Page 9 - Subyek Encyclopedia - Encyclopedia of Separation Science

4 I / AFFINITY SEPARATION / Derivatization

The matching pair must be identiRed, and one afRnity method is not restricted to protein separ-
of them isolated in a pure form. Covalent bond- ations; nucleic acids and whole cells can also be
ing onto an inert matrix in a stable manner must separated.
always allow the ‘docking’ surface of the protein to The simplicity of the chromatographic process is
be positioned to make it available to the target pro- shown in Figure 1. The ligand of interest, covalently
tein. The whole also has to be achieved at an accept- bonded onto the inert matrix, is contained in the
able cost. column, and a solution containing the target (the
This technique has resulted in many successful ap- ligate) is passed through the bed. The ligand recog-
plications, often using antibodies as the afRnity nizes the ligate to the exclusion of all other molecules,
medium (immunoafRnity chromatography), but with the unwanted materials passing through the col-
large scale separations using these ‘natural’ ligands umn packing while the ligate is retained. Once the
are largely restricted by cost and regulatory reasons. bed is saturated with the target molecule (as mea-
Although immunoafRnity chromatography is still sured by the breakthrough point), contaminating spe-
widely practised, in recent years the evolution of cies are washed through, followed by collection of the
design technologies has provided powerful new ap- target molecule as a very pure fraction using an
proaches to mimic protein structures, resulting in the eluting buffer solution. Finally, the column is
development of synthetic ligands able to work in cleansed from any strongly adsorbed trace materials,
harsh operational environments and at low cost. usually by regeneration with a strong alkali or acid,
making it available for many more repeat runs. An
outstanding advantage of the afRnity process is
The Af\nity Process
an ability to concentrate very dilute solutions while
The afRnity method is critically dependent upon stabilizing the captured protein once adsorbed onto
the ‘biological recognition’ existing between species. the column. Many of the in-demand proteins manu-
By permanently bonding onto an inert matrix a mol- factured by genetically engineered microorganisms
ecule (the ligand) that speciRcally recognizes the mol- are labile, allowing only minute quantities to be pres-
ecule of interest, the target molecule (the ligate) can ent in the fermentation mix before they begin to
be separated. The technique can be applied to any deteriorate. An ability to capture these very small
biological entity capable of forming a dissociable quantities while stabilizing them in the adsorbant
complex with another species. The dissociation con- phase results in maximization of yield, making mass-
stant (K d ) for the interaction reSects the comp- ive savings in total production costs.
lementarity between ligand and ligate. The optimal Although the technical processing advantages are
range of K d for afRnity chromatography lies be- clear there is a major difRculty in the appli-
tween 10 4 and 10 mol L . Most biological cation of afRnity chromatography as understood
8
1
ligands can be used for afRnity purposes provid- by most practitioners today. Most ligands described
ing they can be immobilized, and once immobilized in Table 1 suffer from two primary disadvan-
continue to interact successfully with their respective tages: a lack of stability during use; and high
ligates. The ligand can be naturally occurring, an cost. Fortunately these problems have now been over-
engineered macromolecule or a synthetic molecule. come, and afRnity chromatography is now accep-
Table 1 provides some examples of immobili- ted as the major separations technology for
zed ligands used to purify classiRed proteins. The proteins.

Table 1 Affinity ligands and purified proteins
Immobilized ligand Purified protein

Divalent and trivalent metal ion Proteins with an abudance of his, tryp and cys residues
Lectins Glycoproteins, cells
Carbohydrates Lectins
Reactive dyes Most proteins, particularly nucleotide-binding proteins
Nucleic acids Exo and endonucleases, polymerases, other nucleic acid-binding proteins
Amino acids (e.g. lys, arg) Proteases
Nucleotides, cofactors substrates and inhibitors Enzymes
Proteins A and G Immunoglobulins
Hormones, drugs Receptors
Antibodies Antigens
Antigens Antibodies