Real-time Mycoplasma Contamination

Detection for Biomanufacturing

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Mycelia of Mycoplasma contamination growing on a CHO cell*
Biomanufacturing contamination due to Mycoplasma is a very real concern, as it poses a
potential health risk for patients. As such, regulatory agencies require biopharmaceutical
companies to test for the presence of mycoplasma both during the manufacturing process and in
the final product. While any contamination can be extremely challenging and costly, mycoplasma
contamination is particularly difficult.

The presence of mycoplasma in cell culture has been described as pervasive. According to the
publication” The scope of mycoplasma contamination within the biopharmaceutical
industry, “contamination rates in established cell cultures have been reported between 15 and
35% with considerably higher occurrence cited in certain selected populations.” Its pervasiveness
could be due to several different factors, with one certainly being the fact that mycoplasmas can
have a broad range of hosts including humans, animals, insects and plants. The small size of
mycoplasmas also makes them problematic to detect and remove via filter. Mycoplasmas thrive
in cell culture environments because they find nutrients in medium and can grow to high
concentrations without initially causing obvious problems to the host cell or culture productivity.

Release Testing Requirements

As discussed, biopharmaceuticals must be completely free of mycoplasmas and regulatory
authorities require release testing as part of the manufacturing process. Traditional methods for
mycoplasma testing as described in the USP (U.S. Pharmacopeial Convention) and EP
(European Pharmacopoeia) involve the culture method and/or indicator cell culture testing. These
testing methods, while well defined and widely accepted, are difficult to administer because they
require highly trained personnel to administer and can require up to 28 days for results. In
addition, difficult to cultivate or non-cultivable mycoplasma species can result in a mycoplasma
contamination going undetected. As a result, many regulatory agencies now also accept rapid
nucleic acid amplification techniques (NAT) such as real-time quantitative polymerase chain
reaction (qPCR) for mycoplasma testing. In addition to final release testing, qPCR testing for
mycoplasma enables a more robust in-process control strategy.
qPCR Solution
PCR testing for mycoplasma has been gaining acceptance for some time and Roche
CustomBiotech recently presented a poster detailing the validation of their MycoTOOL
Mycoplasma Real-Time PCR Kit (MycoTOOL RT) conducted by Roche Pharma (Penzberg,
Germany) based on EP2.6.7 NAT validation guidelines. The United States, European, and
Japanese pharmacopeias list nucleic acid tests (NAT) as a mycoplasma testing option provided
that they are properly validated.

Poster Highlights
The poster nicely outlines workflow for manual and automated processes and presents data on
the validation study results.

MycoTOOL RT Workflow
The MycoTOOL RT is compatible with either manual or automated protocols and testing can be
conducted in less than 5 hours for both (Figure 1). This is a significant advantage that PCR has
over more traditional methods.

Figure 1. MycoTOOL RT work flow using either a manual or automated DNA extraction method. The automated work flow
based on the MagNA Pure 96 and LightCycler 480 II systems shown as the red marked process procedure has been fully
validated by Roche Pharma Biotech as it is presented in this poster. *Both the MagNA Pure 24 and the QC
Preparation Kit are functionally tested, but not validated.
Another advantage for MycoTOOL RT is that it utilizes highly specific TaqMan® probes, which
permit the detection of both cultivable and non-cultivable mycoplasma species. This reduces the
risk of a contamination going undetected.

Validation Study Results

The study outlined in the poster below, demonstrates compliance of the MycoTOOL RT with the
EP2.6.7 NAT validation guideline, showing that the assay is sensitive, specific, robust, precise
and comparable to compendial methods for CHO manufacturing processes.

In the poster, Roche CustomBiotech presents data on limits of detection (LOD), test specificity,
test robustness, precision, absence of cross contamination and comparability as part of their
validation process. Please refer to the poster for specific details and methods.

A critical part of the data was the results of the comparability study, which concludes that all three
of the mycoplasma detection methods: culture method, indicator cell culture method, and
MycoTOOL RT were able to detect mycoplasma contaminations with a sensitivity ≤10 CFU/mL.
However, MycoTOOL RT was also able to detect strains that are non-cultivable.

Conclusion
It makes sense that biopharmaceutical companies are moving toward a more rapid mycoplasma
detection method. With mycoplasma testing that is now quick and efficient, it is a tool that can be
more widely used and can serve more purposes. For instance, mycoplasma detection testing
could now be used as an inbound quality control step for raw materials or as an in-process early
detection approach to avoid further contamination, loss of time, and resources.

This article is sponsored by Roche CustomBiotech.