The critical role of mobile phase composition in size exclusion chromatography of protein pharmaceuticals

Size exclusion chromatography (SEC) is the most widely used method for aggregation analysis of pharmaceutical proteins. However SEC analysis has a number of limitations, and one of the most important ones is protein adsorption to the resin. This problem is particularly severe when using new columns, and often column preconditioning protocols are required. This review focuses on the role that addition of various cosolvents to the mobile phase plays in suppressing that protein adsorption. Cosolvents such as salt, amino acids, and organic solvents are often used for this purpose. Because the protein interaction with the resin surface is highly heterogeneous, different cosolvents affect the protein adsorption differently. We will summarize the various effects of cosolvents on protein adsorption and retention and describe the mechanism of the cosolvent effects.