Downstream processing - Pharmaceutical Biotechnology: Concepts and Aapplications - page 151

Figure 6.14 Schematic representation of the principle of biospecifi c affi nity chromatography. The chosen
affi nity ligand is chemically attached to the support matrix (agarose bead) via a sui table spacer arm. Only
those ligands in solution that exhibit biospecifi c affi nity for the immobilized species will be retained

out all unbound molecules. An appropriate change in buffer composition, such as inclusion of a
competing ligand, will result in desorption of the retained proteins.
Elution of bound protein from an affi nity column is achieved by altering the composition of the
elution buffer, such that the affi nity of the protein for the immobilized ligand is greatly reduced.
A variety of non-covalent interactions contribute to protein-ligand interaction. In many cases,
changes in buffer pH, ionic strength, inclusion of a detergent or agents such as ethylene glycol
(which reduce solution polarity) may suffi ce to elute the protein. In other cases, inclusion of a com-
peting ligand promotes desorption. Competing ligands often employed include free substrates,
substrate analogues or cofactors. Use of a competing ligand generally results in more selective pro-
tein desorption than does a generalized approach such as alteration of buffer pH or ionic strength.
In some cases, a combination of such elution conditions may be required. Identifi cation of optimal
desorption conditions often requires considerable empirical study.
Affi nity chromatography offers many advantages over conventional chromatographic tech-
niques. The specifi city and selectivity of biospecifi c affi nity chromatography cannot be matched
by other chromatographic procedures. Increases in purity of over 1000-fold, with almost 100 per
cent yields, are often reported, at least on a laboratory scale. Incorporation of an affi nity step
could thus drastically reduce the number of subsequent steps required to achieve protein purifi ca-
tion. This, in turn, could result in dramatic time and cost savings, which would be particularly
signifi cant in an industrial setting. Despite such promise, biospecifi c affi nity chromatography does
display some practical limitations:

• many biospecifi c ligands are extremely expensive and often exhibit poor stability;

• many of the ligand coupling techniques are chemically complex, hazardous, time consuming

and costly;

• any leaching of coupled ligands from the matrix also gives cause for concern for two reasons, as

(a) it effectively reduces the capacity of the system and (b) leaching of what are often noxious
chemicals into the protein products is undesirable.