Enantioselective Affinity Chromatography of a Chiral Drug by Crystalline and Carrier-Bound Antibody Fab Fragment

An antibody Fab fragment, ENA5His, capable of enantioselective affinity chromatographic separation of a chiral drug, finrozole, was stabilized against organic solvents by chemical cross-linking. High concentration of methanol is needed to release the bound drug from the antibody fragment. However, in native form the antibody fragment is unstable at these conditions. We used cross-linked protein crystal technology to stabilize the antibody fragment molecule. Glutaraldehyde cross-linked ENA5His crystals (CLAC) packed in a column separated pure enantiomers from the racemic mixture of the drug. CLAC was totally stable at the elution conditions, enabling reuse of the immunoaffinity column packed with CLAC. However, the specific drug enantiomer binding capacity of CLAC was only 50% of the corresponding capacity of immobilized ENA5His. We were also able to cross-link immobilized ENA5His by glutaraldehyde. This method produced a protein matrix with high activity and stability in the elution conditions.