Widespread mycoplasma contamination of cell cultures in the biopharmaceutical industry and the potential economic consequences associated with it have lead to the development of an extensive range of commercial test kits aimed at research laboratories and bioreactor operators.

Regulatory authorities in Europe and North America require that cell cultures used in pharmaceutical production should be free of mycoplasmas, so that testing must be carried out on all cultures at various stages in the production process. For example, the US Food and Drug Administration has published stringent guidelines for mycoplasma testing in cell cultures used for vaccine development. It is also strongly recommended that research laboratories using cell cultures should perform regular tests for mycoplasma contamination, but it is reported that some labs have still not implemented adequate testing regimes.

Prescribed methods for industry are set out in the European Pharmacopeia and in the US Code of Federal Regulations. Two test methods are used - a culture method and an 'Indicator cell culture' test – as not all mycoplasmas can be successfully cultured. The culture method involves inoculating cell culture supernatant into a broth medium, which is then incubated and periodically subcultured onto agar media. The agar cultures are then incubated and examined for mycoplasma colonies. The test takes at least 28 days to produce a negative result. The indicator cell culture test is performed by transferring samples into a Vero cell culture, which is then grown for 3-5 days. The indicator cells are then stained with Hoechst DNA stain and examined under the microscope for surface fluorescence indicative of mycoplasma contamination. Although this method is more rapid than culture, it is less sensitive and still takes several days to complete. When both methods are used together the test can take up to five weeks to complete. It is therefore not surprising that rapid methods have been developed to enable laboratories and manufacturing sites to run screening tests on cell cultures and media for quality assurance and early warning purposes. The great majority of these methods are PCR-based and properly validated PCR-based detection methods are now accepted, or are under consideration, by regulatory authorities around the world as an alternative to conventional methods.

PCR Since there are at least 6-8 common mycoplasma species likely to be present as contaminants in cell cultures, tests need to be able to detect a relatively wide spectrum of species and not be too specific, yet minimise the number of false positive results. Most of the commercially available tests achieve this by targeting sections of the 16S rRNA coding region of the mycoplasma genome at loci that are well conserved in all Mollicutes. The same loci are not well conserved in other bacterial species, thus reducing the chances of a false positive detection. This approach enables some commercially available assays to detect up to 100 species of mycoplasmas, although most are only validated against the species most likely to be found in cells cultures.

PCR assays for mycoplasmas are capable of achieving high sensitivity and some are able to detect just a handful of genome copies in a single assay. The technique is also extremely rapid in comparison with prescribed methods. Stated detection times for commercial test kits range from 2.5 to 5 hours. The main disadvantage remains higher rates of false positives than for culture methods. But the enormous time savings available with PCR-based methods make them ideal for rapid screening of cell cultures, media, raw materials and for monitoring contamination at different points in industrial production processes.

Both endpoint PCR and real-time PCR (RT-PCR) have been used to develop commercial test kits, but there is a trend towards RT-PCR because it can be made quantitative and is capable of greater sensitivity than endpoint PCR. This allows faster detection of low level contamination.

Many PCR-based commercial test kits are aimed at the research market and are designed primarily for monitoring contamination in laboratory cell lines. Examples include the ATCC™ Universal Mycoplasma Detection Kit, which detects over 60 species including the eight most common, and Agilent Technologies MycoSensor RT-PCR assay kit, which claims to detect the eight commonest cell culture contaminant species in less then 2 hours.

Other products are intended for more industrial applications and can be used to monitor raw materials and for industrial process control applications, testing samples at different stages in the manufacturing process, as well as finished products. Examples include Life Technologies' MycoSEQ® Mycoplasma Detection System, capable of detecting up to 90 species in four hours, and the MycoTOOL test developed by Roche, which is validated for 11 mycoplasma species and has been approved by the European Medicines Agency and the FDA for release testing of certain pharmaceutical products.

A rather different approach has been taken by Millipore in the development of the Milliprobe® Real-time Detection System for Mycoplasma. This too is designed for process control applications, but is one of the few systems that does not target the 16S rRNA gene. Instead it targets ribosomal RNA, which is much more abundant in mycoplasma cells than genomic DNA, using Gen-Probe Real-time Transcription Mediated Amplification (TMA) technology. The result is a system validated for 13 mycoplasma species that is able to produce results in less than four hours. The Milliprobe system is semi-automated and includes sample preparation steps so that laboratories lacking high levels of molecular biology expertise can use it.

Other methods Although PCR-based assays are widely available, they not the only option. Other technologies have also been used to develop rapid mycoplasma detection tests.

A number of immunological ELISA-based tests have been developed into commercial products, but these have largely been superseded by PCR-based methods. However, Roche has developed a hybrid Mycoplasma PCR-ELISA assay based on the amplification of a mycoplasma-specific DNA sequence by PCR, and the subsequent detection of the amplicon by ELISA. R&D Systems® MycoProbe® Mycoplasma Detection Kit is another hybrid assay using microplate technology to detect mycoplasmas. It incubates samples with two labelled DNA probes targeting the 16S rRNA gene. The labelled probes are then captured on a microtitre plate and colorimetric detection is then accomplished in a manner similar to ELISA. MycoProbe was developed to combine some of the sensitivity advantages of PCR with the convenience of ELISA and is able to detect the eight commonest species of mycoplasmas within 4.5 hours.

Other technologies developed into commercial products for detecting mycoplasmas in cell cultures include enzyme activity detection by bioluminescence (Lonza MycoAlert® Mycoplasma Detection Kit) and using engineered live cells to detect mycoplasmas in an easy-to-use colorimetric assay (InvivoGen PlasmoTest™).

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