Screening for protein-protein interactions with asymmetrical flow field-flow fractionation

We describe a new method for screening protein-protein interaction of biopharmaceutical molecules at dilute concentrations to predict development issues at high concentration. The method is based on Asymmetrical Flow Field-Flow Fractionation (AF4) measurements using well known effects of protein-protein attraction on the fractionation profile due to elevated protein concentrations occurring close to the membrane. We explore the effect for 4 different monoclonal antibodies and show that the profiles obtained are quite different. Interestingly, we find that the recovery in AF4 correlates with the diffusion interaction parameter, which is a standard method for the analysis of protein-protein attraction. The results are insensitive to the protein concentration and buffer composition of the sample solution and only depend on the absolute amount of protein loaded and on the running buffer. This makes the method highly suitable for developability assessment in a compound discovery workflow.