title: Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques.
abstract: Mycoplasmas, particularly species of the genera Mycoplasma and Acholeplasma, are known to be occasional microbial contaminants of cell cultures that produce biologics. This presents a serious concern regarding the risk of mycoplasma contamination for research laboratories and commercial facilities developing and manufacturing cell-derived biological and biopharmaceutical products for therapeutic use. Potential undetected contamination of these products or process intermediates with mycoplasmas represents a potential safety risk for patients and a business risk for producers of biopharmaceuticals. To minimize these risks, monitoring for adventitious agents, such as viruses and mycoplasmas, is performed during the manufacture of biologics produced in cell culture substrates. The "gold standard" microbiological assay, currently recommended by the USP, EP, JP and the US FDA, for the mycoplasma testing of biologics, involves the culture of viable mycoplasmas in broth, agar plates and indicator cells. Although the procedure enables highly efficient mycoplasma detection in cell substrates and cell-derived products, the overall testing strategy is time consuming (a minimum of 28 days) and requires skilled interpretation of the results. The long time period required for these conventional assays does not permit their use for products with short shelf-lives or for timely 'go/no-go' decisions during routine in-process testing. PCR methodology has existed for decades, however PCR based and other alternative methods for mycoplasma detection have only recently been considered for application to biologics manufacture. The application of alternative nucleic acid-based, enzyme-based and/or recombinant cell-culture methods, particularly in combination with efficient sample preparation procedures, could provide advantages over conventional microbiological methods in terms of analytical throughput, simplicity, and turnaround time. However, a challenge to the application of alternative methods for detection of mycoplasmas remains whether these alternative methods can provide a limit of detection comparable or superior to those of the culture methods. An additional challenge is that nucleic acid amplification technique (NAT) methods do not allow for accurate discrimination between viable and non-viable mycoplasma contaminants, which might lead to false-positive results (e.g. from inactivated raw materials, etc.). Our review provides an overview of these alternative methods and discusses the pros and cons of their application for the testing of mycoplasmas in biologics and cell substrates.
authors.affiliation: U.S. Food and Drug Administration, Center for Biologics Evaluation and Research, Office of Vaccine Research and Review, Division of Viral Products, Laboratory of Methods Development, HFM-470, 1401 Rockville Pike, Rockville, MD 20852, USA. dmitriy.volokhov@fda.hhs.gov
authors.forename: Dmitriy V
authors.initials: DV
authors.lastname: Volokhov
authors.forename: Laurie J
authors.initials: LJ
authors.lastname: Graham
authors.forename: Kurt A
authors.initials: KA
authors.lastname: Brorson
authors.forename: Vladimir E
authors.initials: VE
authors.lastname: Chizhikov
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D009174:Mycoplasma / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D009175:Mycoplasma Infections / Q000175:diagnosis* / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012329:RNA, Bacterial / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial; D012329:RNA, Bacterial