Predicting protein retention time in ion-exchange chromatography based on three-dimensional protein characterization

The isoelectric point (pI), molecular weight (MW) and aqueous two-phase partitioning coefficients of a set of model proteins were related to retention time in cation-exchange chromatography using partial least squares regression. A three-dimensional method which combined hydrophobic partitioning and two-dimensional electrophoresis was used to determine those three properties for a mixture of proteins. The regression models fit well (R2 = 0.913 and 0.873 for two resin types) considering the limited property basis, and were able to predict results for a small test set of proteins. The models showed that greater size and charge increased retention time, while the net influence of hydrophobicity depended on the base matrix type. This establishes the potential for the intended application to complex mixtures of host cell proteins.