Modification of formed-in-place microfiltration membranes for specific protein separations and fundamental studies of affinity membrane separation processes and protein-ligand interactions

Formed-In-Place (FIP) titania microfiltration membranes were coated with poly (ethylene imine) (PEI) and subsequently crosslinked with multifunctional glutaraldehyde and 1,4-butanediol diglycidyl ether to a stable layer. These membranes were first studied as anion exchange membranes for protein separations using sequential separation procedures. The effects of operating parameters, pH and concentrations of protein solutions on binding and purification properties were determined. In order to achieve more specific protein separations, an affinity ligand Cibacron Blue F3GA (CBF3GA) was immobilized on the PEI layers. The separation properties of the affinity membranes were investigated with human serum albumin (HSA) solutions, including the effects of pH and ionic strength of the protein solution on the binding capacity of the membrane and the influence of the crossflow velocity on the frontal elution profile.; A mathematical model was developed to analyze the frontal performance of the affinity membranes for the protein separations. Predictions by the mathematical model using equilibrium batch adsorption data dissociation constant, K{dollar}sb{lcub}rm d{rcub}{dollar}, and maximum amount adsorbed, q{dollar}sb{lcub}rm m{rcub}{dollar}, and characteristics of the membrane for the breakthrough of HSA were described.; The interaction mechanisms between the albumins and Cibacron Blue F3GA and its immobilized form, Blue Dextran, in aqueous solution was studied by spectrophotometric titration. The results showed that the association of CBF3GA to HSA is not influenced by the attachment of the dye to dextran. However, the binding of the dye molecule to dextran chain apparently hindered the accessibility of the dye to the highest affinity bind sites on the bovine serum albumin (BSA). BSA interacts with free dye with much higher affinity than with immobilized dye. The competitive titration of the dye-albumin complex with bilirubin and potassium palmitate revealed that the bilirubin, in contrast to the results of Leatherbarrow and Dean (44), did not compete with the dye for the same binding sites on both HSA and BSA. However, palmitate significantly dissociates the binding between the dye and BSA but did not appreciably affect the interaction between the dye and HSA.