Mycoplasma is a group of very small (~ 0.2-0.3µm) prokaryotic microorganisms without cell walls, they are usually nonmotile, and are often pathogenic or parasitic, and can cause disease (e.g., pneumonia and urinary tract infections) in animals and humans. Mycoplasma contamination (e.g., genera Mycoplasma and Acholeplasma) is a serious concern in biopharmaceutical manufacturing facilities. Potential undetected mycoplasma contamination of the products or process intermediates poses potential safety risks for patients and business risks for biopharmaceutical manufacturers. To minimize these risks, carefully monitoring for adventitious agents, such as viruses and mycoplasmas, is performed during the biologics manufacturing processes.

Cell banks and pre-harvest cell culture fluids are considered as growth promotion solutions and have nutritive properties to mycoplasma, they are often subject to mycoplasma testing. To be specific, cell banks (MCB, WCB, or EPCB) need to be well-characterized to demonstrate they can permit a consistent source of production cells (identity) throughout the life of a product but also decrease the likelihood of contamination by other cell lines, endogenous and adventitious microbial contaminants (bacteria, fungi, mycoplasmas, and viruses). Pre-harvest cell culture materials from production bioreactors are checked for mycoplasma contamination to ensure product safety.

Mycoplasma testing methods using microbiological assays are recommended by the USP, EP, JP, ChP and the US FDA, usually involves the culture of viable mycoplasmas in broth (typically applies to EOPC to recover cells first), agar plates and indicator cells. The testing usually takes 28 days or more, and results are usually reported as “Detected” or “Not detected”. Simpler and faster testing method is also available through DNA PCR checking. Many contract testing laboratories offer Mycoplasma testing services, namely Wuxi Biotech., Eurofins Lancaster, Charles River, Bionique Testing Laboratories, Incell, Cell Line genetics, etc.

From manufacturing process perspective, mycoplasma control of a biological product may be addressed in the following, but not limited to, common technical documents (CTDs) to define testing requirements and sampling points.

1. MCB and/or WCB testing and release specifications

2. End-of-production cell bank (EPCB) testing specification

3. Process FMEA

4. Process description and process control strategy

5. Master sampling plan (MSP)

6. Mycoplasma risk assessment (if applicable, not required)

7. Mycoplasma testing method SOP(s).   

Reference:

1. USP 33/NF 28 <63> Mycoplasma tests, 2010.

2. USP. Public notices; 2010. http://www.usp.org/USPNF/jan12Recall.html.

3. USP 33/NF 28. <1226> Verification of compendial procedures, 2010.

4. Test for Mycoplasma. 21 CFR Sect 610.30.

5. US FDA points to consider in the characterization of cell lines used to produce biologicals,1993. http://www.fda.gov/downloads/BiologicsBloodVaccines/SafetyAvailability/U… .

6. EP 2.6.7 Mycoplasmas, 6th ed.; 2010.

7. Historical overview of mycoplasma testing for production of biologics

8. Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques