Affinity Chromatography

Affinity chromatography (AF) is a separation method that has greater selectivity than ion-exchange chromatography. It encompasses chromatography techniques such as immunoaffinity chromatography, heparin chromatography etc.

The technique is one of the steps used generally in downstream processing.

Affinity chromatography began in the 1960s and progressed as a biochemical separation technique through the 70s. Cuatrecasas (1970) conducted a great deal of early work into developing methods for derivatizing (chemically binding) ligands onto matrices such as agarose and polyacrylamide. Of interest are the methods of covalent attachment of ligands through amino, carboxyl, phenolic, or imidazole groups of the ligand.

An Example Using Affinity Chromatography

One example is the isolation and purification of lysozyme from hen egg white (Chiang et al., 1993). This was a two-step procedure. The egg white is diluted up to 9 fold using a sodium phosphate buffer. It is processed by sequential dilution diafiltration using an ultrafiltration membrane with a molecular-weight cut-off of 300 kDaltons.

The membrane process is only so good for producing crude enzyme preparations with limited specific activity. It is however an excellent approach to removing substantial amounts of impurities that could ruin a purification as well as preparing the feedstock for the 2nd process step of affinity chromatography. raised the specific activity of the lysozyme 6-fold and there was recovery of 96% of the enzyme. This membrane process is also typical for operations preparing the solution containing the material to be selected for. 

The material selected, lysozyme was purified to an exceptionally high level using affinity chromatography. The enzyme can be bound to the matric either using a batch mixing process or by packing the matrix into a column. Both have their merits! The objective here is to optimise the elution process. It is the case that using a column generally uses less eluant and more enzyme is invariably recovered. The approach also applies to other types of chromatography as well as AF.

To achieve this, the permeate collected from diafiltration was passed through an affinity chromatography column using chitin as the adsorbent. The enzyme was eluted using 0.1M phosphate buffer at pH8 followed by a 2nd elution buffer of 0.1M acetic acid. This second step meant the product had an extremely high specific activity of 70,400 units/mg protein. The overall lysozyme recovery was 79% which is extremely high for any purification process and made possible by using AF.

References

Chiang, B. H., Su, C. K., Tsai, G. J., & Tsao, G. T. (1993). Egg white lysozyme purification by ultrafiltration and affinity chromatography. Journal of Food Science, 58(2), pp. 303-306 (Article).

Cuatrecasas, P. (1970) Protein purification by affinity chromatography: Derivatizations of agarose and polyacrylamide beads. J. Biol. Chem. 245 (12) pp. 3059-3065 (Article)