DNA and RNA tend to be highly negatively charged at just about any physiological pH. Thus, anion exchange resins are often used to remove these nucleic acids from antibodies, with conditions usually selected so that the nucleic acids bind while the antibody flows through.

In addition to the techniques mentioned in the initial capture step, there are a number of other techniques aimed at removing specific contaminants. For example, materials are available commercially that are intended specifically for the removal of impurities such as endotoxins, viruses, DNA and RNA, and albumin. In one way or another, these products take advantage of specific attributes of the impurity to remove them.

Some general examples of secondary purifications are discussed in this section. These include both techniques covered in the initial capture section and special polishing techniques.

The removal or inactivation of viruses must be validated for any animal-sourced or animal cell-sourced products, including antibodies, intended for in vivo use (12,82,83). All the techniques mentioned to date have the potential for removing virus. A specific attribute that is applicable to all viruses is their size. Being significantly larger than antibodies, they can be removed by a size basis.

Although this can be done by SEC, a higher degree of virus removal is typically seen by ultrafiltration, with a membrane specifically selected for a molecular weight cutoff appropriate to retain virus and pass antibody (82,84,85).

In addition to virus clearance, virus inactivation is often performed. The most common techniques used are detergent treatment, low pH, or high temperature (82). Needless to say, the antibody must be tested to be resistant to these conditions before their routine use.

As mentioned earlier, one characteristic of DNA and RNA is the high negative charge. As well as anion IEC, HAP is well known to be effective at binding nucleic acids because of this high charge (76,86,87). HIC also may remove nucleic acids, because nucleic acids do not bind, whereas antibodies do bind to HIC resins (72).

Other properties of nucleic acids are the long, thin nature of the polymer, and, of course, the nucleotide sequence itself. Various precipitation techniques take advantage of this to remove DNA and RNA. Also, complementary nu-cleotides bound to chromatography supports can be used as an affinity adsorbent. Finally, as alluded to in the "Introduction," DNA and RNA can be enzymatically digested using nuclease enzymes (e.g., Benzonase®) to generate very small fragments that can be readily removed from antibodies by sizing techniques (9,10).

Endotoxins are lipopolysaccharides. Many can be removed by anion exchange chromatography (6,53,88). As with the nucleic acids, HIC has been reported to be able to remove endotoxins for antibodies (72). In addition, both precipitative and chromatography techniques focusing in on the lipid portion of the endotoxins have been developed.

Albumin is probably the most typical and highest concentration contaminant in most feedstocks, whether serum, ascites, or cell culture media. As with the other contaminants, the techniques already mentioned have the potential for removing albumin. However, if additional specific removal is required, antialbumin affinity chroma-tography is possible. Also, many serum proteins, including albumin, can be removed using dye-ligand chromatogra-phy. A well-known technique in laboratory chromatogra-phy, it has not been much used in preparative-scale work. The dyes used were originally taken from the textile industry and were not particularly pure. In addition, there was concern about ligand leakage. However, modern dye-ligand resins are made with high-purity dyes prepared especially for this purpose; coupling procedures have improved, minimizing leakage; and assays are often available to track what leakage there may be (89-93).

Perhaps the greatest challenge for the polishing steps is the removal of form variants. The easiest form variant to remove is aggregate, which can be removed by SEC, a step that may be needed for final formulation (see next section) anyway. In addition, there may be light or heavy chain variants if a cell-line producing antibody is not stable and specific. There is the possibility of host antibody from serum, ascites, or serum-supplemented cell culture. And there can be intramolecule variants caused by variations in glycosylation, deamidation, proteolytic digestion, and a number of other possible causes. It is difficult to make a blanket statement in regard to how or even if such variants need be removed. If these variants prove to be extremely difficult to remove, and the product can be proved to be safe and effective with their presence, the economics of the situation may dictate that they not be removed.

Nonetheless, variants may need to be removed, and some examples of possible techniques follow. For chain variants and other source antibodies, if the physical characteristics are significantly different compared to the target antibody, they probably can be removed by IEC, HIC, etc. But, if not, one possibility is HAP. This technique has long been known to sometimes be able to separate different idi-otypes (76,78) (Fig. 7). Dye-ligand chromatography has also been shown to be capable of separating subclasses (93). In addition, the different affinity of different antibodies for protein A and protein G allows these resins to be used for variant removal at times. Immunoaffinity directed against the undesirable variant heavy or light chain(s) is a powerful technique. Glycosylation variants may sometimes be removed by lectin or immunoaffinity chromatog-raphy directed against specific sugars. If physical changes such as deamidation do not change the antibody's charge enough for separation by IEC, preparative electrophoresis or chromatofocusing may sometimes be successful (94).

But, basically, this is a struggle that must be developed on a case-by-case basis and often requires a unique solution.

In general, the difference between initial capture and additional purification steps may be summarized in one word: resolution. In initial capture, the feedstock is a moderately to highly complex mixture. The feedstock may be dirty, that is, contain particulates, if sample preparation is not performed. On the production scale, large volumes are the norm. The goals in initial capture are to remove most of the impurities, with good yield, and to concentrate the product. With secondary purification, the feedstock is relatively clean, most of the protein is the target antibody, and some residual impurities may be physically very similar to the target antibody. Thus, the goal in secondary purification is to achieve high-resolution separation.

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