Protein A Mimetic (PAM) Affinity Chromatography

Abstract

Antibodies of the G class can be conveniently purified, even at large scale, by affinity chromatography using immobilized protein A or G. Because specific and cost-effective ligands are not available, scaling up purification of immunoglobin (Ig)M, IgA, and IgE still presents several problems. 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, and E classes well and is not used to capture and purify these immunoglobulins from crude sources. Recent works pointed out the possibility of using alternative ligands for the affinity purification of IgM. Immobilization of mannanbinding protein (MBP) on solid supports led to 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 room temperature-dependent elution of adsorbed immunoglo bulins in the presence of ethylenediaminotetraacetic acid (EDTA). This ligand shows low binding affinity for IgG, but binds to bovine and human IgM with reduced 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 Ig in serum (4), can be purified by classical chromatographic approaches with an acceptable degree of purity. But several steps, such as ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration, are usually required (5,6). 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.