The immunoglobulin G class can be conventionally purified by immobilized Protein A or G affinity chromatography. However, Protein A does not recognize immunoglobulins of other classes, such as IgM, IgA, and IgE. These immunoglobulins have been purified by a combination of fractionation techniques, such as precipitation, gel permeation and ion-exchange, or by immunoaffinity chromatography such as using anti-IgE antibodies immobilized on solid supports. These processes are complex, laborious, time consuming and of high cost. Recently, organic synthetic mimetic affinity ligands by mimicking the key motifs on fragment B of Protein A, which is known to play an important role in the interaction with the Fc fraction of IgG, have been developed for immunoglobulins purification. The Protein A mimetic ligands (PAM), such as ApA which comprises one triazine ring with one anilino and one tyramino substituents, displayed satisfactory specificity to IgG . But the binding capacities of the PAM media to the antibodies (2-7 mg /ml gel) are low compared with that of Protein A (ca. 20 mg IgG/ml gel), though the PAM density on the support is higher than that of Protein A. This is probably because one PAM ligand is too small to provide strong enough affinity interactions with a huge antibody molecule. Several PAM molecules on the support might capture only one molecule of antibody. This project is to develop new Protein A mimetic ligands (PAM) by linking twice as many functional groups as before. The resulting PAMs are expected to exhibit stronger affinity binding and provide a high binding capacity to antibodies.
In addition, most of the immunoglobulins purification was carried on particle-based affinity matrices. These matrices present several drawbacks; for example, matrices are compressible, and purification is operated at low flow rates and high back pressure, which limit its application on an industrial scale. Recently emerged membrane-based affinity chromatography offers several unique advantages, including high flow rates at lower back pressure, faster binding kinetics, higher efficiency, ease of scale up, lower cost and less processing time. Affinity membrane chromatography, therefore, will be studied.
