Mycoplasma are the smallest known self-replicating prokaryotes. They parasitize a wide range of host organisms, including humans, animals, plants and insects and are common contaminants of mammalian cell culture. Due to their small size, mycoplasmas can readily pass through 0.2 μm filters used to maintain sterility. Sources for the introduction of mycoplasmas into cell culture systems include: cell culture media and additives, the use of previously infected cells and laboratory personnel.
Although visual signs indicative of mycoplasma contamination are often lacking, mycoplasma contamination of cell lines used to produce biopharmaceutical products can disrupt cellular growth and metabolism and lead to changes in gene expression. These adverse cytopathological events can result in decreased product quantity and quality.
More importantly, mycoplasma infections have been associated with respiratory illness, urethritis and arthritis, and they act as a co-factor in numerous infectious diseases. It is for these reasons that world-wide regulatory agencies require that biotechnological products produced in cell substrates be tested to ensure the absence of mycoplasma contamination.
Compendial mycoplasma testing procedures for the detection of viable mycoplasma contaminants are culture based and have three components: direct cultivation on agar plates, enrichment in broth followed by detection on agar plates and detection in indicator cell culture.
The current procedures are time consuming as they are 28 days in length. This time requirement is not amenable for obtaining the rapid lot release testing results needed for biopharmaceutical products that have short half-lives or for which there is high market demand.
The lengthy assay period is also not conducive to the rapid screening of raw materials intended for use in future production, nor to the rapid in-process screening of intermediates for the purpose of detecting and containing contamination events. Eurofins Lancaster Laboratories now offers a rapid mycoplasma test comparable in sensitivity and specificity to the 28-day compendial method.
The EMD Millipore MilliPROBE® Real Time Detection System for Mycoplasma couples membrane-based sample filtration and magnetic bead-based nucleic acid purification, resulting in straight-forward and representative sample preparation. Nucleic acid is purified from the sample in three steps, using a dual-membrane filtration device in a swinging bucket centrifuge. During the first step of sample processing, mammalian cells are captured on a prefilter and mycoplasma are retained on a 0.1 μm EMD Millpore Express® SHR membrane. In the second step, lysis buffer is added through a lateral channel only to the mycoplasma retentive membrane, and the device is heated to lyse the mycoplasma and release the nucleic acid. In the third and final step of the process, the device is centrifuged to collect the lysed nucleic acid in a removable microcentrifuge tube. The closed design feature of the sample preparation device protects the sample from cross-contamination and the mycoplasma nucleic acids in the lysis buffer are further purified and concentrated using automated, magnetic based purification instrumentation. The sample preparation is coupled with EMD Millipore collaborator Gen-Probe’s Target Capture and Real-Time TMA technology using Gen-Probe’s newly developed technology called Background Reduction Technology (BRT). BRT protects the assay from false positive results by ensuring that contaminating nucleic acid or mycoplasma cells that enter the assay after the closed, sample-preparation step cannot be amplified or detected. The combined technologies of a closed, high-volume sample preparation and the BRT lead to a very robust assay with very low false-positive rates. Each assay run includes an external positive and negative control to monitor amplification efficiency and specificity. In addition, an internal control, present in lysis buffer, is introduced to each sample prior to nucleic acid purification and amplification. The use of internal control serves to monitor overall efficiency of nucleic acid purification and amplification and prevents the report of false negative results.
An important advantage of the MilliPROBE System is that it has the capability of processing a volume of material comparable to that tested in the compendial method, making it preferable to rapid mycoplasma detection methods that have volume limitations. More specifically, the MilliPROBE System can process 10-20 mLs of a sample, whereas PCR and RT-PCR methods typically are limited to testing sample volumes that are 1-2 mL. Finally, the membrane-based sample preparation device effectively removes inhibitory substances that can interfere with nucleic acid amplification technologies. After several months of working with the EMD Millipore MilliPROBE® Real Time Detection System for Mycoplasma, Eurofins Lancaster Laboratories’ scientists have unequivocally demonstrated the value of the method for the rapid testing of cell banks, raw materials and manufacturing samples, including unprocessed bulk harvest where matrix interference effects often prevent the use of other rapid mycoplasma methods.
In addition, the system has two features that allow it to preferentially detect viable mycoplasmas. The first feature is the 0.1 μm EMD Millpore Express® SHR membrane retention filter, which retains mycoplasma cells but allows free nucleic acids to be rinsed away into waste. Secondly, the EMD Millipore MilliPROBE Mycoplasma Detection System targets and amplifies mycoplasma rRNA from mycoplasma cells captured and lysed within the sample prep device. RNA is not only a better viability marker than DNA, but it increases the sensitivity of the method as a single mycoplasma contains 1000 or more rRNAs compared to only 1-2 DNAs. Furthermore, the use of rRNA as the target directly relates the limit of detection to culture methods as it is not dependent on the genomic copy to colony forming unit ratio (GC:CFU) of mycoplasma stocks to establish the limit of detection.
EMD Millipore has validated the specificity and sensitivity of the system using the 13 mycoplasma species listed in Table 1, which include the eight species specified in E.P. 2.6.7. In addition, EMD Millipore has performed feasibility testing on approximately 30 additional mycoplasma species and strains outside of the formal validation study. Eurofins Lancaster Laboratories has performed a user verification of the limit of detection using the six mycoplasma species listed in Table 2. Each of the six species was successfully detected in six of six independent assay runs. As all six species generated positive results with inoculum levels <10CFU/mL, the LOD has been determined to be 10 CFU/mL in keeping with the required LOD for compendial mycoplasma detection methods.
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