Optimization in Affinity Chromatography

Introduction to Resin Design and Method Development in Affinity Chromatc raphy 

The application and preparation of afEnity resins was first described in a paper by Cuatrecasas in 1968 [1]. In this work, nearly aU features of this technique were explored. Affinity chromatography is the youngest of the four major purification methods used in biochromatography. In 1968, ion exchange (lEX), hydrophobic interaction (HIC), and gel filtration (GF) were already well established. What new opportunities in the purification of biomolecules were then added by affinity chromatography  

A major target in downstream processing is the isolation of a specific biomolecule from a solution that may contain several thousand substances. The ideal situation is a single-step purification procedure, but the statistical probability of isolating a pure molecule from such a mixture by one chromatography step is determined by a Poisson distribution and falls exponentially with the number of compounds [2]. The only way to improve this situation is to increase the affinity of the resin for the target molecule. The affinity constant is defined as [3]  

There is also the problem of elution of tightly boimd biomacromolecules from a resin, so it is a good compromise to use affinity ligands with dissociation 

HPLC Made to Measure A Practical Handbook for Optimization. Edited by Stavros Kromidas Copyright 2006 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim ISBN 3-527-31377-X 

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Optimization in Affinity ChromatographY 415 Table 2. Comparison of different epoxy-activated resins. 

Some kinds of chromatography require relatively little optimization. In gel permeation chromatography, for example, once the pore size of the support and number of columns is selected, it is only rarely necessary to examine in depth factors such as solvent composition, temperature, and flow rate. Optimization of affinity chromatography is similarly straightforward. In RPLC or IEC, however, retention is a complex and sensitive function of mobile phase composition column type, efficiency, and length flow rate gradient rate and temperature. 

As is common to all affinity chromatography protocols, only the desired protein is supposed to bind to the column. The target protein then has to be eluted off the column, in this case with 0.5 m imidazole which displaces the imidazole rings of the histidine residues of the target protein. Non-specific binding is often observed, which results in several intermittent washing steps being required as well as optimization of the protocol for each protein. 

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