Chromatographic purification.

Subsequent purification steps are tailored to suit the requirements and characteristics of the particular product. Some of the more commonly-used techniques are discussed here, with particular reference to the purification of IgM and IgG, as these classes of monoclonal antibody were used in this study. IgM has a high molecular weight of around 900,000 Daltons and consists of five units, each with two light and two heavy chains. IgGs are approximately 150,000 Daltons in weight, with only one unit.

A widely-used chromatographic technique is affinity chromatography, which separates the product on the principle of adsorption of the required protein by biospecific selection (Schmidt, 1989). The ligand, a molecule with a specific affinity for the product, is bound to a matrix or backbone. The product is selectively bound to the ligand and is thus

removed from the feed stream as it flows over the matrix. It is later eluted by changing the column conditions. The specific interactions that can be exploited by this method include those between an enzyme and its inhibitor or substrate, an antibody and its antigen, and a hormone and its receptor (Scawen & Hammond, 1986). These examples are of true 'specific' ligands. Also available are 'pseudo-specific' dye ligands, which imitate the characteristics of other ligands, but which are cheaper to manufacture (Dean, 1986).

A ligand attracting much interest for mammalian cell products is Protein A, a soluble protein made by mutant strains of

Staphylococcus aureus (Rosevear & Lambe, 1988) . It binds with

several IgG antibodies of interest and is one of the principle methods for their purification on a sub-kilogram scale. Other bacterial proteins, such as Protein G, have a wider specificity for immunoglobulins, including IgM.

Immunospecific methods are useful for the purification of monoclonal antibodies that have only a weak affinity for Proteins A and G, but depend on the availability of either the pure antigen to the antibody of interest, or a second antibody raised against it. Given the high affinity of such molecules, harsh conditions may be needed to elute the product (Hill et

a l , 1986) . IgM has been purified from cell culture fluid using

such immunospecific methods.

To maximise the benefits of an affinity technique, it should be used as early as possible in the purification process, otherwise its high resolving power is not fully utilised

(Bonnerjea et a l , 1986). Protein A has been used to purify IgG, directly from clarified concentrated culture supernatant, giving >95% purity and >90% yield (Birch et a l , 1987a; Rhodes, 1989) . This level of purity would be sufficient for many diagnostic applications.

The technique of affinity chromatography can be run either as a batch operation or using a chromatographic column; the latter is more usual. One drawback of the technique is the leaching of ligands from the matrix, a problem that reduces capacity and contaminates the product. It occurs through cleavage of the matrix-ligand bond, dissolution of the matrix

material or degradation of the ligand (Hill et a l , 1986). This is thought to occur in most applications and is a particular problem when the affinity step is being used in the later stages of purification. Ligands such as Protein A or protease inhibitors are potentially dangerous contaminants and thus must be removed. This is most easily achieved by the use of an ion-exchange step (Rosevear & Lambe, 1988). The problem can be minimised by prewashing the column prior to use.

Because of the expense of an affinity chromatography step, a pretreatment column is often placed just prior to the affinity column to protect it from fouling by removing the last of the cell debris and lipids which would otherwise attach irreversibly to the ligands. Such a step need not give high purity, but should give a high yield. Options include ion exchange or hydrophobic interaction. Ion exchange chromatography works on the principle of electrostatic binding, in which the protein displaces the counterion and binds to the ion exchanger. Anion exchange has been used to remove contaminants, particularly albumin and transferrin, by binding them to the matrix. However, this results in loss of activity of the column matrix and more extensive regeneration requirements than for cation exchange (Schmidt, 1989) . Cation exchange has been used to purify a wide range of antibodies, leaving the contaminants in the flow-through, and gives high purity and recovery levels. Hydrophobic interaction chromatography separates molecules according to differences in their hydrophobicity. All immunoglobulins are hydrophobic, and are often different enough from their contaminants to give a high degree of purification in one step. Problems include the fact that some proteases are co-pur if led with the immunoglobulin, and the conditions of high salt concentration that must be used.

To finish the chromatographic separation stage, a gel filtration step can be used to remove protein dimers and any affinity ligands that may have leaked from the column, and to desalt and buffer-exchange the product prior to storage. Gel filtration separates molecules according to size, and has the effect of diluting the product with buffer.