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Protein A Chromatography

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 native to the cell wall of the bacterium 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, the recombinant form (rProtein A) produced in E. coli is 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. 

Applications

Protein A resins are the most frequently used affinity resins in biomanufacturing. Protein A chromatography is a very robust purification procedure and is used as a capture step due to its specificity. Today Protein A chromatography is the standard technique for capturing recombinant monoclonal antibodies. 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 three Protein A affinity resins, all based on alkaline stable, animal-origin free, recombinant ligands coupled to the proven TOYOPEARL® polymethacrylate matrix. They are derived from one of the IgG binding domains of protein A. For the ultra-high capacity TOYOPEARL® Super A resin, this domain was further optimized and expressed as a hexamer to further increase IgG binding capacity. Multipoint attachment of the ligands to the TOYOPEARL® matrix enhances the chemical and thermal stability of the resins.

All three resins are based on the TOYOPEARL® HW-65F base bead with a particle size of 45 μm and exhibit excellent pressure-flow characteristics. The new ultra-high capacity TOYOPEARL® Super A exceeds all other commercially available protein A media with regard to its IgG binding capacity of >70 g IgG/L resin.

 


TOYOPEARL® Super A resin provides a cutting-edge experience in protein A media, blending top-tier critical performance attributes with exceptional operational flexibility.

It offers industry-leading critical process performance in eluting at a milder pH with low elution volumes and minimizing the process impurities to make the process robust, safe, and compliant performance parameters in addition to the state-of-the-art operational flexibility to support high titers with high dynamic binding capacity (DBC), enhanced alkaline stability to withstand rigorous CIP and excellent pressure flow behavior to make process more efficient.

The ultra-high capacity TOYOPEARL® AF-rProtein A HC-650F  exceeds all other commercially available protein A media with regard to its IgG binding capacity of more than 65 g IgG/L resin at residence times of 5 minutes. Its IgG capacity still exceeds 50 g/L at 2 minutes residence time with feed stock concentrations from 1.0 g/L to 10.0 g/L. Improved mass transfer characteristics allow it to maintain a larger percent of its capacity at lower residence times relative to agarose based, caustic stable resins.

The standard TOYOPEARL® AF-rProtein A-650F resin binds human and mouse immunoglobulin G, IgM, and Fab fragments. It provides a static IgG binding capacity of more than 45 g/L and a typical dynamic IgG binding capacity of more than 30 g/L at 2 minutes residence time (1 g/L protein load). Fast mass transfer kinetics support high binding capacities at high flow rates.

TOYOPEARL® AF-rProtein A-650F is also stable in ethanol, 6 mol/L urea, 6 mol/L guanidinium chloride, and 1% phosphoric acid, respectively. Static binding capacity of the resin is not impaired when heated for 30 minutes to temperatures of up to 90 °C.