Genetic Engineering & Biotechnology News

MAY1 2015

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28 | MAY 1, 2015 | | Genetic Engineering & Biotechnology News Bioprocessors have a huge stake in detecting mycoplasma wherever the organism exists in their facilities, but especially in fnished prod- ucts. At present, bioprocessors detect myco- plasma by utilizing a month-long culture- based methodology. Although this methodol- ogy has been considered the gold standard, rapid polymerase chain reaction (PCR) meth- ods augment—and in some cases exceed—the capabilities of culturing. The MycoSEQ ® mycoplasma detection system from Thermo Fisher Scientifc 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 pro- vide results in hours. "PCR assays make sense for in-process testing," notes Michael Brewer, head of phar- maceutical analytics at Thermo. "It provides rapid answers, for example, before passing cultures to the next bioreactor during cell ex- pansion. 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 bio- manufacturers 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 signifcant percentage of samples testing posi- tive 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 frst inoculating a test sample into mycoplasma culture medium, followed by plating samples onto mycoplas- ma 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 mea- sured 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 myco- plasma 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 un- able to carry out extensive cellular processes such as synthesizing nucleotides and amino acids. Rather, they hijack the host cells' bio- chemical machinery, thus altering host cell metabolism. In instances where a massive host cell death does not occur, negative effects on product quality may be signifcant. 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 man- ager at the ATCC Laboratory Testing Service, provides mycoplasma testing for both inter- nal and external customers. "Internal" refers to release testing of ATCC's own products. "External" refers to the organization's web- site-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 re- sponded, "samples come from everywhere." For in-process mycoplasma detection, ATCC uses PCR-based assays because re- sults are available within one day. "When you have cells taking up space in a quaran- tine lab, or awaiting integration into produc- tion, 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 manufac- turing environment tests depends on their ISO certifcation, according to Dr. Kiss. But she notes that production personnel are rare- ly 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 dif- ferent mycoplasma species. "The advantage of PCR is turnaround time, but the method is limited by primer availability and specifc- ity," Dr. Kiss tells GEN. Direct culture de- tects 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 "diffcult" culture patho- gens such as minute virus of mouse and my- coplasma, 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 pres- ents signifcant and unique challenges. "Species of the class Mollicutes can cause various problems in living organisms and in in vitro cell cultures," they write. "Some spe- cies are pathogenic, introducing changes in cell metabolism and phenotype. Mycoplasma infections can be asymptomatic or of subclin- ical 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 flters used in cell culture, thus raising the po- tential for high contamination rates. How prevalent is mycoplasma contami- nation in biomanufacturing? Judging from the number of reported incidents involving failed batches and top-to-bottom facility disinfection, not very. But there is good evi- dence that less-than-catastrophic infections occur with some regularity. A group from the University of Pennsyl- vania last year published data on an unbi- ased assessment based on genetic sequences published from hundreds of labs. After ana- lyzing sequence data from nearly 10,000 rodent and primate samples, investigators found that 11% of series were derived from contaminated cultures. Their designated cut- off was ≥100 reads/million that mapped to mycoplasma in one or more samples. These researchers also examined the re- lationship between mycoplasma contamina- tion and host gene expression, fnding that 61 host genes contained fragments from my- coplasma. 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 au- thors calculated that mycoplasma contami- nation could waste about $350 million each year. That estimate would probably have been higher if costs related to follow-on ex- periments and results skewed by unnoticed infections had been factored in. Even When Less Than Catastrophic, Mycoplasma Infestations Are More Than Inconvenient Bring Mycoplasma Under Control BIOPROCESSING Perspectives 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.

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