Probing Submicron Protein Aggregation Using Asymmetrical Flow Field-Flow Fractionation (AF4) and Light Scattering
Presented by: Kim Williams, Ph.D., Department of Chemistry, Colorado School of Mines
Presented Live: December 11, 2018
Protein aggregation in biotherapeutic drugs is a major concern as aggregates affect the effective dosage and may cause immunogenic responses in patients. However, a lack of reliable analytical methods has hindered the quantification of submicrometer (0.1 to 1 mm) protein aggregates and a detailed understanding of their formation kinetics. In this study, a simple asymmetrical flow field-flow fractionation (AF4) method is developed and used to investigate nanometer (<0.1 mm) and submicrometer (0.1-1 mm) aggregates of heat-stressed anti-streptavidin (anti-SA) IgG1. The Lumry-Eyring nucleated polymerization model for non-native protein aggregation is fit to AF4 data and kinetic analysis is performed to identify the mechanism of aggregate formation. This work showcases the importance of a separation technique when studying complex and dynamic systems.
