Affinity Chromatography
Affinity Chromatography (AFC) makes use of specific binding interactions between molecules. This specificity makes it attractive as a capture step to isolate the target molecule from crude extracts. A particular ligand is chemically immobilized to a solid support so that when the crude extract is passed over the column, those molecules having a specific binding affinity to the ligand become adsorbed. After the non-specifically binding components are washed away, the target molecule is desorbed from the support, resulting in its purification from the original sample.
In AFC the mechanism of binding and elution varies depending on the immobilized ligand used. In Protein A chromatography elution is accomplished by decreasing the pH. In other cases such as with Chelate resins (for Immobilized Metal Affinity Chromatography or IMAC) the binding and elution mechanism may change through the use of different metals or chemical modifiers.
Applications
Dynamic binding capacity (DBC) values for AFC materials vary with the ligand and are typically on the low side. However because most of the capacity is used for the target, with feedstock impurities remaining unbound, AFC resins are typically used for the Capture and Intermediate purification steps of a manufacturing process.
Protein A resins are the most frequently used affinity resins in biomanufacturing. Protein A specifically adsorbs immunoglobulin G and today Protein A chromatography is the standard technique for capturing recombinant monoclonal antibodies.
TOYOPEARL and TSKgel Affinity Resins
TOYOPEARL affinity resins are composed of hydrophilic, dimensionally stable base resins that exhibit excellent pressure-flow characteristics. These resins use the TOYOPEARL HW-65 SEC resin as a base bead.
There are many custom designed affinity ligands available to the chromatographer. TOYOPEARL affinity resins are functionalized with chemically active groups or group-specific ligands. Resins with activated functional groups are ready to directly couple a protein or other ligand. Resins with reactive groups require carbodiimide coupling or reductive amination to achieve a stable covalent linkage.