Distribution of Average Aggregate Density from Stir-Stressed NISTmAb Protein

Abstract

Quantifying the heterogeneous nature of protein aggregates is important to understanding the impact aggregates may have on the performance of antibody therapeutics. The spatially averaged density ρp of aggregates, defined as the total mass, including water, divided by the volume, is a parameter that can be used to relate size distributions measured by orthogonal methods, to characterize protein particles, and perhaps to estimate the amount of aggregated protein in a sample. We report measurements by two methods on the distribution of density values for different aggregate sizes, where the aggregates were produced by stir-stressing fluorescently labeled monoclonal antibody (NISTmAb). A fluorescence microscope was used to image particles. Each particle was analyzed for brightfield equivalent circular diameter (ECD) and fluorescence intensity and the results converted to average density. Measurements were also obtained using video holography. The aggregates were highly porous with median density decreasing from 1.07 g/cm3 to 1.02 g/cm3 as the size increased from 0.9 μm to 6 μm by fluorescence, and similar results by video holography. The distribution in density for a given particle size was asymmetrical and broad. For example, particles with an ECD of 2.5 μm ranged in density from 1.005 g/cm3 to 1.1 g/cm3.