Protein separation using affinity ultrafiltration with small charged ligands

by Rao, Suma.

There is a critical need to develop new technologies that provide high resolution protein purification at a price, scale, and throughput needed for the production of high value protein products. Affinity chromatography can provide very high selectivity, but throughput is often reduced by mass transfer limitations. Affinity ultrafiltration is a potentially attractive alternative, but the high cost and practical limitations of the large macroligands used in previous studies has limited the viability of this technique. The overall objective of this work was to demonstrate the feasibility of affinity ultrafiltration using small charged affinity ligands, with the change in protein charge exploited for high resolution separation using an electrically charged membrane. Experiments were performed using a model system of bovine serum albumin and ovalbumin with the dye Cibacron Blue chosen as the charged affinity ligand. The equilibrium binding characteristics between the two proteins and Cibacron Blue were evaluated using a simple ultrafiltration technique over a wide range of solution conditions. Protein filtration experiments were performed to evaluate the affects of Cibacron Blue on protein transmission using essentially neutral and negatively-charged versions of a composite regenerated cellulose membrane. The addition of only 1 g/L of Cibacron Blue to an 8 g/L BSA solution reduced the BSA sieving coefficient through the negatively-charged membrane by more than two orders of magnitude, with this effect being largely eliminated at high salt and with the neutral membrane. Protein sieving data were in good agreement with model calculations based on the partitioning of a charged sphere in a charged pore accounting for the change in net protein charge due to ligand iii binding and the increase in solution ionic strength due to the free ligand in solution. These results clearly demonstrate that the addition of small charged ligands can be used to control protein transmission during ultrafiltration. The high affinity of BSA for Cibacron Blue was exploited to enhance the selectivity for the separation of BSA from ovalbumin. The membrane selectivity was a complex function of the solution conditions, Cibacron Blue concentration, and membrane charge. The addition of Cibacron Blue caused a 30-fold increase in selectivity due to the strong electrostatic repulsion of the highly charged BSA-Cibacron Blue complex. Protein separations were accomplished using a diafiltration process, giving a BSA product with a purification factor of more than 90-fold and a yield greater than 90%. An ovalbumin product was generated in the filtrate with a purification factor of over 10 and a yield of nearly 100%. Subsequent experiments used a tangential flow filtration device that was linearly scalable to commercial manufacturing operations, demonstrating that this process should be feasible even at large scale. The results clearly demonstrate the ability to use small charged affinity ligands with bio-specific binding characteristics to achieve high selectivity protein separations by high performance tangential flow filtration. iv