Abstract
Particle characterization is vital in ensuring the safety and efficacy of biopharmaceutical products throughout their development and manufacturing processes. Rotational motion of irregularly shaped particles, such as protein aggregates, causes larger scattering amplitude fluctuations than that of spherical particles. In this study, we demonstrated that protein aggregates can be differentiated from silicone oil droplets in the submicron size range (0.1-1.0 µm) based on fluctuation in scattering amplitude determined by three-dimensional homodyne light detection (3D-HLD) at single-particle level with no sample pretreatment. Monoclonal antibody aggregates were differentiated from silicone oil droplets using the fluctuation in scattering amplitude as a parameter with a misclassification rate below 8%. The average refractive indices of classified particles were estimated by simultaneous acquisition of the scattering amplitude and hydrodynamic particle size, providing complementary information about the features of the classified particles. These results demonstrate the effectiveness of 3D‑HLD as a powerful characterization method for submicron particles.