Problems and Consequences
Of all the threats that keep quality managers awake at night, mycoplasmas rank high. In a career performing quality control or quality assurance for biopharma manufacturing anywhere in the world, a QC or QA specialist will probably encounter mycoplasmal contamination of a production facility at least once. The resulting devastation to the site’s output and schedules, as well as to its staff’s peace of mind, make it urgent to avoid any repetition of the experience.
Mycoplasmas are defined as “a genus of nonmotile, parasitic, pathogenic microorganisms, whose member lack a true cell wall, are Gram-negative, and require steroids such as cholesterol for growth.”1 They are considered the “smallest free-living organisms known to infect humans.”2
These microorganisms are smaller than bacteria, but larger than viruses. Unlike viruses, they don’t need cells to survive. Because of their small size and lack of cell walls, mycoplasmas occasionally penetrate the filters used to “sterilize” cell culture media and sera. Thus low levels of these organisms are accidentally introduced into cultures during routine nutritive activity.
These same characteristics make mycoplasmas difficult and time-consuming to detect, unless they are screened on a regular basis using sophisticated methods. Researchers note that mycoplasmas “can be as small as 0.2-0.3 µm, and can achieve very high densities in cell culture (107-108 organisms/ml) without discernable pH changes or turbidity.”
There’s often no sign of cytopathic effect (CPE). Titer may be reduced, but the organisms simply adhere to and feed off cells without leaving evidence in the form of large numbers of dead cells. Cellular morphology may be affected, with the presence of abnormally large cells.
Some production facilities grow cell cultures in media supplemented with antibiotics, to reduce microbial load to acceptable levels. But antibiotics don’t typically affect mycoplasmas. Further, this methodology may mask poor aseptic technique, and lead to accidental introduction of mycoplasmas along with other microorganisms.
As a result of all these difficulties, mycoplasmas are distressingly common contaminates in cell cultures. Contamina-tion by mycoplasmas can:
- Affect cell growth and morphology
- Inhibit cellular metabolism
- Disrupt DNA/RNA synthesis
- Produce aberrations in normal chromosomal contents of cells
- Change the antigenicity of cell membranes, reducing the effectiveness of immune response
- Mimic viral infections
- Alter virus yields
- Destroy an entire cell line
However, to date, release into the population has been rare. Almost all instances of contamination are eventually detected during the medication manufacturing process. The greatest risks mycoplasmas pose are to the efficiency, speed, and cost-effectiveness of that process itself.
In a production environment, cells are cultured in relatively large-capacity vessels. As they successfully grow, batches of cultures are moved to other large vessels to multiply in their turn. If long-duration testing eventually demonstrates that there was mycoplasma contamination in an original stock, everything since produced from that stock has in the meantime been infected as well.
Also, once present in a laboratory or production facility, mycoplasmally infected cultures often cross-contaminate other cell lines being used.
The consequences are grave.
Quality managers face serious setbacks, starting with the cost of expensive raw materials that go into making each batch. They also lose all the time invested to date. The original vessel must be meticulously decontaminated. And since the tainted stock has been transferred to other vessels, these in turn must be decontaminated as well. In a typical mycoplasmal contamination event, eventual losses can easily mount into the tens of thousands or hundreds of thousands of dollars. Additionally, testing, investigation and decontamination may consume months or even a year of invaluable production time.
This is bad news for manufacturers of vaccines, cancer and diabetes therapies, and other products whose creation depends on growing high volumes of living cells in culture media. They can face ruinous outcomes in yield, throughput, and turnaround times from start of manufacture to product release.
Finally, it is also bad news for all the patients depending on the timely provision of safe, effective biomedications.
A variety of testing methodologies have been used with some success in detecting mycoplasmal contamination. These include the following:
- Direct culture (for cultivable species)
- Indirect culture (DNA fluorochrome staining — for noncultivable species)
- DNA probe
- Enzyme-linked immunosorbent assay (ELISA)
- Real-time PCR
However, as noted, mycoplasmas are not easily cultivated. In past years, the paucity of simple, easy-to-use, reliable detection methods has resulted in a lack of definitive test results until late in the production process. Many cell lines brought into a lab from outside sources — although appearing normal and healthy — are in fact already infected with mycoplasma.
