Cell and gene therapies are poised to revolutionize healthcare, however manufacturing of these products poses new challenges for quality and safety assurance strategies. This is especially true in testing for mycoplasmas, an ubiquitous and practically invisible bacterial contamination of cell cultures. These challenges were a common thread in presentations at the 2019 PharmaLab Pre-Conference Workshop on Mycoplasma qPCR Testing, especially given the anticipated revision of European Pharmacopoeia chapter 2.6.7. Consensus points included, the importance of using cellular matrices for testing and the potential of an implementation strategy that positions NAT as a fast first line of detection, followed by results verification via culture-based compendial methods.

In an effort to investigate some of the most frequent challenges and provide useful answers to frequently asked questions, we spoke with experts and highlighted several consensus points from conference presenters and audience members.

Frequently asked questions and answers are below.

How are advanced therapy medicinal products (ATMP) based on genes, cells, and tissues, creating challenges in mycoplasma testing?

One challenge is turnaround time. It is a critical issue for mycoplasma testing of biopharmaceuticals because delays in manufacturing can be expensive and restrict the availability of much-needed medications. With cell and gene therapies, it is even more crucial because these therapies have shorter shelf lives and are designed for patients with high medical need. The challenge stems from commonly used ‘classical’ compendial methods, that are culture-based and take weeks to generate results. The demand for faster detection of mycoplasmas and the regulatory agencies acceptance of alternative testing methods has moved nucleic acid amplification techniques (NAT) into the spotlight. NAT can detect the presence of mycoplasma DNA via a polymerase chain reaction (PCR), which slashes time-to-results from weeks to hours.

Turnaround time, however, is only the tip of the iceberg. Other considerations are also critical in implementing NAT-based methods to ensure product safety. “A major challenge,” points out Jan-Oliver Karo of the Paul-Ehrlich-Institut Karo, “is that ATMPs are widely diverse, for example, in terms of the origin of starting materials and final biological matrix.” This diversity will also increase with time as further innovation expands the spectrum of approaches and applications for ATMPs.

What stages in the manufacturing process are appropriate testing points?

Experience has shown that not all time points in the production of a biopharmaceuticals or ATMPs are adequate for mycoplasma testing. Testing points should be selected based on the point where risk for mycoplasma contamination is the highest. For ATMPs, in addition to the pre-processed harvest for release testing, in-process testing points should be evaluated to be included, such as testing of the source material or initial cellular starting material after a few passages.

Which sample types are most suitable for the test?

Culture supernatant or other cell-free matrices can be insufficient for testing because mycoplasmas typically attach to or even invade cells. Clearly, sample selection is a critical parameter for a validated testing strategy. Additionally, the sample preparation steps prior to testing need to be carefully considered to ensure the sample type used for testing is still representative of the product. “It is therefore recommended that cell-free matrices be avoided or thoroughly justified,” stated Karo. In fact, most commercial test developers are moving toward cellular matrices and looking at ways to overcome technical hurdles caused by high cell counts.

How do you construct a streamlined validation design that meets regulatory requirements?

From a validation standpoint, it is imperative to work with complex samples or “worst-case products,” as Christiana Schnitzler called them in her presentation. She is leading a team at Boehringer Ingelheim through the generic validation of Roche CustomBiotech’s MycoTOOL Mycoplasma Real-Time PCR Kit. The kit was designed to work with native samples, and Schnitzler is examining if the assay is robust to PCR inhibitors, to DNA extraction variation, and to reagent and sample handling. Ultimately, her goal is to demonstrate that the sensitivity of the assay is at least on par with ‘classical’ compendial methods. The team’s data on the limit of detection are still being collected, but preliminary results indicate that the regulatory requirement for sensitivity (≤10 CFU/mL) can be achieved for the ten tested mycoplasma species.

