Christopher C. Smith, Manager Mycoplasma Services, Eurofins BioPharma Product Testing
Jonathan M. Demick, PhD, Principal Scientist, Eurofins BioPharma Product Testing
Mycoplasmas are commensal parasites that can be contaminants of cell cultures and animal-derived products. These contaminants are not as visually obvious during culture of cells as are other microbiological contaminants. Mycoplasma contaminations are not as easily detected because they do not change the pH of the media, or increase the turbidity of the cell culture. They also do not tend to cause the cells to die off, as other types of microbiological contaminations can. However, mycoplasma contamination can influence cell cultures. For example, a mycoplasma contamination can alter protein synthesis, change cellular metabolism, or even alter cellular morphology. Mycoplasma can also be pathogenic to humans under certain conditions. M. pneumoniae infections of the respiratory tract, commonly known as walking pneumonia, can be persistent and hard to cure with traditional antibiotic courses. Due to these concerns, if a mycoplasma contamination is discovered in a biopharmaceutical product, an immediate and decisive response is required. The response can include discarding any affected batches or stopping production for cleaning and decontamination. In addition, all raw materials, such as cell cultures, serums, biological media components, or cell dissociation reagents that were used in process with the sample that is found to be contaminated, must be tested and recertified as clear of any contamination. By testing for mycoplasma in the biological reagents, the in-process materials, and the final product, the safety, quality, and efficacy of these products can be preserved.
Testing for mycoplasma within these products can be difficult to perform. Traditional testing for mycoplasma is performed as per the compendia via two methods, commonly referred to as the direct culture method and indicator cell culture method.1-3 Both methods are well established and reliable, but do come with limitations. Both methods require that the sample matrix does not interfere with the growth of viable mycoplasma during the process of that method. This can be a severe limitation in the case of certain animal sera, viral vaccines, or sample matrices that are outside of the physiological pH range that supports mycoplasma growth. The direct culture method is used to detect viable mycoplasma. This test requires 10.5 mL sample volume and requires 28 days for completion to generate a valid result. The indicator cell culture method must also be performed in order to detect fastidious mycoplasma that are non-cultivable using the direct culture method. This test requires 1 mL of sample and is also time consuming, requiring 6-7 days to complete. For some advanced therapy medicinal products, this timeline is greater than the lifetime of the product, and the 11.5 mL required for testing in both methods is more than what is produced for treatment of the patient.
The use of nucleic acid-based techniques (NAT) for the detection of mycoplasma is one method of minimizing the time required to test products. NAT assays are also able to detect the cultivable and non-cultivable mycoplasma within a single test. NAT assays work by amplifying a specific amplicon (DNA region of interest) to achieve a sufficient level of detection. Detection of the amplified target is measured via real-time increased fluorescence of DNA binding dyes. All NAT-based methods must be validated according to EP 2.6.7, EP 2.6.21, USP <1223> and USP <1071> to prove equivalency or superiority to the compendial methods. One such PCR method is a DNA based upon detection with SYBR® Green. Where the compendial testing timelines do not align with a customer’s timeline, this assay can be completed within 3-5 days. The primers utilized are optimized to provide sensitive, specific and comprehensive Mycoplasma species detection. Using a multi-parameter evaluation, the assay results will ensure confidence while avoiding false positives and false negatives.
Eurofins BioPharma Product Testing has validated the PCR assay to a limit of detection that meets all compendial requirements. The assay also showed specificity by not displaying cross-reactivity to gram-positive bacteria with a close phylogenetic relation. An initial interference test is performed upon the sample submission to ensure that the sample does not negatively affect the assay. Within this interference test, the sample is mixed with a discriminatory positive control (DPC) prior to DNA extraction. This DPC is a control included with the PCR kit at a concentration of 1000 copies/μL. The DPC is a synthetic amplicon that contains additional inserts to differentiate DPC signal from true positive signal, through the melting temperature (Tm) parameter. With this insert, the Tm is raised above the expected Tm range for mycoplasma DNA. After the DPC signal is seen in the PCR, the sample matrix can be tested for the presence/absence of mycoplasma with confidence that the PCR assay operates as expected.
