Residual DNA Testing

If host cells are infected with a genome-integrating virus, such as a retrovirus, the DNA of this virus can remain in the cell's genome, becoming part of the host cell lysate. This contaminated DNA can then be inadvertently incorporated into the biopharmaceutical product. As a downstream consequence, cells interacting with the contaminated residual DNA in the product could potentially transcribe viral genes and become infected themselves.

DNA infectivity should be considered when conducting a safety assessment of host cell residual DNA, especially when manufacturing biologics in continuous cell lines.

Cells immortalized for manufacturing purposes often harbor genes that can lead to cancer (oncogenes), such as rat sarcoma (RAS) and myelocytomatosis oncogene (MYC). These oncogenes, if introduced to normal cells through residual DNA contamination, could potentially lead to cancer. Oncogenic RAS, for example, promotes rapid growth by suppressing cyclin-dependent kinase inhibitors, and overexpressed MYC promotes cell growth and survival as a transcription factor. Another, less likely, source of oncogenic potential occurs if these residual DNA fragments integrate into the recipient cell genome. In that case, they also have the potential to cause a frameshift mutation with the potential for oncogenic consequences.

The risk of downstream cancer-promoting activity further underscores the necessity of conducting thorough residual DNA testing and quality control on manufactured biologics.

The US FDA guidelines state that for biologics, residual DNA from host cells cannot exceed 100 pg/dose. For high-dose biologics such as monoclonal antibodies, residual DNA cannot exceed 10 ng/dose with certain special considerations for the source of the residual DNA, and the route of administration.

For most biological products, the European Pharmacopeia limits residual DNA to 10 ng/dose, but in some cases, this is much more stringent, and several vaccines (e.g., hepatitis A) have limits of 100 pg/dose.

The WHO guidelines recommend that in finished products, residual DNA should not exceed 10 ng/dose and cannot be over 200 bp in length.

Residual DNA testing is simply the term given to the practice of quantifying the amount of host cell DNA in a biological sample or product where host cell DNA does not belong.

This residual DNA ends up in a biological product in circumstances where host cells are necessary for the production of biological material (e.g., the production of a virus or a plasmid requires host cells).

According to the FDA, residual DNA from host cells cannot exceed 100 pg/dose for regular biologics, and for high-dose biologics, residual DNA cannot exceed 10 ng/dose. However, special considerations are sometimes made in cases where the type of host cell or the route of administration of the product are exceptional.

The base pair length of residual DNA, or the length of contaminating DNA fragments, matters because the longer a piece of DNA is, the more information it encodes. Suppose a fragment is long enough to be transcribed and translated into an entire protein. In that case, the associated potential risk of its presence in the product is much higher because proteins are what carry out physical changes in living systems.

This is also why the World Health Organization guidelines limit DNA fragment length to 200 bp.

In cases where a particular gene is concerning.

For example, if a scientist was using an immortalized cell line to grow a virus for a vaccine, that cell line expressed oncogenic mutant RAS GTPase, but was otherwise a normal cell line. It might be wise to focus on testing specifically for mutant RAS in their residual DNA, because this gene is what is likely to have more negative downstream consequences than the others if it is present in the vaccine product in high concentrations.