Mycoplasma are an unusual type of bacteria that can infect animal, insect, and plant cells. They can be a very costly pest for biopharmaceutical researchers and manufacturers. These little organisms (0.2-2 micron) are the smallest and simplest free-living parasite. They can impact the safety and quality of cell-derived biopharmaceutical products. This puts patients at risk and could cause untimely and costly delays to manufacturing. To minimize these risks, routine testing for mycoplasma should be performed throughout the product research, development and manufacturing process.

There are several ways to test for mycoplasma.  These include direct culture, Hoechst DNA staining, PCR-based testing, and  hybridization-amplification by ELISA. For the production of biopharmaceuticals, direct culture-based approaches assess the presence of viable cells using indicator cell lines exposed to mycoplasma. However, this type of testing requires weeks and costs approximately $5000 per sample. An alternative approach to identifying mycoplasma contamination is through PCR-based or hybridization ELISA testing. The molecular-based PCR method or the hybridization-ELISA method is ideal for research laboratories. These methods are rapid, highly sensitive, specific, reliable, and cost-effective. The PCR-based method and hybridization method are designed to amplify or bind to the conserved 16S rRNA region of the mycoplasma genome. Eight species of mycoplasma are responsible for approximately 95% of cell culture contamination events and are detected using these techniques. There are numerous PCR based protocols (for example see ATCC.org kit offering) that use primers recognizing the 16S rRNA and can detect mycoplasma contamination by measuring a distinct PCR product. For the hybridization technique, (i.e. MycoProbe, Mycoplasma detection kit by R&D Systems) samples are hybridized in a microplate containing biotin labeled capture oligonucleotide probes and digoxigenin-labeled detection probes that map to the 16S rRNA sequence. The hybridization product is detected with anti-digoxigenin alkaline phosphatase.  PCR and hybridization techniques have been demonstrated to be extremely sensitive, comparable to direct culture, are rapid, and cost significantly less than direct culture (approximately $500 per sample).

Enhanced regulated testing with an overlay of quality assurance systems to comply with GMP requirements adds to the cost. For GMP lot release, Europe and the USA-FDA have established guidances to follow. For research and development, PCR and hybridization-ELISA techniques are ideal for routine, rapid screening. Cell lines can be tested every few months or screened immediately after receipt or before/after cryopreservation, therefore, allowing cell lines and biological products to be used after a short quarantine. BioTether Sciences performs both PCR based, and hybridization-ELISA based mycoplasma testing to support your research and development. This service is included free with cell based assay and stable cell line development projects placed at BioTether Sciences. We can also provide rapid turn-around and reporting on client supplied samples (cell line supernatant, cell pellets, reagents). Don’t let those little parasites derail your biopharmaceutical program!

Reference: Drexler HG, Uphoff CC. Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology 39: 75-90, 2002. PMCID: PMC3463982