Characterization of Protein Particles in Therapeutic Formulations Using Imaging Flow Cytometry.

Quantitation of particles >10 μm in therapeutic protein formulations is required by pharmacopeia guidelines, and characterization of particles <10 μm is increasingly expected. Established methods offer limited ability to detect or characterize small particles; consequently, new methods are needed to measure the sub-10 μm size range. Here, we evaluate imaging flow cytometry (IFC) as a new method for detection of protein aggregates, taking advantage of key enabling attributes including rapid multi-modal high-resolution imaging of individual particles, low sample volume, high sampling efficiency, wide dynamic size and concentration range, and low clog risk. IFC sensitivity was compared with dynamic imaging, a "gold standard" technique for analysis of particles in protein formulations. Both techniques yielded similar results for polystyrene beads ≥2 μm. However, IFC demonstrated greater protein particle detection sensitivity, especially for the sub-10 μm size range. Interestingly, for an aggregated lysozyme sample, IFC detected protein particles using fluorescence images, whereas dynamic imaging failed to detect even large particles >25 μm due to high transparency. The results corroborate implementation of IFC as an advanced technique for protein particle analysis, offering in-depth characterization of particle physical and chemical properties, and enhanced sensitivity for sub-10 μm and transparent particles.