Measurement of Average Aggregate Density by Sedimentation and Brownian Motion Analysis
Richard E. Cavicchi, Jason King, Dean C. Ripple
The spatially averaged density of protein aggregates is an important parameter that can be used to relate size distributions measured by orthogonal methods, to characterize protein particles, and perhaps to estimate the amount of protein in aggregate form in a sample. We obtained a series of images of protein aggregates exhibiting Brownian diffusion while settling under the influence of gravity in a sealed capillary. The aggregates were formed by stir-stressing a monoclonal antibody (NISTmAb). Image processing yielded particle tracks, which were then examined to determine settling velocity and particle size based on mean square displacement (MSD) analysis. By using MSD analysis on tracks to determine particle size instead of imaged sizewhich is limited by diffraction and out-of-focus errorswe were able to accurately measure the hydrodynamic diameter down to 1µm. A relationship between size derived from the imaged particle area and MSD diameter was observed, suggesting a future method for correcting size distributions based on imaging. Stokes law was used to estimate the density of each particle. Polystyrene calibration microspheres ranging in size from 1µm to 5µm were used to verify the measurement method. It was found that the aggregates were highly porous with density decreasing from 1.079 g/cm3 to 1.030 g/cm3 as the size increased from 1.4µm to 5µm.
, King, J.
and Ripple, D.
Measurement of Average Aggregate Density by Sedimentation and Brownian Motion Analysis, Journal of Pharmaceutical Sciences, [online], https://doi.org/10.1016/j.xphs.2018.01.013
(Accessed May 18, 2022)