(465a) Protein Separations by Ultrafiltration: Exploiting Small Charged Affinity Ligands | AIChE

(465a) Protein Separations by Ultrafiltration: Exploiting Small Charged Affinity Ligands

Authors 

Rao, S. - Presenter, Pennsylvania State University
Zydney, A., The Pennsylvania State University


Recent studies have demonstrated that it is possible to control protein transport in ultrafiltration by the addition of a small charged ligand which preferentially binds to the protein of interest. Binding of the ligand causes a significant change in the net protein charge and in turn the rate of protein transmission through a charged membrane because of differences in electrostatic interactions. The objective of this work was to demonstrate the potential of using a small charged ligand for protein separations using affinity ultrafiltration, with the specificity of the ligand binding used to dramatically enhance the selectivity of the membrane separation. Experiments were performed using a model system employing mixtures of Bovine Serum Albumin (BSA) (MW 67 kD, pI 4.8) and ovalbumin (MW 44 kD, pI 4.5). Cibacron Blue F3GA (CiB) (MW 0.77 kD) was used as the affinity ligand. CiB has three negatively-charged sulphonic acid groups and is known to bind at multiple sites on the surface of BSA. Data were obtained with negatively charged versions of a Composite Regenerated Cellulose (CRC) membrane prepared by covalent attachment of a sulphonic acid functionality to the membrane surface. Binding experiments indicate that approximately 11 moles of CiB bind per mole of BSA, with significantly weaker interactions with ovalbumin. Sieving experiments were performed over a range of pH, ionic strength, and CiB concentrations using CRC membranes with different surface charge densities to identify appropriate conditions for protein separations. The selectivity for the separation of BSA and ovalbumin increases by 35-fold upon addition of 1.4 g/L of CiB to a binary mixture containing 10 g/L BSA and 6 g/L ovalbumin. This dramatic increase in selectivity is due to the significant increase in BSA retention arising from the strong electrostatic repulsion between the negatively charged protein-dye complex and the negatively-charged membrane. Ovalbumin transmission was largely unaffected by the addition of CiB due to the low level of ligand binding. The optimal concentration of CiB was a complex function of the protein, solution, and membrane properties. Excess levels of CiB reduced the selectivity due to the increase in electrostatic shielding provided by the free (unbound) ligand. These results demonstrate the potential of using a small charged ligand to enhance protein separations by ultrafiltration, and they provide a framework for the development of this type of novel membrane process.

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