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Purification of Proteins Using Displacement Chromatography 73
Increasing affinity
16
10
14
9
Protein conc (mg/ml)
12
10
8
6
4
2
Protein A
Protein B
Displacer
8
7
6
5
4
3
2
1
Displacer conc (mM)
0
0 2 4 6 8 10 12 14 16
Volume (ml)
0
Fig. 1. Sample chromatogram from displacement chromatography.
separated on a given column with the purified components recovered at
significantly higher concentrations. In addition, the tailing observed in nonlinear
elution chromatography is greatly reduced in displacement chromatography
due to the self-sharpening boundaries formed in the process. In displacement
chromatography, the displacer suppresses the adsorption of feed components in
the displacer zone and thus prevents tailing of the most strongly retained feed
component. In a fully developed displacement train, each of the components
displaces the component ahead of it, leading to a suppression of tailing in all
solute zones. This makes displacement chromatography less sensitive to feed
loads resulting in high throughputs without sacrificing resolution and purity.
Displacement chromatography exploits the nonlinear, multi-component competition
amongst the components to be separated, resulting in higher resolution,
particularly among closely related species. In addition, product recovery is
possible under relatively low mobile phase modifier concentrations (e.g., salt).
This combination of high throughput and high resolution in a single process
makes displacement chromatography an attractive mode of operation for preparative
separations.
Displacer affinity and its utilization are the critical components of
displacement chromatography. It has been accepted that retention in ion
exchange systems is not purely based on electrostatic interactions (12–15), and
there are a few reports in the literature concerning the relative importance of