Schnitzler’s implementation approach couples the assay validation with comparability studies to determine if the PCR method is as sensitive, specific, and robust as the ‘classical’ compendial Culture and Indicator Cell Culture Methods. At this point, the initial NAT-based method can substitute the more laborious, sample-intensive, and lengthy gold standard methods only if this equivalency can be demonstrated. Yet, a strong implementation strategy would be one that uses NAT as a first line of detection and culture-based compendial methods for results verification if necessary. Roche Pharma, for one, has been using its MycoTOOL Mycoplasma Real-Time PCR solution on its manufacturing floors for years and leverages the culture-based methods for verification purposes. “Our experience so far has been that this strategy is welcome by regulatory bodies and results in successful submissions,” declared Alexander Bartes from Roche Pharma. The right constellation of technologies for a fast yet comprehensive testing strategy can also leverage existing facilities. Schnitzler, for example, opted for another method to verify NAT results. “We are in the fortunate situation,” she described, “of having a sequencing lab next door. We confirmed all species identifications through sequencing.”

How do you implement NAT-based methods into a process workflow?

Commercial mycoplasma tests based on NAT offer the convenience of optimized assay architecture with broad species coverage and high sensitivity, plus the option of high-throughput testing and automation.

Several presenters shared assessments of tests on the market and lessons learned that could make implementing NAT-based methods easier for others. For example, Andrej Steyer from the Institute of Microbiology and Immunology of the University of Ljubljana Faculty of Medicine underscored the need for well-characterized validation standards with a low genome copy to CFU ratio (GC:CFU).

Steyer compared a SYBR Green mycoplasma test and the probe-based MycoTOOL Mycoplasma Real-Time PCR Kit. Having opted to focus on the probe-based kit because it exhibited slightly better sensitivity and less variability in CT values, Steyer examined its performance on two different cyclers and with automated DNA extraction. A ten-fold difference in GC:CFU ratio between the two mycoplasma reference standards used in the study re-emerged in the resulting PCR and sequencing data. Steyer advised to “bear in mind the quality of mycoplasma reference standards and the impact that these might have.”

The robustness of a commercial solution to adaptations that enable its integration into existing automated lab setups was the focus of another presenter. Christie English from Mycoplasma Experience Ltd. described input volume adjustments her team made to allow automated DNA extraction for the MycoTOOL Mycoplasma Real-Time PCR Kit on the Roche MagNA Pure Compact Instrument (the kit was developed on the larger MagNA Pure 96). The adaptations were successful, and a comparison with the mycoplasma testing solution already used in the lab revealed complementary benefits of the two solutions. “In fact, we are now using both kits,” concluded English.

What impact will the anticipated revision of European Pharmacopoeia chapter 2.6.7 have on for using NAT-based methods for mycoplasma testing?

Implementing NAT-based methods for mycoplasma testing requires comprehensive validation and demonstration that performance is at least equivalent to the ‘classical’ compendial methods. The topics discussed at the workshop accentuated the need for more detailed guidance in validating and implementing NAT-based methods. However, it will be essential to ensure that concrete recommendations do not distract from creating guidelines that remain relevant for future products.

A case in point was an exchange about the mycoplasma reference strains required as test organisms for validating NAT-based methods and as positive controls in routine testing. The species coverage of existing PCR-based mycoplasma tests is quite comprehensive. For example, the MycoTOOL Mycoplasma Real-Time PCR Kit theoretically has coverage for roughly 150 culturable and nonculturable strains. This broad coverage can be leveraged for generic validation. Among others, Prof. Dr. Renate Rosengarten of the Institute of Microbiology at the University of Veterinary Medicine in Vienna and Director of Mycosafe Consulting recommended expanding the list of mycoplasma species required for testing with NAT-based methods so the selection depends on product-relevance based on growth ability following potential entry determined based on a risk assessment. Karo agreed with the suggestion and underscored that a corresponding discussion is within the scope of guideline revisions. In this context, he pointed out that validation strategies must capture the specific range of mycoplasma species that can potentially contaminate a product. “We endorse the use of validated NAT,” stated Karo. “However, in addition to a generic validation, we emphasize the need for a product-specific validation supported by a thorough risk assessment of the product.”

For more information on Rapid Mycoplasma Testing, please see full conference coverage at 2nd International Mycoplasma qPCR Testing User Day

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