What is affinity chromatography?

Exploiting highly specific biorecognition phenomena makes this method ideally suited to the purification of biomolecules. This is done by first identifying an affinity ligand specific for the target molecule and then binding it to a solid support. The sample mixture containing the target molecule can then be passed over this affinity ligand to allow the target molecule to bind its ligand and the rest of the sample to be washed away. The target molecule can then be stripped from its bound ligand by altering the pH of the column to inhibit binding, yielding a purified solution.

This simple ‘load-wash-elute’ strategy combined with the exquisite specificity of biorecognition and high yields has made affinity chromatography the gold standard for protein purification. A single pass of a cell lysate sample through an affinity column can give more than a 1000-fold increase in concentration of a given protein.

Affinity chromatography column

Developed almost 50 years ago by Cuatrecasas, Wilchek and Anfinsen (link is external) this method still represents one of the most powerful tools for studying biologically active compounds. Academic and industrial researchers alike now use affinity chromatography to isolate proteins of interest. The proteins can then be studied to determine their structures or analysed to identify their binding partners. The method has proven an indispensable tool for studying different biological processes, such as the mechanism of action of enzymes, hormones and protein–protein or cell–cell interactions. Its importance was verified by the award of the 1987 Wolf Prize in Medicine to Cuatrecasas and Wilchek for the invention and development of affinity chromatography and its applications to biomedical sciences. In drug development and medicine affinity chromatography can remove toxic impurities from a biopharmaceutical solution or from a solution such as blood, where the removal of heme peptides by binding immobilised serum albumin (link is external) in affinity chromatography formed the basis of haemoperfusion.

Historically the most common use of affinity chromatography within the biotechnology sector has been for the purification of antibodies using standard affinity resins like protein A and protein G, which are robust enough to withstand the harsh conditions used in affinity chromatography and for cleaning the affinity columns for repeated use and possess high affinity for antibody proteins. These antibodies are used as research reagents, as part of diagnostic assays and increasingly as therapeutics. However, the increase in number and variety of alternative biological products (link is external) entering the market is driving the development of alternative affinity ligands to allow the purification of these alternative biologics. In 2010 the sales of protein-based therapeutics across Europe and the USA reached $108 bln (link is external), less than half of which derived from monoclonal antibodies.

Clearly with such high revenues at stake the purification of therapeutic proteins by affinity chromatography is of great interest to the biotechnology and biopharmaceutical industries. A study (link is external) by Genetic Engineering and Biotechnology News have shown that even in the purification of monoclonal antibodies there is keen interest in the industry in alternatives to the standard affinity resin of protein A. Although protein A is associated with high affinity for antibodies, mechanical robustness to the harsh conditions of affinity chromatography and column cleaning, and it is accepted by regulatory bodies, the high costs associated with use and the bottleneck in capacity mean that users are starting to look elsewhere (link is external) for their affinity chromatography needs.

Despite the interest in new and different affinity ligands that can isolate the diverse range of biologics entering the market use across industry has shown that this technique is not the chromatography nirvana (link is external) it was first presumed to be. Affinity ligands must be highly specific for the target protein, show high affinity to allow its isolation from a complex sample yet reversibly release it under the appropriate conditions, be robust to the extremes of pH needed to elute proteins from the columns and that are part of the cleaning-in-place procedures required to meet GMP regulations. Even with this list of essential criteria as a mere starting point for any affinity resin new affinity ligands are emerging for the range of desired targets. A new review (link is external) from the New University of Lisbon covers recent case studies of proven alternative affinity chromatography ligands, including Affimer proteins, that offer advantages such as lower production costs, rapid ligand development and high robustness. So perhaps affinity chromatography is not chromatography nirvana yet, but potentially in the future it could be.