Assessment of Imaging Flow Cytometry for the Simultaneous Discrimination of Protein Particles and Silicone Oil Droplets in Biologicals

Purpose Silicone oil droplets in biopharmaceutical products can originate from sources such as siliconized surfaces of primary packaging materials, potentially triggering the formation of protein–silicone oil particles. To better understand this phenomenon, there is a need for particle detection devices that cannot only distinguish between protein particles and silicone oil droplets but also determine particle sizes ranging from nanometers to micrometers. Method In this study, we conducted a systematic assessment of imaging flow cytometry (IFC) using the FlowSight® instrument. Our first step was to investigate specific instrument settings using protein particle samples spiked with silicone oil for particle classification. Based on these findings, we established suitable, harmonized working templates. Next, we evaluated the instrument’s accuracy and precision for particle sizes within the range of 0.5 to 100 µm and their respective concentrations. Finally, we investigated any constraints in particle concentration within this size range. Results This study demonstrates that IFC can effectively distinguish protein particles from silicone oil droplets when the latter is labeled with a specific fluorescent dye. Our findings suggest that fluorescently labeled particles ≥ 0.5 µm can be reliably detected. Through our research, we determined the particle concentration limits for each particle size in the range of 0.5 to 10 µm, with a precision deviation of less than 15%. However, our study also revealed that IFC exhibited insufficient accuracy for the tested particle concentrations within this size range. Additionally, we showed that the measurements were significantly influenced by the instrument settings. Conclusion Although we addressed numerous new aspects to enhance the experimental procedure of IFC measurements, we conclude that IFC is not an ideal technique for quantifying sub-visible particles. Instead, it should be employed to provide supportive characterization data in conjunction with commonly used sub-visible particle detection methods. If distinguishing between protein particles and silicone oil droplets is essential, IFC is an option, as long as the fluorescent dye is carefully selected. Graphical Abstract