Affinity Purification of Immunoglobulins Using Protein A Mimetic (PAM)

Abstract

While antibodies of the G class can be conveniently purified by affinity chromatography using immobilized protein A or G even at large scale, scaling up purification of IgM, IgA, and IgE and IgY still presents several problems, as specific and costeffective ligands for these classes of immunoglobulins are not available. Protein A (1), which is widely used for the affinity purification of antibodies from sera or cell culture supernatants, does not recognize immunoglobulins of the M, A, E, and Y classes well and is not used to capture and purify these immunoglobulins from crude sources. Moreover, these two proteins are obtained from microorganisms or genetically modified bacteria, which carries the risk of affecting the safety of the purified antibodies through the presence of contaminants such as viruses, pirogens, or DNA fragments. As a result, the availability of alternative ligands for the affinity purification of antibodies is highly important from an industrial aspect. After immobilization on solid supports, the mannan binding protein (MBP), an affinity ligand for IgM, provides affinity media useful for IgM isolation based on a temperature-dependent interaction of the ligand with the immunoglobulins (2). The use of immobilized MBP for the purification of IgM is based on the adsorption in the presence of calcium at a temperature of 4°C, and the elution at room temperature of adsorbed immunoglobulins in the presence of ethylenediamineotetraacetic acid (EDTA). This ligand shows low binding affinity for IgG, but binds to bovine and human IgM with lower affinity than murine IgM. However, in addition to the complexity of MBP isolation, functional binding capacities of MBP columns are limited to 1 or 2 mg of IgM per milliliter of support. IgA, which is involved in the first specific defense against natural infection (3) and represents the second most abundant immunoglobulin in serum (4), can be purified through the combination of different fractionation techniques such as ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration (5,6). All these procedures are time consuming, labor intensive, and are not compatible with industrial scaling up. Lectin jacalin, isolated from jackfruit seeds (7), binds to IgA and can be conveniently used for the affinity purification of IgA from colostrum or serum (8). However, several aspects limit the use of this lectin for large-scale purification of monoclonal IgA from cell culture supernatants. First, jacalin is a biologically active lectin, being a potent T cell mitogen and a strong B cell polyclonal activator (9), thus requiring a careful control for ligand leakage into the purified preparation. Second, jacalin binds to the carbohydrate moiety of IgA, and D-galactose is required to elute IgA from affinity columns, which is costly and impractical for large-scale operations.