the extensive utilization in, for example, reaction engineering. The workflow aims to facilitate the adoption of 3D printing as a tool to achieve process intensification of crystallization processes through structurization.

Second, the performance of a pneumatically agitated airlift crystallizer (ALC) is characterized and compared to a mechanically agitated stirred tank crystallizer (STC) for the crystallization of the model protein lysozyme. The desupersaturation profile is substantially shorter in the ALC compared to STC for all tested conditions, which can be an advantage for processes with slower crystallizer kinetics. The shorter desupersaturation profile in ALC may be attributed to the gas-liquid surface of the bubbles lowering the free energy barrier for nucleation. Furthermore, larger, and less agglomerated crystals are obtained from the ALC. Additionally, the biological activity of the product indicates that the functionality of the protein is preserved during the process in the ALC. Protein crystallization in an ALC has not previously been reported. Process intensification is achieved in this work through structurization by the adoption of an ALC for protein crystallization resulting in higher throughput, along with larger and unagglomerated crystals with high activity.

Third, the 3D printed ALC and STC are characterized for continuous crystallization of lysozyme with a focus on the attainable solid-state forms from a mixed-suspension mixed-product-removal (MSMPR) crystallizer. The metastable needle-like form of lysozyme can be obtained from continuous crystallization in MSMPR crystallizers for conditions at which the stable tetragonal form is obtained from conventional batch crystallization. The needle-like crystals appear after nucleation of the tetragonal form. An earlier transition from the tetragonal form to the needle-like form is observed in ALC compared to STC, which is possibly caused by reduced attrition and the presence of a gas-liquid interface in the ALC. Furthermore, a long residence time and high precipitant concentration favor the formation of the needle-like crystals. This work presents the first experimental study on protein crystallization in an MSMPR crystallizer, which reveals a dramatic influence of the mode of operation on the attainable solid-state form of a protein.

Finally, three 3D printed tubular crystallizers, a standard Kenics static mixer (sKTC), a novel gapped Kenics static mixer (gKTC), and a hollow tube (HTC), are designed and characterized numerically and experimentally. This work aims for process intensification through structurization by the adoption of a novel design for the static mixer in a tubular crystallizer. Numerical results indicate a shorter mixing length can be achieved in the sKTC and the gKTC compared to the HTC and, therefore, reduced supersaturation gradients can be expected when applying the former crystallizers. The residence time distribution (RTD) from the sKTC and gKTC is similar and closer to plug flow compared to the HTC. A lower shear rate is present in the gKTC compared to the sKTC, which can result in a lower secondary nucleation rate in the former. Experimental characterization of the crystallizers shows that both the sKTC and the gKTC can avoid segregation of the crystals due to gravity at relatively low flow rates compared to the HTC, while a substantially lower pressure drop is observed in the gKTC compared to the sKTC for the same flow rate. The crystallizers are characterized for seeded crystallization of lysozyme, which indicates that the continuous re-orientation of flow in the sKTC can promote settling of smaller crystals on the walls through shear-induced migration of larger crystals towards the center of the tubular crystallizer. This study shows that the novel gKTC outperforms the HTC in terms of mixing length, RTD, and solid suspension and the sKTC in terms of pressure drop, solids suspension, and lower shear while achieving the same RTD and mixing length, which has important implications for practical applications of continuous protein crystallization.

Process intensification is achieved through structurization by employing the airlift crystallizer and the static mixer-based tubular crystallizer and continuous operation by MSMPR crystallizer and plug flow crystallizer. The work presented in this thesis can provide a basis for the development of innovative concepts for process intensification through structurization and continuous operation. These concepts include an integrated multi-stage airlift crystallizer and a hybrid static mixer-based crystallizer. The workflow developed in this work can aid in the fabrication of these innovative crystallizers with a custom-designed geometry.