The application of continuous mammalian cell lines for the production of recombinant proteins might result in the presence of oncogene-bearing DNA-fragments in the final protein product (Lo¨wer, 1990; Walter and Werner, 1993). A stringent purification protocol that is capable of reducing the DNA content to a safe level is therefore necessary (Berthold and Walter, 1994). There are a number of approaches available to validate that the purification method removes cellular DNA and RNA. One such approach involves incubating the cell line with radiolabeled nucleotides and determining radioactivity in the purified product obtained through the purification protocol. Another method is dye-binding fluores-cence-enhancement assay for nucleotides. If the presence of nucleic acids persists in a final preparation, then additional steps must be introduced in the purification process. The question about a safe level of nucleic acids in biotech products is difficult to answer because of the lack of relevant know-how. Transfection with so-called naked DNA is very difficult and a high concentration of DNA is needed. Nevertheless, it is agreed for safety reasons that final product contamination by nucleic acids should not exceed 100 pg to 10 ng per daily dose depending on the kind of culture system (Eur Pharm III, 1997; WHO, 1998; Kung et al., 1990).
As mentioned before, trace amounts of “foreign” proteins may appear in biotech products. These types of contaminants are a potential health hazard because, if present, they may be recognized as antigens by the patient receiving the recombinant protein product. On repeated use the patient may show an immune reaction caused by the contaminant while the protein of interest is performing its beneficial function. In such cases the immunogenicity may be misinterpreted as being due to the recombinant protein itself. Therefore, one must be very cautious in interpreting safety data of a given recombinant therapeutic protein.
Generally, the sources of protein contaminants are the growth medium used or the host proteins of the cells. Among the host derived contaminants, the host species’ version of the recombinant protein could be present (WHO, 1998). As these proteins are similar in structure, it is possible that undesired proteins are co-purified with the desired product. For example, urokinase is known to be present in many continuous cell lines. The synthesis of highly active biological molecules such as cytokines by hybridoma cells, might be another concern (FDA, 1990; Schindler and Dinarello, 1990). Depending upon their nature and concentration these cytokines might enhance the antigenicity of the product.
Known” or expected contaminants should be monitored at the successive stages in a purification process by suitable in-process controls, e.g., sensitive immunoassay(s). Tracing of the many “unknown” cell-derived proteins is more difficult. When devel-oping a purification process other, less specific analyses such as SDS-PAGE are usually used in combination with various staining techniques.