Selection of chromatographic protein purification operations based on physicochemical properties

The charge characteristics of four proteins (conalbumin, ovalbumin, apotransferrin, and soybean trypsin inhibitor) were determined over a range of pH using an electrophoretic technique (titration curve). HPLC anion-exchange chromatography for each of the proteins was performed to investigate whether electrophoretic and chromatographic results could be correlated. It was found that charge density, estimated from the electrophoretic titration curves (as net charge divided by the protein molecular weight) in most cases correlates extremely well the retention of the protein in terms of time needed for the protein to be eluted from the column. This correlation was far better than that of net charge or that of surface charge (net charge/surface area). The performance of anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration using preparative columns on an FPLC were compared. The ability of each method to separate the components of the mixture of the four proteins was assessed by calculating the resolution of the chromatographic results obtained for each pair of proteins. Using the concept of the deviation factor (DF) between individual properties of pairs of proteins (charge density, molecular weight, hydrophobicity), the separation efficiency factor (eta s) could be calculated. Resolution results showed that anion exchange was the best technique for separating the proteins followed by hydrophobic interaction chromatography and gel filtration. Calculations for efficiency, eta s, confirmed the superiority of anion-exchange chromatography as a separation method over gel filtration. For hydrophobic interaction chromatography, it was not possible to find a suitable parameter to express DF and, consequently, eta s. An appropriate way to express the hydrophobicity of a protein, as a physicochemical property, that can be used in the expression of DF should be investigated.