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Purification of Proteins Using Displacement Chromatography 75
(SOS) (35) have been employed as displacers for anion exchange systems.
While most of these separations were carried out on ion exchange resins, the
use of displacement chromatography on reversed phase and hydroxyapatite
(HA) resins was also demonstrated. Viscomi et al. (36) used the combination of
reversed-phase and ion exchange displacement chromatography for the purification
of a synthetic peptide, the fragment 163-171 of human interleukin-B.
In the reversed-phase displacement chromatography step, the displacer was
benzyltributyl ammonium chloride, whereas in the ion exchange displacement
step, the displacer was an ammonium citrate solution. The use of displacement
to separate proteins in immobilized metal affinity chromatography (IMAC)
has also been reported (37,38). Freitag et al. (39) presented the application of
displacement chromatography on HA stationary phases.
The remainder of this chapter aims to provide the reader with all of the
tools necessary to determine the best operating conditions for a successful
displacement experiment for ion exchange systems. However, knowing that
sometimes material and/or time requirements may not allow the reader to
go through all of the steps described in this chapter, where appropriate an
abbreviated version of methods development will be described.
2. Methods
2.1. Identification of Stationary Phase and Operating Conditions
for Selectivity
Linear elution chromatography can be employed to select an appropriate
stationary phase with sufficient selectivity as well as an operating condition
(buffer pH, mobile phase additives such as salt type and concentration) that
provides a sufficient resolution between the feed components.
2.2. Constructing the Adsorption Isotherms
If pure feed components are available, the next step will be obtaining the
adsorption isotherms for the feed components. If pure feed components are not
available, proceed to the steps in Subheading 2.3 for the ranking and selection
of displacers. This chapter will focus on the use of the steric mass action (SMA)
isotherm as a tool to define operating conditions for a successful ion exchange
displacement chromatography (see Note 1). The SMA model (40) has been
shown to be a convenient methodology for examining the chromatographic
behavior of proteins in ion exchange systems. In this model, adsorption has
been described using three (SMA) parameters: characteristic charge (), which
is the number of interaction sites each molecule has with the stationary phase
material; the equilibrium constant (K) of the reaction between the solute and