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complicated mixture. For large lots, and increasingly for small lots, monoclonal antibodies are produced in an artificial reactor, using at least a semidefined culture media. Again, historically, culture media could be considered only semidefined because it was supplemented with serum. However, even in this case, the concentration and number of macromolecular contaminants to be removed are lower than those in serum. Also, the current trend for monoclonal antibody production is toward use of serum-free or even protein-free media.

The second reason that polyclonal antibodies may be more challenging to purify is intrinsic to the antibody itself. Monoclonal antibodies are not necessarily absolutely homogenous. Often, there are differences in glycosylation. And there will be aging differences, such as in degree of deamidation. They will, however, all be of the same isotype, directed against the same epitope. This is not the case for polyclonal antibodies. Polyclonal antibodies tend to have a broader pI range than monoclonal antibodies, making IEC and related techniques such as HAP more complicated (77,86,94). They also tend to give broader peaks on hydrophobic interaction (72).

The same techniques used for purification ofmonoclonal antibodies are used for the purification of polyclonal antibodies. Indeed, most of the techniques were developed before the advent of monoclonal antibodies. As with monoclonal antibodies, the key to selecting the right purification techniques lies in knowing the desired yield, the desired purity, and the nature of the contaminants. Success at producing high-purity polyclonal antibodies centers around two points: extensive preparation and the use of affinity techniques.

In regard to preparation, characterizing all the individual impurities in a crude polyclonal mixture would be out-

landishly time consuming. However, a valuable approach is to run standard IEC, HIC, and HAP conditions, where most if not all the proteins initially bind. Analysis of the elution fractions by an analytical technique such as two-dimensional electrophoresis will indicate which column technique can remove which impurities. The limitation to this approach is the well-known fact that the binding conditions used in chromatography affect the resolution ofelu-tion.

The affinity and pseudoaffinity techniques such as dye-ligand, IMAC, and thiophilic chromatography can be important in several roles. These techniques can be selected not only to bind to the target antibody, but also to remove specific impurities. For instance, one of the most vexing problems in antibody purification is removal of host antibody. If a specific isotype is desired, affinity techniques that bind to the other isotypes can be used to remove these unwanted host antibodies. Generic affinity techniques such as protein A and G are still very powerful tools. And, if practical to use, immunoaffinity has excellent selectivity.

In summary, there is nothing unique in purifying poly-clonal antibodies compared to monoclonal antibodies. To reach a given degree of purity, the polyclonal antibody will probably take more planning; more preparation; and for high purity, use of more powerful techniques, especially affinity techniques.

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