Mycoplasma Contamination Testing
Cell substrates used in the manufacture of biologics must be shown to be free of adventitious agents, including mycoplasma. Testing for mycoplasma contamination must be performed at various phases of product development, including raw materials, cell banks, viral seed stocks, unprocessed bulk harvest materials and final products. Spiroplasma testing is also suggested for biopharmaceutical products that have been in contact with plant or insect materials.
Regulatory-compliant Mycoplasma Test Methods
Charles River performs mycoplasma testing of biologic products according to worldwide regulatory guidance documents, including those from the US, EU and Japanese Pharmacopoeia (USP, EP, JP) and Points to Consider (PTC), as well as combination protocols covering multiple regulations:
- Combined EP/USP/JP Protocol
- Combined EP/USP/PTC Protocol
- Combined EP/USP/JP/PTC Protocol
We additionally offer testing for the presence of mycoplasma using the large volume method and quantitative PCR (qPCR) assays for rapid receipt of results. The mycoplasma assays developed at Charles River incorporate the use of many positive controls including M. pneumoniae, M. orale, M. gallisepticum, M. hyorhinis, M. synoviae, A. laidlawii and S. clarkii. Mycoplasma clearance studies and spiroplasma testing are also available upon request.
Test items are inoculated onto agar plates and into broth, and maintained under the atmospheric conditions according to the specified guidelines. The broth is subsequently subcultured onto agar plates and observed for evidence of mycoplasma contamination.
Indicator Cell Culture Method
For non-cultivable mycoplasma species, the test material is cultured in the presence of an indicator cell line, like Vero cells. The cells are subsequently stained with a DNA binding fluorochrome stain (DAPI) and evaluated microscopically for the presence of a mycoplasma infection. Some test materials may be cytotoxic to the indicator cells, and must be diluted before inoculation.
In some cases, the test item may interfere with the growth of mycoplasma in the culture or indicator cell culture assay. For this reason, the EP and USP request verification of the method by a mycoplasmastasis study, testing for inhibitory substances via spiking positive controls into the test material. This procedure is normally performed only once for each test matrix. If the test item interferes with the growth of mycoplasma, the inhibitory substance must be neutralized or the test item further diluted.
We use the following qPCR methods to detect mycoplasma contamination:
- Amplification of specific nucleic acids and fluorescent probe technology: This assay detects the presence of a particular nucleic acid sequence, but does not necessarily indicate the presence of viable mycoplasma.
- Broth culture growth enrichment and RT-PCR: This assay includes a growth enrichment step prior to the nucleic acid test in order to delineate viable organisms from non-viable organisms and residual environmental sources. Samples are tested at day 0 and day 7.
- Growth enrichment by cell co-cultivation with RT-PCR: This assay includes a growth enrichment step prior to the nucleic acid test to distinguish viable organisms from non-viable organisms and residual environmental sources. Samples are tested from a cell co-cultivation at day 0 and day 5.
- Spiroplasma testing: This assay includes a growth enrichment step prior to the nucleic acid test in order to delineate viable organisms from non-viable organisms and residual environmental sources. Samples are tested at day 0 and day 7.