ABSTRACT
For the pharmaceutical development of proteins, multiple methods of analysis are recommended for evaluating aggregation, and the development of more quantitative and simpler analytical techniques for subvisible particles is expected. This study introduces the Pinched-Flow Fractionation (PFF)-Coulter method, which combines the Pinched-flow fractionation (PFF) and Coulter methods to analyze the concentration of submicron-sized particles. The PFF method separates the particles by size. Separated particles were individually detected using the Coulter method. We have utilized the PFF-Coulter method to quantitatively analyze particle concentrations using standard particles, evaluate detection limits, variability, and correlation between theoretical and measured values, and analyze mixtures of different particle sizes. The PFF-Coulter method allows for quantitatively analyzing of particle sizes from 0.2 to 2.0 µm. The quantifiable weight concentration range was 2.5 × 10-2 - 50 µg/mL, and the number concentration range was 104-1010 particles/mL. The sample volume was small (<10 µL). The PFF-Coulter method is capable of quantitative analysis that complements data from conventional measurement techniques, and when used in conjunction with existing submicron-size particle analysis techniques, will enable more accurate particle analysis.
