Downstream processing - Pharmaceutical Biotechnology: Concepts and Aapplications - page 150

OH

(a) SEPHAROSE

O CH 2 CH CH 2 O

OH

(b) SEPHAROSE O CH 2 CH CH 2 O (CH 2 ) 7 CH 3

Figure 6.13 Chemical structure of (a) phenyl and (b) octyl sepharose, widely used in hydrophobic interac-
tion chromatography

octyl- and phenyl-sepharose gels, which contain octyl and phenyl hydrophobic groups respectively
( Figure 6.13).
Protein separation by hydrophobic interaction chromatography is dependent upon interac-
tions between the protein itself, the gel matrix and the surrounding aqueous solvent. Increas-
ing the ionic strength of a solution by the addition of a neutral salt (e.g. ammonium sulfate or
sodium chloride) increases the hydrophobicity of protein molecules. This may be explained
(somewhat simplistically) on the basis that the hydration of salt ions in solution results in an
ordered shell of water molecules forming around each ion. This attracts water molecules away
from protein molecules, which in turn helps to unmask hydrophobic domains on the surface
of the protein.
Protein samples, therefore, are best applied to hydrophobic interaction columns under condi-
tions of high ionic strength. As they percolate through the column, proteins may be retained via
hydrophobic interactions. The more hydrophobic the protein, the tighter the binding. After a wash-
ing step, bound protein may be eluted by utilizing conditions that promote a decrease in hydro-
phobic interactions. This may be achieved by irrigation with a buffer of decreased ionic strength,
inclusion of a sui table detergent, or lowering the polarity of the buffer by including agents such as
ethanol or ethylene glycol.
Reverse-phase chromatography may also be used to separate proteins on the basis of differen-
tial hydrophobicity. This technique involves applying the protein sample to a highly hydrophobic
column to which most proteins will bind. Elution is promoted by decreasing the polarity of the
mobile phase. This is normally achieved by the introduction of an organic solvent. Elution condi-
tions are harsh and generally result in denaturation of many proteins.

6.6.4 Affi nity chromatography

Affi nity chromatography is often described as the most powerful highly selective method of pro-
tein purifi cation available. This technique relies on the ability of most proteins to bind specifi cally
and reversibly to other compounds, often termed ligands ( Figure 6.14). A wide variety of ligands
may be covalently attached to an inert support matrix, and subsequently packed into a chromato-
graphic column. In such a system, only the protein molecules that selectively bind to the immobi-
lized ligand will be retained on the column. Washing the column with a sui table buffer will fl ush