Generalized flow imaging microscopy instrument settings for measuring subvisible particle sizes and concentrations in protein therapies

Flow imaging microscopy (FIM) is a common imaging-based technique for measuring the subvisible particle content of protein therapies. FIM measurement accuracy strongly depends on particle segmentation, the process of detecting particles in digital images. Some FIM instruments like FlowCam allow users to adjust capture settings, parameters that influence segmentation algorithm performance. However, it has been challenging to identify settings that simultaneously yield accurate measurements of the various particle types found in protein formulations and common particle standards used for instrument qualification and validation. The present study describes generalized capture settings that allow FIM instruments to accurately measure the size and concentration of common particle types encountered in protein therapy development and manufacturing. The settings were developed using a mixture of qualitative and quantitative optimization strategies, as well as using both particle standards and representative samples. At these settings, FIM instruments yielded accurate measurements on opaque (e.g., polystyrene beads) and translucent (e.g., ethylene tetrafluoroethylene particles) particle standards. Protein formulations were also analyzed using these settings to investigate the impact of these settings on the measured subvisible particle content. These settings yield accurate measurements of typical particle types in protein formulations and aim to aid in standardizing FIM as an analytical technique.