Methodology for predicting the separation of proteins by hydrophobic interaction chromatography and its application...

Hydrophobic interaction chromatography (HIC) is widely used in the downstream processing of proteins. Resolution of HIC is very good, but sometimes not as high as expected. Resolution values could be increased if good operating conditions were selected. In this paper we present a methodology for selecting good operating conditions. First, it is necessary to predict the dimensionless retention time (DRT) of each protein in the mixture. We use a correlation such as DRT = A + Bphi + Cphi2, where phi is the superficial hydrophobicity of the protein, which is calculated considering the hydrophobicity of the superficial amino acids using the Miyazawa-Jernigan scale. Considering that there was little interaction amongst proteins in a mixture at the concentrations investigated (2 g/l of each protein), the behaviour of the proteins in the mixture was considered to be similar to that of the individual proteins. Using simulations it was possible to test different operating conditions for the purification of a target protein from a mixture of proteins and it was possible to select ideal conditions. The methodology developed was also tested for the purification of a recombinant protein from a fermentation extract of yeast producing human superoxide dismutase and the results have been satisfactory.