Light-scattering detection within the difficult size range of protein particle measurement using flow cytometry.

The phenomenon of protein aggregation is a prominent challenge that impacts biopharmaceutical development at every stage. It may have a number of deleterious effects on protein drugs, including the loss of efficacy, induction of immunogenicity, altered pharmacokinetics and reduced shelf life. At present, multiple methods are available for counting and sizing particles over a broad range of sizes. However, there remains a conundrum in the measurement of particles in the submicrometer range, from 100 nm to 2 μm. In this study, the capability of our new laboratory built FCM system to detect model polystyrene (PS) and silica (SiO) submicrometer microspheres was evaluated and benchmarked against flow field-flow fractionation (FFF). The FCM system showed its advantages on sensitivity, selectivity, reproducibility and speed. The laboratory-built FCM system can readily analyze model PS and SiO microspheres down to 200 nm, covering much of the difficult range from 100 nm to 2 μm. Our data also showed that this machine was able to monitor the distribution of antibody aggregates ranged between 200 nm and 10 μm, suggesting its usability for characterizing protein aggregation in future.