Ultrafiltration Membrane Charge Property And Its Effects On Protein Separation

It has been shown that the cost of downstream separation and high extra-value protein purification process can achieve up to 90 percentage of total cost in the production of biotechnology products. Although ultrafiltration has been widely used in downstream process, since traditional ultrafiltration was viewed as a purely size-based separation, the lower selectivity and the conflict between selectivity and hydraulic permeability limit the applications of ultrafiltration in protein separation. It has been shown that the electrostatic interaction has direct effect on the retention of protein and the operation behavior of the whole membrane system. Membrane charge modification can be an effective way to enhance ultrafiltration membrane selectivity with relatively high permeability at the same time and separate molecules of similar molecular weights. Therefore, research on ultrafiltration membranes charge, and its effect on the membrane properties and protein separation behavior are significant useful to effectively improve the yield of high value protein, reduce cost and develop biologic technology.Negative and positive charge-modified regenerated cellulose (RC) membranes with different surface charge densities can be obtained through chemical reaction. The membrane charge was characterized by zeta potential which was evaluated from streaming potential measurement. The application of charged ultrafiltration membranes were conducted in the following two aspects:First, effect of charged ultrafiltration membrane on protein separation and purification was studied. Effect of surface charge on solvent flow and sieving coefficient of bovine serum albumin (BSA) was evaluated quantitatively. The separation of lysozyme and bovine serum albumin (BSA) on positively, negatively charged regenerated cellulose membranes (RC membranes) and Biomax? polyethersulfone membrane were also studied.Second, effect of ultrafiltration using charged membranes on small charged molecules was studied. Ultrafiltration experiments were performed to evaluate the transmission of three model impurities of Cibacron Blue, Rose Bengal and Vitamin B12 (MW~1kD) with different electrical charges through ultrafiltration membranes (MWCO=10kD) having different surface charge characteristics.The following main results were obtained:(1) Zeta potential calculated from streaming potential measurement can be effectively used to represent the variety of membrane charge. For example, zeta potential of RC membrane decreases from -0.55mv to -11.03mv after negative charge modification, indicating the method of modification used in this study is effective.(2) Charged ultrafiltration membrane can provide better combination of permeability and selectivity. For example, the sieving coefficient of BSA using 12h charged ultrafiltration membrane in 10mM decreases 76.4% while hydraulic permeability only decreases 5.9%. Separation rate and retention can be improved at the same time during actual separation process of protein. This can largely explore the application areas of ultrafiltration in downstream process (such as protein concentration and purification) of biotechnolgy.(3) Electrostatic interaction can be optimized to achieve effective separation of proteins when suitable charged membrane and solution environment (pH and ionic strength) are selected. For example, electrostatic repulsion exists between positive charged RC membrane and positive charged lysozyme molecules, which causes the smallest observed sieving coefficient among native, negative and positive charged RC membrane. Compared with pH=7, observed sieving coefficient of lysozyme at pH=4 would decrease since the lysozyme at pH4 is farther away from the isoelectric point of 11 and has more positive charge. Meanwhile, high ionic strength increased the electrostatic shielding and thus enhanced observed sieving coefficient of lysozyme.(4) Electrostatic interaction strongly affects the clearance of small charged molecules during ultrafiltration with charged membrane. For the lower ionic strength of 10mM, more than 95% Cibacron Blue molecules will be retained on Biomax? 10kD, but about 40% retained for higher ionic strength of 500mM. Therefore, choosing suitable ionic strength and increasing diafiltration multiple can realize effective and economical clearance of high charged small molecules compared with non-charged ultrafiltration membrane.