PCR test for mycoplasma rivals culture methods

Scientists at US biotechnology company Genentech have developed a rapid testing method for mycoplasma contamination in Chinese hamster ovary cells, one of the most common mammalian cell lines used in biological drug production.

The presence of mycoplasma - a unique group of microorganisms that fall in the category between the bacteria and viruses - in cell cultures can have a dramatic impact on the growth and metabolism of the cells, and a knock-on effect on the yield - and possibly the activity - of the recombinant protein.

The new testing method is based on the polymerase chain reaction (PCR) that detects and amplifies DNA in a sample so that it can be identified. At present, European and US regulations demand that CHO cells used to make recombinant therapeutic proteins are routinely tested for mycoplasma, a process that can take four to six weeks using conventional approaches such as broth/agar cultures and DNA staining of indicator cell cultures.

With the new PCR approach, the time required to carry out this testing is reduced to 1-2 days, and because PCR is such a widely used technology, the reagents required for the test are plentiful and relatively inexpensive, reducing the cost of the procedure, according to Genentech's Joyce Eldering, who led the team that developed the test.

Other testing protocols based on PCR have been developed, she noted, but these tend to suffer from a lack of sensitivity, failing to pick up contamination of less than 1,000 colony-forming units/ml, while culturing and DNA staining can detect down to 1-10cfu/ml. Other PCR approaches have also tended to have a risk of false-positive and false negative results, preventing them from replacing the traditional culture methods in the industrial setting, she said.

The new PCR assay for mycoplasma detection "appears to resolve these issues while being sufficiently simple and inexpensive for routine use," according to the team, which published its findings in the journal Biologicals, the official journal of The International Association for Biologicals (IABs).

The team improved the sensitivity of the PCR probe by targeting it to a DNA sequence that is a feature of mycoplasma strains that infect mammalian cell cultures. It is also based on the combination of 8-methoxypsoralen and ultraviolet (UV) light treatment to decontaminate the PCR reagents of DNA, the use of hot-start Taq DNA polymerase to reduce non-specific priming events, and so-called touchdown PCR to increase sensitivity, while also reducing non-specific priming events.

"In extracts of mycoplasma DNA, the limit of detection for eight different mycoplasma species is 10 genomic copies. In CHO cell production cultures containing gentamicin, the limit of detection for a model organism, gentamicin-resistant Mycoplasma hyorhinis, is 1 cfu/ml," they write.

This level of sensitivity seems equivalent to the cell culture methods currently used by the biopharmaceutical industry, they concluded.