Spiroplasma and Mycoplasma Detection Assays
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 Testing Methods
Mycoplasma testing is a regulatory requirement and mycoplasma test methods are described in several compendia, regulations, and guidance documents worldwide. The traditional culture method and the indicator cell culture method are not fully harmonized and may vary between different jurisdictions.
We offer all relevant compendial test methods and perform mycoplasma testing compliant with the United States Pharmacopoeia (USP) General Chapter <63>, the European Pharmacopoeia (EP) General Chapter 2.6.7, and the Japanese Pharmacopoeia (JP), along with combination protocols.
Spiroplasma testing is also available. The test methods incorporate the use of control strains including M. pneumoniae, M. orale, M. gallisepticum, M. hyorhinis, M. synoviae, A. laidlawii, and S. citri.
We additionally offer mycoplasma testing using the large volume method and quantitative PCR assays for rapid receipt of results. Mycoplasma clearance studies can also be performed to demonstrate the capacity of the downstream process to inactivate or remove mycoplasma contaminants.
Mycoplasma Detection Test Methods
- Test samples are inoculated directly onto agar plates as well as into liquid medium.
- Agar and liquid media chosen are shown to have satisfactory nutritive properties to support the growth of a broad range of mycoplasma species.
- Negative and positive controls are included in each mycoplasma detection assay to check the system suitability.
- During the incubation period the liquid cultures are subcultured at multiple time points onto agar plates to visualize mycoplasma growth.
- At the end of the incubation period, the agar plates are examined microscopically for the presence of mycoplasma colonies.
- Total test time is 28 days.
Indicator Cell Culture Method
There are fastidious mycoplasma strains which are called “non-cultivable” because they do not grow in the standard media used for the culture method. Cultures of Vero or NIH3T3 cells are used to grow mycoplasmas. After an incubation period of one week, the cells are fixed and stained with a DNA-binding fluorescent dye and evaluated microscopically.
In the case of a mycoplasma infection there is a characteristic pattern of bright fluorescence on the surfaces of the cells and in surrounding areas when the contamination is heavy.
We use the following qPCR methods for mycoplasma detection:
- Amplification of specific nucleic acids and fluorescent probe technology: This mycoplasma detection 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 mycoplasma detection 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 to delineate viable organisms from non-viable organisms and residual environmental sources. Samples are tested at day 0 and day 7.
Even though PCR testing is used more often for the detection of mycoplasmas, there is no general approval for PCR-based mycoplasma testing by the regulating agencies. It is possible to use PCR as a complementary method for in-process controls and product-specific validation; however, a comparability study is required against the traditional method to demonstrate the methods are equivalent.
Our experts can help develop a mycoplasma testing strategy specific to your product.
Frequently Asked Questions (FAQs) about Mycoplasma Testing
What mycoplasma testing methods are available at Charles River?
We perform 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
Additional mycoplasma testing includes:
- Large volume and quantitative PCR (qPCR) assays
- Mycoplasma clearance studies
- Spiroplasma testing
In which cases are the mycoplasmastasis study method utilized?
Mycoplasmastasis study methods are used to demonstrate that a test sample does not contain inhibitory substances. These test samples must demonstrate the ability of different mycoplasma species to grow. The test for mycoplasmastasis should be performed at least once per individual sample matrix and must be repeated whenever there is a change in the composition that may affect the detection of mycoplasmas.
What regulatory requirements are recommended for mycoplasma testing?
The testing guidelines that address mycoplasma testing for biopharmaceutical products include the following:
- European Pharmacopoeia 2.6.7
- United States Pharmacopeia <63>
- ICH Q5D, Quality of Biotechnological Product (1997)
- CBER/FDA, Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993)
- CBER/FDA, Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (1997)
- FDA, Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications (February 2010)