While NAT assays are robust and specific, there are some limitations. Extraction of nucleic acids can be time-consuming and a source of error. Within the validated method at Eurofins, extraction and PCR encompass over 50 unique steps that can take between 8-10 hours to complete. While some of these steps are performed with a magnetic bead automated extraction system, there are still stages within the sample preparation when an error could occur. Additional evaluation of automation to minimize this error is continually underway.
With the current PCR assay, the template is DNA, which does not provide information on the viability of mycoplasma (if detected), only that the organism was present at some point in the material production process. Another limitation is the use of SYBR® Green dye. While binding to double-stranded DNA, SYBR® Green can also bind to other compounds that can carry over through the extraction process. This competitive binding of the dye can lead to an interference of the assay. This also has prevented the PCR assay from functioning as expected with samples that contain high DNA content (plasmids) and some cell lines (CHO K1).
When RNA is used as the template for PCR, it can be a way of determining if a positive signal is from viable mycoplasma, or residual from a past contamination, as RNA is prone to faster degradation than DNA. A new technology on the horizon is a cartridge-based PCR assay. This system is a closed system that extracts RNA from samples. Within a closed system, all chemistry required to isolate, amplify and detect a target amplicon is contained within a single unit. The RNA extraction is immediately followed by reverse transcription to provide the initial template for PCR. Subsequent nested PCR expands the initial template generated (stage 1), followed by a more precise amplification for the targeted amplicons (stage 2).4 A different dye is used to bind to the double-stranded DNA amplicon (LCGreen).4 This dye has been shown to experience less interference than SYBR Green.5 By extracting RNA within a closed system, this assay will reduce handling time, minimize handling errors (as there are less steps involved), and provide assurances that any positive detected can be from a viable organism. As with all fluorescence dyes, the possibility of competitive binding between the dye and a compound other than DNA is still a risk.
To mitigate competitive binding of dyes, utilization of probes within PCR reactions is also being evaluated. A probe is a covalently linked dye to a primer that anneals to the central region of the amplicon. One such assay is Droplet Digital PCR (ddPCR). By moving to the ddPCR platform, carry-over contaminants are diluted out so that sensitivity is increased.6 Incorporating a probe, rather than dyes alone, also lowers the potential for competitive binding occurring. The utilization of the probe coupled with the ddPCR method may provide a means to test samples that display inhibition through other methods. As this assay incorporates three stages (extraction, droplet generation with PCR, and droplet data generation), handling increases and time increases must be weighed against the benefits of lower probability of sample interference.
With mycoplasma testing, there is not a single method that works for every one of the large variety of biopharmaceutical samples that are being developed globally. The traditional compendial methods (direct culture and indicator cell culture) require too much time for some sample types. The newer PCR methods, while having a truncated timeline, have limitations based upon the sample matrix type and possible interferences within the sample matrix for that specific method. The development of the evolving rapid mycoplasma testing technologies will provide ever-decreasing turnaround times for our clients’ samples. By continually evaluating new technologies, it will be possible to provide results on sample matrices that encounter challenges with traditional compendial assays or the current available rapid assays. Through our comprehensive suite of assays, both traditional and rapid, Eurofins BioPharma Product Testing can help clients determine the best test methods to satisfy all of the mycoplasma testing needs for their unique products.
References
- United States Pharmacopeia <63>
- European Pharmacopeia 2.6.7
- Japanese Pharmacopeia General Chapter.
- Barry WE, Toxopeus C, Brown J, Kornowske L, Andjelic CD, Cassard S, Montero-Julian FA, Kim, M, Phillips, CL. Performance Evaluation of a Rapid, Fully Automated Mycoplasma Detection System. Poster Presented at 2019 PDA Cell & Gene Therapy Conference; May 6-7, 2019 Long Beach CA.
- Herrmann MG, Durtschi JD, Bromley LK, Wittwer CT, Voelkerding KV. Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping: Cross-Platform Comparison of Instruments and Dyes. Clinical Chemistry 2006 (52) 3:494-503.
- Taylor SC, Laperriere G, Germain H. Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Scientific Reports. 2017, 7 (Article number:2409).
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