Enantioselective antibody fragments: Dissertation

Antibodies have a unique ability to bind to a wide variety of different molecules, ranging from large protein antigens to low molecular weight organic molecules. The specificity and affinity of the binding varies between antibodies and can be utilised in many applications. Recombinant antibody fragments have advantages over traditional poly- and monoclonal antibodies in production and immobilisation and in the optimisation of their properties. Enantiomers of a chiral compound provide a challenge for separation methods and analytics due to their similar chemical and physical properties. Antibodies provide a highly specific way to fractionate enantiomers in both preparative and analytical applications. In this work two different antibody-based approaches to separate enantiomers of a chiral drug candidate were developed. Antibody fragments were cloned, produced in bacteria and immobilised on a solid affinity support. Repeated affinity purification of enantiomers was achieved in optimised conditions. In the other approach antibody fragments were immobilised inside the nanotubes of an alumina membrane and the bionanomembrane was used to fractionate enantiomers of a racemic mixture. In addition a sample preparation method, antibody-based solid-phase extraction, was developed in a way that can be applied to high-throughput format. Recoveries were comparable to those reported for non-specific sorbents, but with the advantage of the enantioselectivity. The method was used to extract an enantiomer from a spiked buffer or serum. The preparatory sample treatment protocols usually used for serum, e.g. protein precipitation, were not needed. A homology model of one of the antibody fragments was constructed and used to design site-specific mutations in order to adjust the affinity of the antibody to be suitable for the preparative and analytical approaches developed in this work. One of the mutants, ENA5His Tyr96Val, had appropriate properties both in preparative and analytical applications.