Cell and gene therapies are the engine powering the future of medicine. They hold the promise of treating many diseases that currently elude researchers and clinicians — including cancer, cystic fibrosis, heart disease, diabetes, hemophilia, and AIDS. However, as with all new technological advancements, some kinks still need to be worked out.
With complex development processes, there are many potential contamination sources for cell and gene therapies that must be carefully monitored along these products’ journey to market. In particular, host cell DNA contamination that is not detected and filtered properly can have dire consequences for the patient who receives a contaminated therapy and the lab that produces it. In addition, since the nature and production of these products are highly sophisticated, they require similarly sophisticated laboratory techniques to ensure their proper standardization and control.
Every host cell line carries risks
When manufacturing new cell or gene therapies, host cells function as factories to produce the viral vectors needed to deliver the final product.1 Scientists can choose from a wide array of host cell lines for this purpose, each with its own advantages and disadvantages.
The human cell lines most commonly used for creating new therapies are HeLa and HEK 293. HeLa cells, derived from cervical cancer cells, were the first immortal cell line to be established. They are prized for their remarkable durability and were used to develop the polio vaccine and techniques like gene mapping and in vitro fertilization. HEK 293 cells are human embryonic kidney-derived epithelial cells, and are the second most widely used line because of their reliable growth and propensity for transfection. Among the most used non-human cell lines are CHO cells, which are derived from Chinese hamster ovaries and are popular due to their low chromosome number.2
Potential fallout of residual host cell DNA contamination
Since cell and gene therapies are biological products delivered directly to patients, it is critical that the viral vector batches be kept completely free of impurities and contaminants, including host cell components. Any level of contamination can have potentially disastrous consequences if it isn’t caught before the treatment is administered.
Should any host cell DNA make it into a patient, there is a risk that it will trigger an adverse reaction, such as an immune or inflammatory response. Since many commonly used cell lines — including HeLa and HEK 293 — are derived from cancer cells, there is a risk of oncogenic DNA transfer, which can cause the development of tumors in the patient.3 Beyond patient concerns, if any lab were to release a therapeutic with residual DNA still in it, there would be legal and regulatory blowback that could inhibit the progress of any future therapeutics making it to market. Such an incident could even impact the long-term viability of the lab or larger organization.
How to meet purity standards
Host cell DNA is removed along with other products and process-related impurities in a downstream purification process typically consisting of a capture step followed by two or more polishing steps, including an anion exchange chromatography step.4 Significant DNA clearance is usually achieved in the capture step. Still, scientists cannot afford a large margin of error, so they must take additional steps to ensure that all but the slightest traces of residual host cell DNA have been eliminated.
The Food and Drug Administration (FDA) published industry guidance for the manufacturing and control of new applications for cell and gene therapies:
“We recommend that you limit the amount of residual DNA for continuous non-tumorigenic cells to less than 10ng/dose and the DNA size to below approximately 200 base pairs. If you are using cells that are tumor derived (e.g. HeLa) or have tumorigenic phenotypes (e.g., HEK293, HEK293T) or other characteristics that may give rise to special concerns, the limitation of specific residual DNA quantities may be needed to assure product safety … Your tests should be appropriately controlled and of sufficient sensitivity and specificity to determine the level of these sequences in your product.”5
Scientists can only obtain such sensitive and specific measurements using a test that provides absolute quantification, reducing measurement errors. To meet these requirements, many labs are switching from quantitative PCR (qPCR) to Droplet Digital PCR (ddPCR) technology for monitoring cell persistence, determining dosages, detecting contaminants, and more. ddPCR testing offers greater precision and sensitivity, as it does not rely on standard curves. Armed with an absolute measurement of nucleic acids in a sample, scientists can be confident that residual host DNA is limited to acceptable levels before allowing a treatment to be used on a patient. ddPCR assays are poised to become the gold standard because the protocol is extraction-free, requiring less sample manipulation and introducing fewer variables into the process overall. This technological advancement is a big leap forward for safety and has become the gold standard for ensuring biologics are safe and as contaminant free as possible.
A promising future makes rigor worthwhile
The excitement around cell and gene therapies has never been higher, with new therapies getting approval and more clinical trials beginning with greater frequency. Although safety remains a concern, researchers are breaking new ground by creating many new treatments and the possibility that some cures may follow. However, researchers must remain vigilant in keeping their products free of contaminants because it only takes one well-publicized error to set research back years.
Learn more about how digital PCR assays can help you detect and quantify contamination.