Development and Workflow of a Continuous Protein Crystallization Process: A Case of Lysozyme

In the present work, a workflow on the development of a continuous protein crystallization is introduced, with lysozyme as a model protein, from microliter screening experiments, to small scale batch crystallization experiments in a shaking crystallization platform, and to batch and continuous crystallization experiments in an oscillatory flow platform. The lysozyme crystallizations investigated were for a concentration range from 30 to 100 mg/mL, shaking conditions from 100 to 200 rpm in the batch shaking crystallization platform, and oscillatory conditions with amplitude (x(0)) from 5 to 30 mm and frequency (f) from 0.1 to 1.0 Hz in the batch oscillatory flow crystallization platform. We propose the use of the Reynold's number (R-e) for scaling up of the process from the shaking batch to the continuous oscillatory flow platform. Additionally, it is shown that the nucleation rate increased with increase in concentration of initial lysozyme solution, or increase in shear rate, inducing smaller size of lysozyme crystals. The properties and qualities of the crystal products indicate that continuous crystallization platforms may offer advantages to the downstream bioprocessing of proteins.