The problem is time.
The “gold standard” direct cultivation method approved by regulators takes 28 days to produce results. Even the indirect culture method takes 3 to 5 days.
Quality managers at most biopharma companies simply can’t wait for these lengthy testing regimens. At significant points in their time-critical production schedules, they need a fast, reliable check that tells them whether it’s safe to move forward with a given cell line or batch.
The Fast-Track Alternative
From the alternative testing methodologies in the list above, many manufacturers are turning to real-time polymerase chain reaction (PCR) testing. Real-time PCR is defined as “[a] technique designed to detect and quantify sequence-specific PCR products as they accumulate in ‘real-time’ during the PCR amplification process.”5
This methodology evaluates DNA in test samples in one continuous, relatively speedy process. DNA is first amplified via PCR, then quantified via various methods. These include interpolating fluorescent dyes into double-strand DNA, as well as the use of modified DNA oligonucleotides (probes) that fluoresce when hybridized with complementary DNA.
Correctly performed, real-time PCR offers the following advantages:
- Provides fast, precise results
- Accurately quantifies DNA and/or RNA
- Furnishes increased sensitivity and specificity
- Detects all known mycoplasma species
- Works whether mycoplasmas in sample are living or dead
The target sequence detected may be living or dead; this can only be only determined when put into culture (directly or indirectly).
State-of-the-art real-time PCR screening routinely offers detection sensitivity down to only a few copies of a target mycoplasma species. This high specificity provides assurance to quality managers that a given test has reliably ruled out mycoplasmal contamination in a given sample, so that further high-volume production can proceed.
This gains critical time. Whereas the traditional direct cultivation method takes 28 days to provide results, modern real-time PCR analysis in an experienced testing laboratory may be accomplished in an actual testing duration of only 24 hours, plus the time needed for sample collection, shipment, preparation, and handling.
The FDA has not yet ruled on real-time PCR. But since the agency mandates its preferred forms of mycoplasma screening — direct or indirect cultivation — only as a precondition for final release, biopharma firms are free to employ real-time PCR at any earlier stage of their manufacturing process.
Additionally, the real-time PCR method has been fully accepted for mycoplasmal screening — including product release testing — at any stage by the regulating authorities of the European Pharmacopoeia (EP). Manufacturers selling only in Europe are thus able to apply real-time PCR testing at any and all stages of their process. The FDA may well be considering moving to approval of real-time PCR for release screening at some future time.
The technology of PCR analyzers and the kits that run on them has improved over the years. Some approaches sensitive enough to routinely detect as few as 10 copies of a target DNA sequence, and in some cases, they can detect a single copy. Contract laboratories are pushing the envelope in this area, with some investigating the development of a real-time PCR capability in virology as well.
These services are especially useful for manufacturers starting a new production process. Initial testing can assure managers that the vessels they use for production, their initial stock of cells, and all the consumables and raw materials they will use are mycoplasma-free.
Mycoplasma testing is also invaluable at the point where manufacturers have just produced their master stock. They want to move into creating production stocks, but don’t wish to propagate any hidden contamination problems. Fast, accurate, realtime PCR screening can produce answers within 24 to 48 hours, not 28 days.
If contamination is detected, the manufacturer can take swift corrective action before committing further time, materials, money, and effort — saving precious resources that would otherwise be completely wasted.
If no contamination is found, minimal time has been lost. Armed with a clean result, the manufacturer can confidently move ahead to the next step in the high-volume production of safe, effective biopharmaceuticals.
- Definition at http://medical-dictionary.thefreedictionary.com/mycoplasma
- Glossary of HIV/AIDS Terms at www.amfar.org/cgi-bin/iowa/abouthiv/index.html
- “Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals.” Attachment #2. FDA. CBER (1993).
- 21 CFR 610.30.
- Perspectives Glossary at www.nature.com/nrg/journal/v6/n2/glossary/nrg1525_glossary.html
Dennis Champagne is director, Laboratory Services at Microtest Laboratories, Inc. He leads Microtest’s microbiology, contract analytical chemistry, laboratory support, and virology departments. He can be reached at firstname.lastname@example.org