Properties of Antibodies Compared to Typical Feedstock Contaminants

Typical feedstock contains serum proteins such as albumin and transferrin as well as cell-degradation products such as DNA and cytosol proteins. In addition, if the antibody is in ascites fluid, the host animal's (typically mouse) antibodies would be present as well. By a combination of chromatographic or precipitation methods, one seeks to purify the desired antibody away from these contaminants. More recently, cell-culture media have become available that are described as "protein-free" and usually contain only low molecular weight digests of proteins or amino acids. If the antibody-secreting cells can grow and produce well in such media, the purification process becomes much easier, because the IgG molecules at a molecular weight (MW) of 150 kDa are readily separated by size from the components of such media.

As described in other articles, IgG antibody molecules are multichain glycoproteins that have been divided into various subclasses based on structure and properties. Their carbohydrate content is only 2% and is invariably located in the CH2 domain of the Fc portion. The carbohydrate is sequestered, in that the carbohydrate chains are directed inward and are the actual contact residues between the heavy chains in this region (3), with the two heavy chains bowed outward to accommodate the carbohydrate. Thus, lectins generally do not bind to IgG, and purification must be directed toward the polypeptide portion. It should be noted that there are occasional reports of a carbohydrate site in the variable region of the IgG (3), in which case a purification method could be directed toward the carbohydrate moiety.

IgM molecules, on the other hand, contain about 9 to 12% carbohydrate, attached at five locations throughout the constant domains (4,5). Therefore, carbohydrate-directed methods have been developed for IgM and will be discussed later.

Specific affinity interactions for IgG and to some extent for IgM have been exploited in their purification. These affinity methods fall into three categories. First are those that rely on the key functional activity of the antibody, that is, its binding to antigen. Thus, a classic method of purification of antibody is binding on a solid-phase absorbent to which antigen has been immobilized. The second category relies on certain proteins that specifically bind to IgG. In this category are the well-known bacterial proteins A and G as well as second antibodies raised to bind against the desired antibody. Third, less-specific but often considered affinity chromatographic techniques include those relying on the interaction of the antibody with immobilized metals (IMAC), dye-ligands, hydroxyapatite, and thio-philic adsorbents.

The isoelectric points vary widely among different antibodies, mostly between about pH 6-9, but within different subclasses the range is generally much tighter. The isoelectric point directly affects the ability to use ion exchange, hydroxyapatite, chromatofocusing, and preparative electrofocusing techniques to purify antibodies. For example, the isoelectric point of serum albumin at 4.5 to 5.0 (6,7) indicates that its removal by ion exchange should be feasible for most antibody-purification schemes. Transfer-rin, at pI = 5.5-6.0 (8), can be more challenging.

The hydrophobicity and solubility of antibodies are important in their purification by such methods as hydropho-bic interaction chromatography and precipitation meth ods. In general, antibodies are more hydrophobic and less soluble in the presence of precipitating salts than feedstock contaminants.

Finally, the size of antibodies, 150 kDa for IgG and 900 kDa for IgM, makes this a key property to exploit in their purification. Size-exclusion chromatography is widely used, as are ultrafiltration methods. Most contaminants are of lower molecular weight than antibodies. The nucleic acids DNA and RNA, which vary widely in size, can be digested using nucleases (9-11) to convert them to small oligonucleotides that are readily removed by sizing methods.

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