Continuous Protein Crystallization in Mixed-Suspension Mixed-Product-Removal Crystallizers

The metastable needlelike form of lysozyme can be obtained from continuous crystallization in mixed-suspension mixed-product-removal (MSMPR) crystallizers for conditions at which the stable tetragonal form is obtained from batch crystallization. A 3D-printed airlift crystallizer and stirred tank crystallizer are used, which both approximate an MSMPR crystallizer well. The influence of residence time, precipitant concentration, and agitation mechanism on the attainable solid-state form is presented. The needlelike crystals appear after nucleation of the tetragonal form. An earlier transition from the tetragonal form to the needlelike form is observed in an airlift crystallizer compared to a conventional stirred crystallizer, which is possibly caused by reduced attrition and the presence of a gas-liquid interface in the airlift crystallizer. Furthermore, a long residence time and high precipitant concentration favor the formation of the needlelike crystals. This work demonstrates how the mode of operation of well-mixed crystallizers affects the attainable solid-state form of a model protein. The results generally show the importance of obtaining a quantitative understanding of the dynamic interplay between stochastic primary nucleation processes and deterministic growth and secondary nucleation processes so that future continuous MSMPR crystallizers can be designed and operated robustly to produce protein crystals of a desired solid-state form.