Protein A Assay

Protein A immobilized on various chromatography media is commonly used to purify antibodies. Even when covalently attached, Protein A can leach off of the chromatography support and co-elute with the antibody. For applications such as the therapeutic use of the antibody, contamination with Protein A must be minimized to avoid any adverse patient effects. This immunoassay method provides sensitivity to detect Protein A contamination to less than 100pg/mL. Leached Protein A is typically bound to the product immunoglobulin present in the sample through its Fc region. This binding of Protein A to the product antibody can interfere in the accurate quantitation of Protein A in some immunoassays by inhibiting the ability of the antiProtein A antibodies used in the assay to bind to the complexed Protein A. This inhibition can result in a significant underestimation of Protein A contamination. Such interference is highly variable from one product antibody to the next but is particularly common with many human IgGs. There are several manufacturers of Protein A and Protein A chromatography supports. In addition to natural Protein A purified from Staphylococcus aureus, there are also various recombinant constructs of Protein A typically produced in E.coli. Some of these recombinant Protein A’s are essentially identical to natural Protein A. However, there are other unnatural recombinant constructs of Protein A that have very significant structural differences when compared to natural Protein A. GE Healthcare sells one such unique construct of Protein A marketed as MabSelect SuReTM. Because of the very different structure of this protein the possibility exists that some antibodies for Protein A will recognize the various constructs of Protein A differently resulting in quantitation inaccuracies. Such is the case with the two older Protein A kits manufactured by Cygnus Technologies, Cat #s F050 & F050H. These older kits recognize natural and as well as conserved recombinant Protein A with essentially the same structure as the purely natural Protein A. However, the F050 & F050H kits will underestimate the true concentration of the MabSelect SuReTM ligand. For this reason, Cygnus has developed the F400 kit. This kit will cross react essentially 1:1 with MabSelect SuReTM relative to natural and highly conserved recombinant Protein A. If you are using natural or structurally conserved, recombinant forms of Protein A and have successfully qualified the F050 or F050H kits you may continue to use those products as they will be manufactured indefinitely. If you are using a recombinant form of Protein A with very significant structural differences from natural Protein A such as MabSelect SuReTM you should use the Cat # F400 kit. If you are evaluating our Protein A assays for the first time, we recommend use of Cat # F400 kit as it offers other robustness and procedural advantages over the older kits, in addition to its ability to more accurately quantitate unnatural forms of Protein A. This kit, Cat # F400, is designed to detect all currently marketed constructs of Protein A. This kit will eliminate most product antibody inhibition and provide accurate quantitation through the use of a carefully qualified sample treatment step (See ‘Limitations’ section). This assay is designed to provide a simple to use, precise, and highly sensitive method to detect Protein A contamination to less than 100pg/mL in the presence of up to mg/mL quantities of humanized monoclonal antibodies. As such this kit can be uses as a tool to aid in optimal purification process development and in routine quality control of in-process streams as well as final product.