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Note: mono, monoclonal; poly, polyclonal.

Note: mono, monoclonal; poly, polyclonal.

their usage more general. A number of these are available commercially.

Ion Exchange. Another very widely used technique is ion exchange chromatography (IEC). Although less specific than proteins A and G, it can often provide purities of more than 80% in one step, which is adequate for many purposes (18,60-64). Both cationic and anionic IEC can be used, in either binding or flowthrough modes. The choice of method and its utility depend on the relative ionic characteristics of the antibody and its impurities. Care must be taken in selecting the resin and conditions to be used, not only from the standpoint of resolution, but also of capacity. If conditions are chosen where both the antibody and impurities bind, the capacity of the resin for antibody could be dramatically reduced.

The pI of a protein is not an exact predictor of a protein's behavior on IEC. The charge distribution on the surface of the protein and a number of other minor effects also play a role. Nonetheless, the pI is a good first approximation. In one study of 15 murine monoclonal antibodies, pIs of 4.9 to 8.3 were reported (65). Bovine albumin has been reported to have a pI of about 4.5 to 5.0 (4,5). The pI of another common impurity, transferrin, ranges from 5.5 to 6.0, depending on the species and degree of iron saturation (66). Thus, it is quite possible to have an antibody that is acidic enough to be inseparable from transferrin and even albumin by IEC. One of the first steps in developing an antibody purification procedure is to determine the pI of the antibody and the contaminants. This may be done quickly and easily and will give a good indication of how useful IEC might be.

Immobilized-Metal Affinity Chromatography. In addition to the widely used techniques described in the previous section, a number of others may be used for the initial purification of antibody. One technique that is growing in use is immobilized metal affinity chromatography (IMAC). Metal ions, usually Ni2 + , are held to a chromatography resin by ligands. In turn, these metal ions can interact with proteins, primarily with histidine groups. Many engineered antibodies and fragments are being produced with a polyhistidine tail, which gives this technique a high specificity. IMAC can sometimes also be used for nonen-gineered antibodies that have naturally occurring surface histidines, although histidine is an uncommon amino acid (67-69).

Hydrophobic Interaction Chromatography. Hydrophobic interaction chromatography (HIC) is another technique that can be used for initial capture. Antibodies tend to be among the most hydrophobic of the proteins in the crude feedstock (70,71). Thus, this technique can be quite powerful (6,8,72,73). An example is given in Figure 2 (72), where bovine serum proteins are separated from antibody. On the down side, experience shows that the loading buffer must be carefully chosen (67). As with IEC, conditions where most of the contaminating proteins bind along with the antibody should be avoided for the initial capture. In such a situation, capacity is decreased and selectivity diminished. Also, selection of an appropriate media can be

Figure 2. HIC separation of mouse polyclonal IgG from fetal bovine serum proteins and bovine polyclonal IgG. Chromatogram 1, bovine polyclonal IgG; 2, mouse polyclonal IgG; 3, partially purified (on ABx®, J.T. Baker) fetal bovine serum; 4, neat fetal bovine serum. Source: Reprinted from Ref. 72 with kind permission of Eaton Publishing.

Figure 2. HIC separation of mouse polyclonal IgG from fetal bovine serum proteins and bovine polyclonal IgG. Chromatogram 1, bovine polyclonal IgG; 2, mouse polyclonal IgG; 3, partially purified (on ABx®, J.T. Baker) fetal bovine serum; 4, neat fetal bovine serum. Source: Reprinted from Ref. 72 with kind permission of Eaton Publishing.

somewhat more tedious than for IEC, because the choice of potential binding strengths is very broad. Not only is there supplier-to-supplier variation in ligand density and surface chemistry, variabilities also seen in IEC, but there are a large number of binding groups, such as ethyl, propyl, butyl, phenyl, ether, phenylether, and others. Even reversed-phase media, such as octyl, can be used in the aqueous HIC mode with weakly hydrophobic proteins.

Because of the variation in manufacturing, very little can be said in a general fashion about the strengths of the various resins. Within an individual manufacturer's line, usually the longer the alkyl chain length, the stronger the binding. In practice, however, tests should be made. It is important to make these tests, because the selection of a resin is an important way to manipulate selectivity.

Another means of altering selectivity is through the choice of buffer. In general, the stronger the lyotropic effect, the stronger the induction of hydrophobic binding by a salt (Fig. 3) (71). An example of this is seen in Figure 4 (74). However, strong salt solutions can have a detrimental effect on the integrity of proteins, so the choice should be limited, if possible, to those with strong salting out effects. Ammonium sulfate is the most commonly used salt, but it has the disadvantage of outgassing ammonia above pH 7.5 (71,75). Sodium sulfate and potassium phosphate are also good choices, but are somewhat limited in solubility (71,75). Because of the limited choice of buffers, the proper choice of resin becomes important.

In addition to salt, the selection of pH can have an effect on retention. In general, above pH 9 and below pH 4, retention increases, probably a result of denaturation of the protein. Between these pHs some proteins show an effect, but others do not. It is not possible to make generalizations; rather, the effect must be tested (71,75). Solution modifiers such as sugars, alcohols, PEG, and urea can also affect both binding and elution and can play a role in special situations (71,75).

HIC is perhaps deserving of more attention than it has received. Not only is it potentially a powerful technique itself, but for multistep procedures, it can be an ideal complement to IEC. IEC uses a low-ionic-strength load and a high-ionic-strength elution. HIC used a high-ionic-strength load and a low-ionic-strength elution. Thus, it is quite possible that conditions can be developed to allow one column to flow directly to the next, without intermediate sample handling.

Hydroxyapatite. Hydroxyapatite chromatography (HAP) has seen considerable use on a small basis, but little use on the large scale. The main reason for this is that until very recently, all versions of this material were mechanically fragile. Low flow rates were typical, and reuse of the columns was limited (53,76,77).

The mechanism of separation is not fully understood, but interaction of the protein with the calcium and phosphate in the resin matrix plays an important role, because a phosphate buffer gradient is most typically used for elu-tion (53,76,77). As with other nonspecific techniques such as HIC and IEC, antibodies exhibit a wide variety of behavior on HAP, and generalizations are somewhat unsafe. However, HAP has been used not only for purifying antibodies (Fig. 5) (78), but also for separating different antibody subclasses (Fig. 6) (77). It is well known for its ability

Increasing salting out characteristics (lyotropic)

SCN~<I~<ClO4~<NO3~<Br~<Cl~<CH3COO~<SO42~<PO43~<Citrate

Ba2+<Ca2+<Mg+2<Li+<Cs+<Na+<K+<Rb+<NH4+<(CH3)4N+

Figure 3. Lyotropic/chaotropic characteristics of salts.

Decreasing salting in characteristics (chaotropic)

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