Cell cultures used in the manufacture of biopharmaceuticals must be tested for Mycoplasma contamination. Mycoplasma contamination does not produce turbidity but can have adverse effects on cell line characteristics/growth rates and virus production. Certain Mycoplasma species are also classed as human pathogens, therefore their presence in a manufacturing process is of serious concern.
We conduct Mycoplasma testing of client test articles in our GMP facility in compliance with pharmacopeial standards. Our portfolio of tests includes a combined assay which meets the requirements of both European Pharmacopoeia Section 2.6.7 and the United States Pharmacopoeia <63> Monograph.
Prior to conducting a mycoplasma assay a mycoplasmastasis assay is carried out.This is a qualification test to examine the test article 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.
International regulatory guidelines vary slightly in their recommended Mycoplasma detection methods. Variations of the following assays are used, all with multiple positive controls:
Mammalian cell culture - The sample is incubated with monkey kidney cells which are then fixed, stained with a DNA-binding fluorochrome (Hoechst Stain), and evaluated microscopically by epifluorescence for the presence of Mycoplasma.
Broth and agar assays- Broth flasks are inoculated with the test article and are incubated aerobically. Samples from the broth flasks are sub-cultured onto agar plates
Plate agar assays - The sample is inoculated directly onto agar plates.
In the above assays agar plates are incubated under appropriate microaerophilic, anaerobic and/or aerobic conditions depending on the regulatory guidance being followed. Plates are examined for Mycoplasma 14 days post-inoculation.