Imaging Flow Cytometry for Sizing and Counting of Subvisible Particles in Biotherapeutics

Area of Science:

• Biopharmaceutical Analysis
• Analytical Chemistry
• Microscopy Techniques

Background:

• Imaging flow cytometry (IFC) offers multi-channel data acquisition (brightfield, fluorescence, side scatter) for particle analysis.
• IFC was introduced to biopharmaceutical subvisible particle analysis approximately ten years ago, aiming to provide additional information on particle origin.
• Accurate particle characterization is critical in biopharmaceutical development and quality control.

Purpose of the Study:

• To systematically develop and optimize masks for imaging flow cytometry (IFC) image analysis.
• To enhance the accuracy of size determination for polystyrene beads and pharmaceutically relevant particles (protein, silicone oil) using IFC.
• To compare particle sizing and counting accuracy of IFC with flow imaging microscopy (FIM).

Main Methods:

• Development and systematic optimization of image analysis masks for IFC.
• Analysis of polystyrene beads, protein particles, and silicone oil particles in brightfield and fluorescence channels.
• Comparison of particle sizing and counting results from IFC with those from flow imaging microscopy (FIM).

Main Results:

• Mask verification using fluorescent polystyrene particles showed good agreement in size determination between IFC and FIM.
• IFC reported lower concentrations than FIM for polystyrene particle standards.
• For fluorescently stained silicone oil and protein particles, IFC fluorescence imaging reported higher concentrations in the low micrometer range.

Conclusions:

• Imaging flow cytometry (IFC) is a suitable tool for supportive data generation in particle characterization and troubleshooting.
• IFC is not recommended as a routine quantitative technique for subvisible particle analysis in drug product development or quality control.
• Optimization of image analysis masks is essential for improving the accuracy of IFC in particle sizing.