Mycoplasma testing

Mycoplasmas are one of the most common type of contaminants, are part of the Mollicutes family, the smallest and simplest self-replicating organisms and a common bacterial contaminant of cell culture samples. Mycoplasmas are more than 90 species, they are very small in size (their dimensions range from 0.2 to 0.8 µm) and, therefore, they can pass through some filters used to remove bacteria. Moreover, they give limited turbidity in cell culture media and some species are cell-invasive.

For the above reasons, Mycoplasma contamination analysis requires an highly sensitive detection technique to be used as part of a routine and regular quality control screening procedure in laboratories that adhere to Good Manufacturing Product guidelines (e.g. in process control and batch release step for cell lines produced in cell factories). Cell lines must be Mycoplasma-free as required by the EU Pharmacopoeia (EP 6.6 chapter 2.6.7) and EMA regulatory requirements.

There are different methods for mycoplasma detection in cell culture supernatant. The most commonly used and approved method by the European Pharmacopeia is the Broth Culture Method that, after 14 days, allows to detect at least 10 CFU/ml of mycoplasma in a cell culture supernatant. Because mycoplasmas grow slowly (the colonies need up to 3 weeks to develop), traditional culture methods are unacceptable for rapid high-throughput testing. However, the European Pharmacopeia suggests that alternative methods (e.g. ELISA, PCR ) can be used to replace the Broth Culture method, but the acceptability criteria of the official method, such as Limit of detection, Sensitivity and Specificity need to be maintained.

Procelltech developed an alternative and innovative method for Mycoplasma screening based on Real Time PCR. This method can be used for quality control of cell cultures released in GMP condition. The advantages of using this method are the following:

• High throughput
• Detection of more than 90 Mycoplasma Species in one single test
• High sensitivity (Limit of detection <10 CFU/ml)
• Specificity for Mycoplasmas (does not detect DNA purified from related or unrelated species)
• High reproducibility and affordability

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