Probing Submicron Aggregation Kinetics of an IgG Protein by Asymmetrical Flow Field-Flow Fractionation

A lack of reliable analytical methods has hindered the quantification of submicron protein aggregates and a detailed understanding of their formation kinetics. In this study, a simple asymmetrical flow field-flow fractionation (AF4) method with good size selectivity (>0.5) is used to investigate nanometer (<0.1 μm) and submicron (0.1-1 μm) aggregates of heat-stressed anti-streptavidin (anti-SA) IgG1. The Lumry-Eyring nucleated polymerization (LENP) model for non-native protein aggregation is fit to the AF4 data, and kinetic analysis shows that aggregates are formed via slow nucleation and aggregate condensation at long stress times. Comparison of centrifuged and uncentrifuged heat-stressed anti-SA IgG1 AF4 results show the removal of high molar mass submicron aggregates and large material (>20 μm) and suggests that centrifugation may influence the aggregation kinetics. Furthermore, qualitative LENP model analysis of centrifuged and uncentrifuged samples suggests that significant aggregate-aggregate condensation occurs even at early stress times and highlights the potential of AF4 to determine aggregation kinetics for species greater than 1 μm.