Quality Control of Protein Crystal Suspensions Using Microflow Imaging and Flow Cytometry

Protein crystallization is an attractive method for protein processing and formulation. However, minor changes in the crystallization setup can lead to changes in the crystal structure or the formation of amorphous protein aggregates, which affect the product quality. Only few analytical tools for qualitative and quantitative differentiation between protein crystals and amorphous protein exist. Electron microscopy requires expensive instrumentation, demanding sample preparation, and challenging image analysis. Therefore, there is a need to establish other analytical techniques. It was the aim of this study to investigate the capability of light obscuration (LO), microflow imaging (MFI), and flow cytometry (FC) in differentiating the amorphous and crystalline states of insulin as a relevant model. Qualitative discrimination of the two populations based on the particle size was possible using LO. Quantitative determination of amorphous protein and crystals by MFI was challenging due to overlapping size distributions. This problem was overcome by particle analysis based on the mean light intensity. Additionally, FC was applied as a new method for the determination of the quality and quantity of amorphous protein by differences in the light scattering. Our results show the potential of MFI and FC for rapid high throughput screening of crystallization conditions and product quality. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3860-3866, 2013