Mycoplasma Testing 〔海外情報〕

Importance of Mycoplasma in the Biotechnology Industry

Regulatory and safety concerns have pushed the biotechnology industry towards stricter guidelines for sterility during recombinant protein manufacture and purification. Careful preparation of cell culture media is of key importance to ensuring sterility of the cell culture batch and subsequent purification processing. Mycoplasma is recognized as the smallest bacterium thriving in cell cultures and mycoplasma is the generic name assigned to microorganisms in the class Mollicutes. These organisms lack a cell wall and are bound by a trilaminar plasma membrane and are the smallest, free-living, self-replicating microorganisms. Since mycoplasma lack a cell wall, they are resistant to many antibiotics often added to cell cultures to prevent microbial contamination.

It has been estimated that between 5 and 35 % of cell cultures currently in use are infected with at least one species of mycoplasma. The nutrient rich media used in cell cultures can lead to propagation of mycoplasma, resulting in diminished cell growth. Mycoplasma infections can also alter host cell characteristics, such as changing cell membrane characteristics, creating chromosome abnormalities and also changing cell enzyme characteristics. More importantly, mycoplasma can affect host cell metabolism and expression levels and ultimately lead to the loss of cell lines. Since the root cause of mycoplasma contamination may only be identified 50% of the time, it is important to implement a comprehensive program within the biotechnology manufacturing process to prevent, reduce and detect mycoplasma contamination.

Where Should I Test for Mycoplasma?

Therapeutic products for both human and animal use are required to undergo mycoplasma testing throughout various steps in the production process including:

Raw materials
Cell banks lines/banks
Virus seeding
Fermented samples
Crude harvests
Bulk vaccines
Purifies biologic products

Preventing Mycoplasma Contamination

(1) Evaluate your raw materials. The bioburden of your raw materials should be characterized including specific screening for mycoplasma.

(2) Use non-animal derived or synthetic media when possible. Animal-based products are more likely to contain mycoplasma contamination then plant based products.

(3) Institute proper aseptic handling procedures, such as training operators on proper handling techniques.

(4) Incorporate cell banking techniques that ensure master and working cell lines are free from mycoplasma contamination.

(5) Use 0.1 µm filtration of process intermediates when possible to ensure the highest level of mycoplasma removal.

(6) Create simple, reliable closed system designs to prevent mycoplasma contamination from the production environment.

Testing for Mycoplasma Contamination

Culturing Techniques
Specific methods are used to detect mycoplasma contamination since they are often not detected in standard bioburden testing. Growth requirements vary depending on the species of mycoplasma which may be recovered. Recovery using standard TSB (Tryptic Soy Broth) most commonly used for bioburden studies does not recover all varieties of mycoplasma and if growth occurs, colonies are small and difficult to detect. Also, liquid culture media with high concentrations of mycoplasma may not cause a pH change or appear turbid making visual checks for contamination unreliable. Media and culturing techniques specific for mycoplasma must be used to ensure recovery of most relevant species of mycoplasma.

A culture test on mycoplasma agar may be performed. In this procedure broth and agar are inoculated with the test sample. The cultures are observed and sub passaged several times and the cultures are incubated at two different temperatures. Positive controls are required to validate and routinely perform testing, therefore testing is not performed within a biotechnology product facility and is often performed at a contract test lab. The duration of the assay is 28-35 days. This test is often referred to as the “FDA Points to Consider” test.

Another culturing technique involves using indicator cell lines. In this test, the indicator cell line is inoculated with the test sample. After incubation of the sample, DNA florescence staining is used to detect presence of mycoplasma in the cell culture. The sample is examined for the presence of extra nuclear florescence staining to determine if mycoplasma is present. The duration of this assay is approximately 7-10 days.

Nucleic Acid Techniques (NAT)
Qualitative Real Time Polymerase Chain Reaction (QPCR) can be used to detect the presence of mycoplasma. This rapid and accurate molecular based method for in-process testing is being driven by the Process Analytical Technology (PAT) initiative, which calls for the manufacturer to audit the process in real-time at multiple points. This method uses universal primers to detect mycoplasma DNA and SPYR Green I as the florescent dye. Specificity of the primers is directed, but not limited to several common mycoplasma species. Limit of detection can range from 1 CFU/mL to 100 CFU/mL depending on the mycoplasma species and or method. Assay duration will depend on the nature of the sample submitted for testing, but results can be obtain in 4 days when a culturing step is included

Regulations Regarding Mycoplasma

EP 2.6.21 “Nucleic Acid Detection Techniques”
EP2.6.7 “Mycoplasmas”
21 CFR 610.30 Code of Federal Regulations 221 CFR Ch.1 Subpart D Mycoplasma Center for Biologics Evaluation and Research, FDA Points to Consider in the Evaluation of Cell Lines Used to Produce Biologicals 1993

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