Protein A Affinity Chromatography
Protein A Chromatography relies on the specific and reversible binding of antibodies to an immobilized protein A ligand. Protein A is a 56 kDa surface protein of Staphylococcus aureus. It is composed of five immunoglobulin-binding domains, each of which are able to bind proteins from many mammalian species, most notably Immunoglobulin G (IgG) through the heavy chain within the Fc region. While the native form of Protein A was used as the ligand for first generation Protein A resins, recombinant forms (rProtein A) produced in E. coli are the most prevalent today. Modifications to the protein structure of the ligand, the advent of ligands composed of single domain multimers, and multipoint attachment have given rise to the caustic stable, high capacity and extremely robust Protein A resins in use today.
Protein A resins are the most frequently used affinity resins in biomanufacturing. Today it is the standard technique for capturing recombinant monoclonal antibodies. Protein A chromatography is a very robust purification procedure and is used as a capture step due to its specificity. Depending on the intended use for the target molecule (antibodies for diagnostic testing) Protein A capturing might be the only chromatographic step required to achieve adequate product purity.
TOYOPEARL Protein A Resins
Tosoh Bioscience offers two Protein A affinity resins, both based on alkaline stable, recombinant ligands coupled to the proven TOYOPEARL polymethacrylate matrix. Both ligands are recombinant protein A variants expressed in E. coli. They are derived from one of the IgG binding domains of protein A. The ligand of TOYOPEARL AF-rProtein A-650F consists of a tetramer of this modified domain. For the ultra-high capacity TOYOPEARL AF-rProtein A HC-650F, this domain was further optimized and expressed as a hexamer in order to further increase IgG binding capacity. Multipoint attachment of the ligands to the TOYOPEARL matrix enhances the chemical and thermal stability of the resins.
The new ultra-high capacity TOYOPEARL AF-rProtein A HC-650M excels all other commercially available protein A media with regard to its IgG binding capacity of >65 g IgG/L resin.