Affinity Chromatography - Department of Molecular and Cellular ...

For any capture step, select the technique showing the most effective binding to the target
protein while binding as few of the contaminants as possible, i.e. the technique with the
highest selectivity and/or capacity for the target protein.
A sample is purified using a combination of techniques and alternative selectivities. For
example, in an IEX-HIC-GF strategy, the capture step selects according to differences in
charge (IEX), the intermediate purification step according to differences in hydrophobicity
(HIC) and the final polishing step according to differences in size (GF).
If nothing is known about the target protein use IEX-HIC-GF. This combination of techniques
can be regarded as a standard protocol. Consider the use of both anion and cation exchange
chromatography to give different selectivities within the same purification strategy.
IEX is a technique which offers different selectivities using either anion or cation exchangers.
The pH can be modified to alter the charge characteristics of the sample components. It is
therefore possible to use IEX more than once in a purification strategy, for capture, intermediate
purification or polishing. IEX can be used effectively in the same purification
scheme for rapid purification in low resolution mode during capture and in high resolution
mode during polishing.
Consider reversed phase chromatography (RPC) for a polishing step, provided that the
target protein can withstand the run conditions. RPC separates proteins and peptides on the
basis of hydrophobicity. RPC is a high selectivity (high resolution) technique, requiring the
use of organic solvents. The technique is widely used for purity check analyses when recovery
of activity and tertiary structure are not essential. Since many proteins are denatured by
organic solvents, RPC is not generally recommended for protein purification because
recovery of activity and return to a native tertiary structure may be compromised. How ever,
in the polishing phase, when the majority of protein impurities have been removed, RPC
can be excellent, particularly for small target proteins that are not often denatured by
organic solvents.
CIPP does not mean that there must always be three purification steps. For example,
capture and intermediate purification may be achievable in a single step, as may intermediate
purification and polishing. Similarly, purity demands may be so low that a rapid capture
step is sufficient to achieve the desired result. For purification of therapeutic proteins, a
fourth or fifth purification step may be required to fulfil the highest purity and safety
demands. The number of steps used will always depend upon the purity requirements and
intended use for the protein.
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