Development of cross-linked antibody Fab fragment crystals for enantioselective separation of a drug enantiomer

A novel immunoaffinity separation material, cross-linked antibody crystals (CLAC), was developed by crystallization and cross-linking of a recombinant antibody Fab fragment ENA5His capable of enantiospecific separation of a chiral drug, finrozole. Crystallization conditions of the antibody fragment were screened by hanging drop vapor diffusion experiments. The small-scale vapor diffusion crystallization was easily scaled up to 10-mL batch crystallization with a 70% yield. Also, the low affinity mutant of ENA5His having one single amino acid mutation crystallized at the same conditions. Simple glutaraldehyde cross-linking of the crystals yielded CLAC, which were insoluble in water, 5−100% methanol, and 2% DMSO in PBS buffer. Batch experiments showed that the CLAC material was functional as it specifically bound the desired enantiomer from the drug racemate.