Figure 5. HPHT chromatography of unpurified ascites fluid containing monoclonal antibody. Source. Reprinted from reference 78 with kind permission of Eaton Publishing.

the sulfur ligand of the resin is thought to interact with the aromatic groups of the proteins. It has been shown that this technique can be used to effectively purify proteins (22,79). Like HIC, this technique generally loads proteins in high-salt buffers. However, unlike HIC, high salt concentrations also promote elution. Needless to say, different salts have different effects (79).

In addition to chromatographic techniques, there are a number of other techniques that can substantially purify antibodies. The precipitative techniques were mentioned earlier in "Sample Preparation." Ultrafiltration and diafiltration may also be used. Certainly, low molecular weight impurities may be removed by this technique. However, in addition, by careful selection of the membrane and buffer used, some contaminant proteins may be removed as well, such as albumin from IgG (80). The technique of preparative electrophoresis has gradually been advancing during the past few years. Currently it is can be considered useful for small-scale (milligram) bench preparations, especially non-GMP. Techniques for true production-scale electropho-resis that can be performed aseptically are being developed (81). Given the resolving power of analytical electropho-resis, success at developing production-scale techniques could add a valuable new tool to the development of purification methods.

In purification of antibodies on a large scale, process time is very important for economic reasons. One way to increase the efficiency of a process is to combine steps. In the past few years, a number of approaches to combining the concentration and clarification aspects of the sample prep step and the initial capture step have been commercialized. These approaches are generally either a fluidized bed, large-diameter bead resins, or wide-channel tangential flow filters. They are all designed so that cells and cell debris will flow through without clogging the setup, thus achieving clarification. The antibody is to bind to the resin or membrane, and after clarification is complete, it can then be eluted, providing both concentration and purification. Although simple in theory, a number of technical challenges had to be overcome before these approaches became practical.

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