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(18b) A Small Molecule Based Affinity Membrane Spin Column for Antibody Purification

Authors: 
Mejia, F., University of Notre Dame
Mustafaoglu, N., University of Notre Dame
Canonico, M., University of Notre Dame
Bilgicer, B., University of Notre Dame
Antibodies have extraordinary specificity and affinity to antigens, which in turn makes them important candidates to be used in numerous applications including clinical, academic and industrial, while new implementations are continuously being explored in academic research. A major contributor to the cost of antibody applications is mostly linked to the downstream production process, or more specifically the usage of Protein A (or G) affinity columns for purification of antibodies. These columns are expensive and have short life cycles with several obstacles that preventing them to be used repeatedly. Here, we design a novel affinity spin column for the purification of monoclonal and polyclonal antibodies from complex protein mixtures such as cell culture media of hybridomas and ascites fluids. This method utilizes the nucleotide-binding site (NBS) that is located on the Fab variable domain of immunoglobulins to enable capturing of antibody molecules on a membrane affinity column via a small molecule, which has a moderate binding affinity to the NBS. A ring structured small molecule was attached to the regenerated cellulose membrane in order to target the NBS for capturing antibodies on the spin column. Antibody capture was accomplished by running of samples in an equilibration buffer (20 mM TRIS pH 7.0) through the spin column at 500 rpm and elution of the antibody was achieved by running a mild elution buffer (0.5 M KCl in 20 mM TRIS pH 7.0). Several pharmaceutical antibodies were tested on the spin column, and the results indicate that the column efficiency for antibodies was >95%, with a purity level of >95%. Furthermore, small molecule based affinity column has a strong binding specificity towards antibodies, and has a dynamic binding capacity of ~48 µg/mg which is greater than any current protein A (or G) spin columns. The quality and the capacity of this small molecule affinity membrane spin column method is further evaluated for a number of parameters such as: injection concentrations, volumes, number of membranes, antibody/protein-contaminant combinations, and column reusability and stability. This study provides a good tool for fast antibody purification at a small scale.