Studies of Loose Protein Aggregates by Flow Field-Flow Fractionation (FFF) Coupled to Multi-Angle Laser Light Scattering (MALLS)

Abstract

Flow FFF was used to study the aggregation properties of an antibody, two different proteins and a protein-polysaccharide conjugate (PPC). Two FFF methods were applied, the standard FFF method with separation (with focus flow and with cross flow) and FFF without separation (without focus flow and without cross flow). The FFF method without separation was found to be less destructive on the aggregates. By comparing the two FFF methods, loose aggregates could be detected for the antibody and the two different proteins. These loose aggregates reversed to mainly monomers during standard FFF analyses. The presence of these aggregates was confirmed by complementary spectroscopic and microscopic techniques. In the case of the PPC, focus flow and cross flow induced the formation of bigger and smaller aggregates. These results document the importance of using different methods to characterize the complexity of protein aggregation.