May 1, 2015 (Vol. 35, No. 9)

Even When Less Than Catastrophic, Mycoplasma Infestations Are More Than Inconvenient

Bioprocessors have a huge stake in detecting mycoplasma wherever the organism exists in their facilities, but especially in finished products. At present, bioprocessors detect mycoplasma by utilizing a month-long culture-based methodology. Although this methodology has been considered the gold standard, rapid polymerase chain reaction (PCR) methods augment—and in some cases exceed—the capabilities of culturing.

The MycoSEQ® mycoplasma detection system from Thermo Fisher Scientific was designed to meet the guidance provided by the European Pharmacopoeia in 2007 of rapid-response tests. MycoSEQ, which utilizes quantitative PCR (qPCR) to detect more than 90 mycoplasma species, can provide results in hours.

“PCR assays make sense for in-process testing,” notes Michael Brewer, head of pharmaceutical analytics at Thermo. “It provides rapid answers, for example, before passing cultures to the next bioreactor during cell expansion. Routine in-process testing is a way to avoid unpleasant surprises.”

The European Pharmacopoeia published guidance on use of nucleic acid-based tests as alternatives to the culture protocol in 2007. For validation studies, regulators ask biomanufacturers to compare PCR and qPCR mycoplasma assays to the long-accepted 28-day culture-based test, particularly when companies are validating with the goal of switching from the longer assay to PCR.

At least three leading manufacturers have presented comparability validation data at major industry conferences showing that PCR/qPCR methods may be superior to the culture-based assay. According to Brewer, a significant percentage of samples testing positive by qPCR were negative in the cell culture assay arm of the studies.

Brewer attributes this phenomenon to “a variety of potential mechanisms.” The culture assay involves first inoculating a test sample into mycoplasma culture medium, followed by plating samples onto mycoplasma agar plates. The readouts are the agar colony counts.

“It appears that some mycoplasma species or strains grow well in liquid media but don’t show up as colonies on plates,” Brewer says. According to investigators who have measured mycoplasma growth kinetics in liquid culture using qPCR, results may vary because the growth stage of the cells in culture must be appropriate for the cells to grow on agar. Brewer, however, offers another explanation: “PCR may simply be more accurate.”

Unlike minute virus of mouse, another biomanufacturing safety target, several mycoplasma species are not only capable of causing serious human illnesses, they can also infect CHO expression systems. As a consequence of their simple genome, mycoplasma are unable to carry out extensive cellular processes such as synthesizing nucleotides and amino acids. Rather, they hijack the host cells’ biochemical machinery, thus altering host cell metabolism. In instances where a massive host cell death does not occur, negative effects on product quality may be significant.

This image shows a number of Gram-negative Mycoplasma hominis and T-strain Mycoplasma isolates that were grown on agar medium. Mycoplasmas that contaminate cell substrates and cell-derived biologics can be detected by traditional means, incubation with specially formulated mycoplasma media, or by means of PCR testing. [CDC/Dr. E. Arum and Dr. N. Jacobs.]

Testing as a Service

Several laboratories provide mycoplasma testing as a service to companies that choose not to conduct tests themselves or who seek a second opinion. Katalin Kiss, Ph.D., a manager at the ATCC Laboratory Testing Service, provides mycoplasma testing for both internal and external customers. “Internal” refers to release testing of ATCC’s own products. “External” refers to the organization’s website-driven process, which customers use to submit orders for testing samples that arrive at ATCC in frozen vials. When asked what industries her laboratory serves Dr. Kiss responded, “samples come from everywhere.”

For in-process mycoplasma detection, ATCC uses PCR-based assays because results are available within one day. “When you have cells taking up space in a quarantine lab, or awaiting integration into production, it is nice to get that data quickly,” Dr. Kiss explains.

End-of-process release testing involves direct cell culture for 28 days. By the time results come in, the product has already been manufactured and preserved.

Whether organizations employ manufacturing environment tests depends on their ISO certification, according to Dr. Kiss. But she notes that production personnel are rarely tested. “Any kind of work on products for human use might require environmental monitoring, or a contract might contain that stipulation.” Workplace environment testing includes sampling air and surfaces through swabbing. Analysis proceeds either through cell growth or PCR methods.

In addition to month-long culturing, ATCC uses its in-house developed PCR protocol that detects approximately 60 different mycoplasma species. “The advantage of PCR is turnaround time, but the method is limited by primer availability and specificity,” Dr. Kiss tells GEN. Direct culture detects more than 60 species but is, as noted, much slower.

Need for Vigilance

Biopharmaceutical product quality is intimately related to the health and quality of the expression system during cell culture. Incidents involving “difficult” culture pathogens such as minute virus of mouse and mycoplasma, although not new, reinforce the need for vigilance.

In a recent white paper, Sven M. Deutschmann, Ph.D., and Alexander Bartes of Penzberg, Germany-based Roche Diagnostics write that mycoplasma contamination presents significant and unique challenges.

“Species of the class Mollicutes can cause various problems in living organisms and in in vitro cell cultures,” they write. “Some species are pathogenic, introducing changes in cell metabolism and phenotype. Mycoplasma infections can be asymptomatic or of subclinical nature, and therefore they are insidious and sometimes hardly detectable.”

Because they lack a cell wall, mycoplasma resist the most common antibiotics, and they may pass through the conventional 0.2 µm filters used in cell culture, thus raising the potential for high contamination rates.

How prevalent is mycoplasma contamination in biomanufacturing? Judging from the number of reported incidents involving failed batches and top-to-bottom facility disinfection, not very. But there is good evidence that less-than-catastrophic infections occur with some regularity.

A group from the University of Pennsylvania last year published data on an unbiased assessment based on genetic sequences published from hundreds of labs. After analyzing sequence data from nearly 10,000 rodent and primate samples, investigators found that 11% of series were derived from contaminated cultures. Their designated cutoff was ≥100 reads/million that mapped to mycoplasma in one or more samples.

These researchers also examined the relationship between mycoplasma contamination and host gene expression, finding that 61 host genes contained fragments from mycoplasma.

As an economics exercise, the authors estimated the cost of NIH-funded research involving cultured cells. For the 2013–2014 period, the cost added up to $3.17 billion. Using the 11% contamination rate, the authors calculated that mycoplasma contamination could waste about $350 million each year. That estimate would probably have been higher if costs related to follow-on experiments and results skewed by unnoticed infections had been factored in.

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