James Zellner, Technical Sales
Mycoplasmas are a fascinating group of extremely small bacteria with unique characteristics and growth habits. There are over 120 species of Mycoplasmas found in and on a variety of animal and plant hosts. They possess no cell walls, are fastidious in their growth requirements, and are the smallest type of self-replicating prokaryotic organism. Their lack of a cell wall makes them resistant to antibiotics like Penicillin, which target cell wall synthesis in other bacterial organisms. Mycoplasmas are parasitic and commensal bacteria that commonly colonize cell lines and tissue cultures in the lab environment, and the epithelial lining of the respiratory and urogenital tracts in humans. Their parasitic nature is a result of their inability to produce a number of factors required for growth, hence their association with living cells and tissues.
The characterization and detection of Mycoplasmas can be difficult, since they do not Gram stain (no cell walls), and when they are viewed microscopically, present with varying cell size and morphology. They can’t be detected using standard sterility test methodology like USP <71>, so specialized techniques are required to screen for Mycoplasmas. Detection of Mycoplasma contamination is critical for cell and tissue line production, biologic therapeutics derived from host and cell lines, and in the production of cell line medias and supplements. Infections of Mycoplasmas in cells and tissues may go undetected for an extended period of time, while simultaneously altering every aspect of the growth and metabolism of the cell line. Visible damage, alteration of cell appearance, or turbidity may not be present in cell products contaminated with Mycoplasma. A 2008 Corning study on cell culture contamination indicated that Mycoplasma contamination was a widespread problem, appearing in a disturbingly high number of cultures in the lab environment. The study also highlighted the very high density of growth possible, and difficulty associated with attempting to filter Mycoplasma out, all due to the extremely small size of these organisms1.
There are a variety of techniques available to screen for Mycoplasma contamination. USP <63> Mycoplasma Tests, serves as the compendial reference chapter. USP states “Testing for Mycoplasma is a necessary quality control requirement to assure reliably pure biotechnological products and allied materials used to generate these products.” The two compendial methods referenced in the chapter are an agar and broth procedure, and an indicator cell line procedure. In the agar and broth technique, a variety of specialized medias and environmental conditions are used to culture Mycoplasma, if present. The alternate method, an indicator cell line procedure, uses an actual cell culture which is exposed to the product being tested to encourage Mycoplasma growth and indicate contamination. Downsides to each of these is the time required to run the tests. The agar and broth method takes several weeks, and the cell line method takes over a week. In addition to these compendial procedures, USP describes the use of validated nucleic acid amplification techniques, such as PCR, to screen test samples. PCR methods are highly beneficial since the turnaround time can be 3 days or less. ARL Bio Pharma offers a validated, formulation specific PCR method with quick turnaround times. In all cases, careful aseptic technique, appropriate laboratory conditions, and a highly trained staff able to properly interpret the results are required.
With the increasing demand for biologic products and patient specific treatment protocols, Mycoplasma testing should be considered as critical as any other quality test during production and on finished products. Mycoplasma’s ability to remain undetected, reach high population density, and evade attempts at removal, make screening paramount. Mycoplasma contamination poses a significant risk to patient safety and potential loss of products made from biologic sources.
- 1John Ryan (2008). "Understanding and Managing Cell Culture Contamination", Technical Bulletin, Corning Incorporated. p. 24.
- US Pharmacopeial Convention (USP). USP 43/NF 38 <63> Mycoplasma Tests. 2022.
- US Pharmacopeial Convention (USP). USP 43/NF 38 <71> Sterility Tests. 2022.
For more information, please contact ARL at info@arlok.com or 800-393-1595.