Improved Method for Evaluating the Dead Volume and Protein–Protein Interactions by Self-Interaction Chromatography

Abstract

Self-interaction chromatography (SIC) is a well-established method for studying protein-protein interactions. The second virial coefficient in SIC is evaluated directly from the measured retention coefficient for the protein using a column packed with resin on which the same protein has been immobilized on the pore surface. One of the challenges in determining the retention coefficient is the evaluation of the dead volume, which is the retention volume that would be measured for a noninteracting solute with the same effective size as the protein of interest. Previous studies of SIC have used a "dead column" packed with the same resin but without the immobilized protein to evaluate the dead volume, but this creates several experimental and theoretical challenges. We have developed a new approach using a dextran standard with effective size equal to that of the protein (as determined by size exclusion chromatography). The second virial coefficient was evaluated for a monoclonal antibody over a range of buffer conditions using this new approach. The data were in good agreement with independent measurements obtained by membrane osmometry under conditions dominated by repulsive interactions. The simplicity and accuracy of this method should facilitate the use of self-interaction chromatography for quantifying protein-protein interactions.