High performance tangential flow filtration using charged affinity ligands

High performance tangential flow filtration is an emerging technology for protein separations. The objective of this work was to examine the use of small charged affinity ligands to control the protein charge and thus extend the potential applicability of this separation technique. Experiments were performed using binary mixtures of bovine serum albumin and ovalbumin, with Cibacron Blue added as an affinity ligand. The addition of Cibacron Blue caused a large increase in the selectivity due to the increase in BSA retention associated with the strong electrostatic repulsion between the negatively-charged protein-ligand complex and the negatively-charged cellulosic membrane. Appropriate conditions for the separation were identified using total recycle experiments in a small-scale tangential flow filtration device. A diafiltration process was designed to separate BSA from ovalbumin, with the Cibacron Blue recovered in a second stage operated with a buffer containing NaSCN to displace the dye from the protein. The results demonstrate the ability to use small charged affinity ligands with bio-specific binding characteristics to enhance separations by high performance tangential flow filtration.