USING the UNCONVENTIONAL NUCLEOTIDE Binding-Site for Affinity Purification of Antibodies

This talk will describe a novel method to purify monoclonal and polyclonal antibodies (immunoglobulins) from cell lysates and ascites fluids using affinity chromatography where a small molecule (nucleotide analog) presented on the immobile phase binds to the unconventional nucleotide binding site that is present on all immunoglubulins.  We targeted the nucleotide binding site present on each Fab arm of imminoglobulins with a small molecule that was discovered using computational methods for in silico screening.  It has a K_d between 10 mM to 1 mM for this site on various IgG & IgE antibodies we tested originating from various species.  Rituximab was used for 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 (2M NaCl in 50 mM phosphate pH 7.0).  Column’s capture efficiency was >95%, with a purified antibody of >98% yield.  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, and effects of injection buffer.  This method provides a superior alternative to the protein-A affinity purification method that is widely used for purification of humanized and chimeric antibodies.