Antibody Purification Via Affinity Membrane Chromatography Utilizing Nucleotide Binding Site Targeting

Current antibody use in therapeutics and research is increasing rapidly in both complexity and demand. Therefore, there is a constant, growing need for more efficient laboratory tools and methods to facilitate antibody engineering, production, and, in particular, purification. As of now, the predominant method of antibody purification is affinity chromatography, wherein the affinity molecule is Protein A or Protein G, proteins derived from bacteria that bind to IgGs via the Fc region. This process delivers a high yield and purity, but has several drawbacks, including high cost, difficulty in isolating the affinity molecules, harsh elution steps, leaching of the affinity molecule, and limited reusability.

Our lab has developed an alternative method. The Nucleotide Binding Site (NBS) is a conserved, underutilized binding site in the Fab variable region of the antibody. We have identified a number of ligands that have moderate affinity for the NBS. For the chromatographic matrix, regenerated cellulose (RC) membranes were selected for their mechanical stability, inexpensive price, and ease of use. Membrane chromatography allows the solute and matrix to interact in the open pores of the membrane, rather than the dead pores of a resin molecule, thereby decreasing the pressure drop and allowing rapid flow-induced mass transport. By conjugating our NBS-ligand to the RC membranes, it is possible to selectively capture IgGs from solutions containing contaminants such as cell culture media of hybridomas or ascites fluids. Furthermore, the elution step is extremely gentle, only requiring 0.5 M KCl salt buffer at neutral pH, as opposed to the highly acidic (pH 2.5-3.0) conditions of the rivaling Protein A method.

After functionalizing RC membranes, they can be packed into columns in a variety of formulations. Previously, our lab has demonstrated their effectivity in HPLC columns. More recently, we have scaled them down and specialized them for use by laboratory researchers. Packing 5-20 membranes into a centrifugal spin column allows unrivaled ease and efficiency in many research settings, with each small-scale purification taking only 5 minutes or so. Such spin columns have proven very effective in our laboratory, with antibody recovery of >90% in samples spiked with contaminants. They also compare very favorably with competing products which utilize Protein A. Not only is their antibody recovery comparable or higher, but also they are highly reusable, cheaper, and require much gentler elution conditions.