Mycoplasma Assays
Testing manufacturing cell cultures for Mycoplasma contamination is critical in the production of reliable biotechnology products. Mycoplasma contamination does not produce turbidity but can have adverse effects on cell line growth rates, characteristics and viral production. Certain mycoplasma species are also classed as human pathogens, therefore their presence in a manufacturing process is a serious regulatory concern.

As such, the US Code of Federal Regulations (CFR), FDA Points to Consider (PTC), International Conference on Harmonisation (ICH) and EU Pharmacopeia (EP) guidelines provide technical documentation on the procedures required for the detection of Mycoplasma during biopharmaceutical production. The EP, PTC, and CFR vary slightly in their recommendations on how to perform the assay. Generally, at least three species of mycoplasma serve as positive controls, and the assays are used to detect the presence of mycoplasma in three ways:

• Mammalian cell culture. The sample is incubated with monkey kidney cells and is then fixed, stained with a DNA-binding fluorochrome (Hoechst Stain), and evaluated microscopically by epifluorescence for the presence of mycoplasma.
• Broth and agar. Broth flasks are inoculated with the test article and are incubated aerobically. Samples from the broth flasks are sub-cultured on appropriate days onto agar plates. Plates are incubated under appropriate microareophilic, anaerobic and aerobic conditions for a period of 7-14 days depending on the regulatory guidance being followed.
• Plate agar assays. The sample is inoculated directly on agar plates and incubated under appropriate microareophilic, anaerobic and aerobic conditions depending on the regulatory guidance being followed. Plates are examined for mycoplasma no sooner than 14 days post-inoculation.

Mycoplasmastasis
Mycoplasmastasis is described in the EP guidance, and represents a qualification test to examine the test sample for product-specific inhibitors that can interfere with the growth of mycoplasma. Normally this test would be required only once for any given product provided there is no change in the manufacturing process.

Regulatory Compliance
All our Mycoplasma assays at a minimum meet the required regulatory standards.

Appropriate documentation links:

US Code of federal regulations

FDA Center for Biologics Evaluation and Research “Characterization and Qualification of Cell Substrates and Other Biological Starting Materials Used in the Production of Viral Vaccines for the Prevention and Treatment of Infectious Diseases”

FDA “Points to Consider in the Characterisation of Cell Lines Used to Produce Biologicals”

Ph.Eur. 2.6.7 European Pharmacopoeia (2.6.7 Mycoplasmas)

Mycoplasma qPCR Assay
The Ph.Eur. 2.6.7 guidance states that Nucleic acid amplification techniques (NAT) can be used to test for mycoplasma when a complementary test is required or can be used as an alternative. For example when the test sample is a cytotoxic viral suspension, or when under special circumstances the regulatory authority has approved this technique for product lot release after the review of special risk-based analysis. In addition, the FDA state that PCR for the detection of Mycoplasma can be applied for products with a limited shelf life.

Vitrology’s regulatory compliant in-house Mycoplasma qPCR is designed to detect the presence of mycoplasma in biological materials. With qPCR testing, results are obtained in less than half the time of culture-based methods. Vitrology’s primer and probe set is designed to a highly conserved Mycoplasma region allowing specific detection of a wide range of mycoplasma species with high sensitivity and specificity.
The qPCR assay is designed to detect, and is not limited to the following species:

Click here for literature references.