Protein Nucleation and Crystallization Process with Process Analytical Technologies in a Batch Crystallizer

[Image: see text] Protein crystallization has drawn great attention to replacing the traditional downstream processing for protein-based pharmaceuticals due to its advantages in stability, storage, and delivery. Limited understanding of the protein crystallization processes requires essential information based on real-time tracking during the crystallization process. A batch crystallizer of 100 mL fitted with a focused beam reflectance measurement (FBRM) probe and a thermocouple was designed for in situ monitoring of the protein crystallization process, with simutaneously record of off-line concentrations and crystal images. Three stages in the protein batch crystallization process were identified: long-period slow nucleation, rapid crystallization, and slow growth and breakage. The induction time was estimated by FBRM, i.e., increasing numbers of particles in the solution, which could be half of the time required for detecting the decrease of the concentration, by offline measurement. The induction time decreased with an increase in supersaturation within the same salt concentration. The interfacial energy for nucleation was analyzed based on each experimental group with equal salt concentration and different concentrations of lysozyme. The interfacial energy reduced with an increase in salt concentration in the solution. The yield of the experiments was significantly affected by the protein and salt concentrations and could achieve up to 99% yield with a 26.5 μm median crystal size upon stabilized concentration readings.