Theory and Development of Affinity Chromatography

packed with starch was used. Presumably the column disintegrated during the process! Attempts were made in the 1950s to link antigens to cellulose columns for the purification of antibodies, but these were not very successful because of the low capacity of the cellulose matrix. Unless the particles used to pack the column are permeable to the proteins, only the surface of the particles is available for attachment. The more successful applications commenced when suitable protein-permeable particulate materials were developed, together with reliable chemical methods that could be used for attaching specific ligands to these materials. Much of the original work was based on the use of cyanogen bromide as a method for activating the matrix, and the use of agarose beads as the support material. Agarose has almost ideal properties, in that the gel formation of the beads has pores permeable to most proteins, so that the internal volume of the beads is all available for attachment, as well as the outside surface. Moreover, agarose in itself has virtually no affinity for any protein (other than agarases), so nonspecific binding is rarely observed. It is still the matrix of choice for most affinity chromatography, though there are now many competing materials, both polysaccharide-based and synthetic.

Activation of the matrix involves treating the material with a chemical that introduces a group (normally reacting with hydroxyls on polysaccharide matrices) which itself will then react with something, usually an amine, in the ligand to be used. All such activating chemicals are extremely reactive and dangerous to handle, especially those that are volatile. Cyanogen bromide reacts to introduce several forms of cyanate derivatives, of which the cyanate ester is the most important. This ester will couple to a primary amine, such as a lysine residue in a protein, to produce the isourea-linked ligand (Figure 1).

Figure 1 Activation of a carbohydrate matrix with cyanogen bromide. The major product is the cyanate ester after reaction with a primary hydroxyl group. This rapidly couples with an amine to produce the isourea structure, which is positively charged at neutral pH.

Cyanogen bromide activation is still widely used, and the dangerous chemistry is overcome by having the already-activated agarose available as a commercial product. However, it does have some disadvantages compared with other methods outlined below - in particular, the instability of the isourea linkage.

Many other activation methods have been developed, and a brief summary of these is given in Table 1. Several of these incorporate a bifunctional reagent, one end of which combines with the matrix, and the other with the ligand. These may cause some cross-linking within the matrix, but this can be advantageous for matrix stability. Bifunctional reagents also introduce a spacer arm (see below) of various lengths depending on the reagent. The activated matrix then reacts with a nucleophile such as an amine, or in some cases a sulfhydryl group in the ligand being coupled (Figure 2A).

The ligands exploited at first were mainly enzyme substrates. In particular, so-called group-specific ligands were developed which could be used for a variety of different enzymes having the same

Table 1 A selection of activation methods, and the properties of the spacer arms introduced after coupling with amino-reactive ligand. A positive charge can result in some nonspecific anion exchange behaviour at low ionic strengths. Cleaning of protein adsorbents is best carried out with alkali, but many linkages are alkali-labile

Reagent Spacerarmlength, Typeoflinkage ChargeatpH7 Alkalilability atoms

Cyanogen bromide 1 Isourea # Yes

Carbodiimide 1 Amide 0 Yes

Epichlorhydrin 3 Secondary amine # No

Bisoxirane 11 Secondary amine # No

Divinyl sulfone 5 Secondary amine # Yes

Tosyl/tresyl 0 Secondary amine # No

Hydroxy-succinimide 8a Amide 0 Yes

Cyanuric chloride 4 Aromatic amine 0 No aDepends on activation reagents.

Figure 2 (A) Activation of a matrix with a bifunctional reagent, which provides a spacer arm. (B) Coupling of a ligand containing a built-in spacer arm to an activated matrix.

substrate - more particularly, a common cofactor. Thus we had, and can still purchase, adsorbents containing nucleotide cofactors such as ATP and NAD, lectins for glycoproteins and nucleic acids for binding either other nucleic acids or enzymes involved in nucleic acid metabolism. The use of affinity chromatography which exploits the interaction between an antibody and its antigen has been extensively developed, and is described in detail elsewhere.

The main problems associated with affinity chromatography soon appeared. These can be summarized as:

1. It took a long time to develop an adsorbent that does the job.

2. Once made, the adsorbent is expensive and has a limited useful life.

3. There is nonspecific binding of unwanted proteins.

4. There is a need for spacer arms.

5. There are difficulties in satisfactory elution.

Many of these problems have now been solved, with a clearer understanding of the important factors involved. It is now not often that a completely new adsorbent has to be developed. The expense parameter is less important on a research scale, but is a major consideration in large scale commercial purification. The related problems of nonspecific binding and spacer arms can usually be overcome by judicious process design, and elution procedures for the more difficult tasks such as antibody-antigen interactions are now better established. Even so, there are many cases in which a workable true affinity method cannot be established, usually because the natural affinity may be very specific, but quantitatively weak.

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