Paper BI+SS+AS-TuM5
An Atomic Force Microscopy Based Method for the Determination of Protein Stability

Tuesday, October 30, 2012, 9:20 am, Room 23

A method for the early detection of instability and aggregation propensity of proteins and other biological macromolecules would be valuable for the rapid development of novel biopharmaceutical formulations. The aim of this study was to investigate the potential of atomic force microscopy (AFM) based adhesion force measurements to meet this need. We report the first key step in demonstrating this approach; a clear relationship between how frequently an AFM probe adheres to a protein coated surface and the fraction of unfolded proteins on that surface. Instability and subsequently protein denaturation are commonly linked with protein aggregation, and hence formulation failure. It was found that for the protein bovine serum albumin (BSA), the adhesion between AFM tips and protein-coated samples occurred much more frequently as either the concentration of a denaturant or temperature was gradually increased. We compared this behaviour with fluorescence based studies of the BSA unfolding in solution. Both methods provided us with almost identical ΔG values of stability and 50% unfolding ([D]^50%) values. The data demonstrates for the first time, an AFM based method for protein stability determination. Interestingly, the method also appears to be a good reporter of the protein solution behaviour. With further development this approach could be utilized to screen for instability and aggregation propensity of a given protein therapeutic, in a range of conditions. The ultimate aim is to create a robust technique that can be performed rapidly and routinely.