(469c) Continuous Crystallisation Oscillatory Flow Platform for Protein Purification and Bio-Separation

Crystallization of proteins could be a potentially cost effective and robust method to purify protein molecules in the pursuit of the next generation of protein based biopharmaceutic therapies. However, the step from traditional small-scale high throughput screening methods, to crystallizers which can produce high yields of pure crystalline protein material, is still in its infancy.

Here, we present a platform for continuous oscillatory flow crystallization (COFC) (Figure 1) and its application for the crystallization of lysozyme[1,2]. A workflow is developed from µl screening experiments, to scaled up batch oscillatory flow crystallization (BOFC) and COFC experiments.

The induction time for lysozyme is found to be inversely proportional to the initial protein concentration, with the same relationship found between the induction time and the frequency and amplitude of the oscillation. However, it is also observed that the decrease in induction time comes at a cost to the crystal size and quality. In turn, the high shear environment from increased amplitude and frequency of the COFC can also damage the fragile protein crystals, resulting in the formation of more fine particles and an undesirable size distribution. This led to further investigation of slug flow mixing in the continuous crystalliser, where the crystal size and shapes are compared to the particles produced from the osciallatory flow reactor. Further work around optimization of the crystallization parameters and crystallizer geometry using the gCrystal platform to find the optimum scale up conditions are also discussed. The use of seeding and potential templating materials is also covered with a view to expanding the use of this crystallizer to less well characterized protein materials.

It is demonstrated that purification of proteins can be achieved by crystallization in a continuous mode. This route offers the possibility to revolutionise downstream separations of biopharmaceuticals

References

[1] H.Y. Yang, W.Q. Chen, P. Peczulis, J.Y.Y. Heng (2019): Development and Workflow of a Continuous Protein Crystallization Process: A Case of Lysozyme, Cryst. Grow. Des; 19, 983-991.

[2] H.Y. Yang, P. Peczulis, P. Inguva, X.Y. Li, J.Y.Y. Heng (2018): Continous Protein Crystallisation Platform and Process: Case of Lysozyme, Chem. Eng. Res. Des. 136, 529-53