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Antibody affinity purification

Liquid chromatography is used to purify antibodies from other molecules in biological samples. Liquid chromatography involves passing mixtures through a solid phase to allow target molecules to bind or pass through. These techniques vary widely and include affinity binding, ion exchange, and size exclusion chromatography.

Affinity chromatography is a separation method that allows the isolation of proteins or other biomolecules through strong but reversible binding to a chromatography matrix that has an appropriate ligand. Affinity chromatography is a popular first step in purifying biologic molecules, because up to 95% purity is possible in a single step, with high recovery. Antibody affinity chromatography is typically performed with a protein A or protein G for research applications. Protein A is the popular choice for biomanufacturing.

Protein A binding and chromatography

Polyclonal and monoclonal antibodies (mAbs) require varying levels of purity based on the downstream use. In biomanufacturing, very high purity is required for the final product. Affinity chromatography using a protein A ligand is a popular choice. Protein A is a cell wall protein that’s derived from Staphylococcus aureus bacteria. Native protein A is typically not used in bioproduction. Instead, protein A is modified to improve binding or stability to cleaning agents, for example.

Protein A affinity chromatography is one of the most common affinity chromatographic separation techniques. It captures recombinant monoclonal antibodies (mAb) or other antibody-derived molecules, traditionally using protein A columns filled with resin. However, Fibro PrismA units filled with a protein A-derived fiber material can offer advantages in specific applications such as drug discovery and scale up of small batch sizes. These units are based on Fibro technology, which allows residence times of seconds.

Protein A purification employs specific binding between immobilized protein A ligands and the antibodies of interest. Protein A can bind a diverse range of antibodies including immunoglobulin G (IgGs).

Antibody affinity FAQs

Below are answers to some frequently asked questions about antibody affinity.

What is antibody affinity?

Antibody affinity is the strength of the interaction between an antibody and a binding partner, such as an antigen or ligand (e.g., protein A or protein G).

What is IgG binding?

IgG is the major type of antibody found in blood and fluid outside of cells. IgG binding refers to the strong but reversible interaction between the molecule and binding parter. In the body, this could be a pathogen or other substance considered to be foreign. IgG binding also refers to the interaction with a different binding partner, such as proteins A, G, and L.

What do antibodies bind to?

Antibodies are proteins that are part of the immune system. They bind to foreign substances, often part of a virus or other pathogen that represents an antigen. Through antigen binding, antibodies help to clear the body of material that could cause infection or other adverse effects. Antibodies also bind to ligands like protein A, a property that is used to separate them from many other impurities when producing a therapeutic molecule.

What is affinity chromatography?

Affinity chromatography is a separation technique that helps isolate specific biomolecules or groups of biomolecules from a mixture, based on specific binding interactions. Examples include antibodies binding to protein A, proteins binding to a dye substance, and histidine-containing proteins binding metal ions as in immobilized metal ion affinity chromatography (IMAC).

While affinity chromatography can be used for single-step purifications due to its high selectivity, it is more commonly used for the first capture step, followed by one or more polishing steps to remove remaining impurities.

Which antibody affinity product should I use?

Cytiva offers several types of affinity chromatography resins and fiber-based chromatography units to purify antibodies. The Purify App will help you find the right product for your needs.