(336f) An Affinity Chromatography Method for Purification of Antibodies Utilizing Nucleotide Binding Site Targeting with a Small Molecule

Mustafaoglu, N., University of Notre Dame
Deak, P., University of Notre Dame
Kiziltepe, T., University of Notre Dame
Bilgicer, B., University of Notre Dame
We describe a novel affinity membrane chromatography technique (m-NBSTryptamine) for the purification of monoclonal and polyclonal antibodies from complex protein mixtures such as cell culture media as well as ascites fluids. m-NBSTryptamine method utilizes the nucleotide-binding site (NBS) that is located on the Fab variable domain of all antibodies to facilitate capturing antibodies on a membrane affinity column via a small molecule, tryptamine, which has a moderate binding affinity to the NBS. Regenerated cellulose membrane was selected as a matrix due to the major advantages over traditionally used resin-based affinity systems. In this study, we used the pharmaceutical antibody Rituximab in our proof of concept experiments. Antibody capture was accomplished by injection of samples while running equilibration buffer (50 mM sodium phosphate pH 7.0) and elution of the antibody was achieved by running a gradient of mild elution buffer (3M NaCl in 50 mM phosphate pH 7.0). Our results demonstrate that the m-NBSTryptamine column efficiency for Rituximab was >98%, with a purity level of >98%. This small molecule affinity purification method is further tested for a number of necessary parameters such as: injection concentrations, volumes, wash/bind time, elution gradient, antibody/protein-contaminant combinations, effects of injection buffer, post-purification antigen binding activity of antibodies, and column reusability and stability. The m-NBSTryptamine method provides a superior alternative to the protein-A affinity purification method that is widely used for purification of humanized and chimeric antibodies. We hope that in the long term, this new method will help to increase the availability of antibody based treatment and diagnostic systems to patients, as well as reducing the cost of antibody molecules used in scientific research.