Separation of proteins via pH parametric pumping with electric field

A new semi-continuous parametric pumping process for the separation of protein mixtures has been developed, based on cyclic variation of pH and electric field. The model system used for this process consisted of a mixture of human serum albumin and human hemoglobin in contact with CM Sepharose cation exchanger. Experimental results show that the protein separation via the pH parapump with electric field is two to three times greater than the results which are obtained in batch chromatography or via either single-column or multi-column parapumps with cyclic variation of pH and ionic strength. The separation in the new process depends on selective adsorption of one protein onto the ion exchanger, and also on a difference in the migration velocities of the two proteins in the presence of an electric field. Protein A (hemoglobin) was stripped from the top stream and enriched in the bottom stream by adsorption at low pH and desorption at high pH. The top stream was thereby enriched in Protein B (albumin) relative to Protein A. Protein B was then stripped from the bottom stream by applying an electric field across the parapump during the desorption stage of each cycle of the process. Various modes of operation and a number of experimental parameters were considered. A low ionic strength organic buffer (0.05M Tris-Maleate + NaOH) was selected in order to maximize field strength with minimum heat generation, and the optimum fluid displacement for this buffer was determined experimentally. The effect of the electric field on the adsorption and desorption concentration waves and pH profiles was examined for different bulk velocities and alternate field polarities. These results were applied to the final process. A mathematical model was developed to verify the experimental data, based on finite mass transfer and constant electric mobility. The adsorption isotherm was assumed to be linear in the region of interest. The transport equations were solved by the finite difference method and also by the Stop & Go model. A pH wave lag was necessarily incorporated into the mathematical model. The pH wave velocity was assumed to be constant. The model agrees well with the experiemental data. Protein separation was optimized mathematically as a function of top or bottom feed rates and as a function of total production rate. Various parametric pumping processes have been reported based on temperature, pressure, and pH. The use of electric field polarity as a thermodynamic variable in a parapumping process is a promising new concept in protein separation technology