In recent years, with the rapid development of biomedicine, the emergence of cell and gene therapies under the COVID-19 pandemic, and the advent of mRNA vaccines, ensuring the safety and reliability of biological products has become a focal point for governments and regulatory bodies worldwide. Mycoplasma contamination is a common but typically challenging type of contamination to eliminate. Regulatory requirements mandate the "ensuring of no mycoplasma contamination" for bioprocesses involving cell culture.
Regulatory Agencies' Requirements for Mycoplasma Testing:
- The FDA's "Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications" stipulates mycoplasma control for raw materials, viral seeds, and unprocessed harvest fluids.
- The 2020 edition of the Chinese Pharmacopoeia Part III "Preparation and Quality Control of Animal Cell Substrates Used for Biological Product Production Testing" requires mycoplasma testing for production cells, including Master Cell Banks (MCB), Working Cell Banks (WCB), and End-of-Production Cells (EOPC).
- The "Technical Guidelines for Pharmaceutical Research and Evaluation of Immune Cell Therapy Products (Trial)" recommend conducting mycoplasma safety-related testing on suitable intermediate samples at key time points or implementing relevant measures for control. Mycoplasma testing is also required as a release test for final products.
Nucleic Acid Testing (NAT) and Traditional Methods:
Before the advent of rapid detection methods such as Nucleic Acid Amplification Technology (NAT), traditional culture methods and indicator cell assays were employed. However, due to lengthy detection cycles or sensitivity issues, these methods often prolonged production or release cycles or necessitated conditional release of cell materials based on risk assessment or the exploration of alternative methods. With the advancement of cell and gene therapies, there's an increasing demand within the industry for mycoplasma detection methods with higher timeliness and sensitivity. The short shelf life of products cannot sustain lengthy testing periods, thus NAT methods have emerged as favorable alternatives.
Currently, the European Pharmacopoeia (EP) <2.6.7>, Japanese Pharmacopoeia (JP), and United States Pharmacopeia (USP) <63> have all included NAT methods as mycoplasma detection methods. However, validation of this method and comparison with traditional methods to ensure its sensitivity is not inferior are prerequisites for its use. Although the 2020 edition of the Chinese Pharmacopoeia (ChP) has not included NAT as a mycoplasma detection method, it mentions the possibility of using "other methods recognized by the national drug regulatory authority." In May 2022, the Drug Review Center of the National Medical Products Administration (NMPA) released the "Technical Guidelines for Pharmaceutical Research and Evaluation of Immune Cell Therapy Products (Trial)," which suggests considering the development of novel sterile and mycoplasma detection methods for release testing under special circumstances when sample volume is limited or rapid release is needed and pharmacopeial methods are inadequate. Therefore, NAT methods are expected to be increasingly utilized by more raw material suppliers and Advanced Therapy Medicinal Product (ATMP) companies as a method capable of supporting rapid mycoplasma release testing.
NAT Method - Method Validation Requirements
The validation process for NAT methods is elaborately described in the European Pharmacopoeia <2.6.7> and Japanese Pharmacopoeia, with their content being essentially consistent. The National Institutes for Food and Drug Control (NIFDC) has also published an article titled "Considerations on Nucleic Acid Detection Methods and Methodological Validation for Mycoplasma Examination," aiming to provide guidance for developers and users of mycoplasma nucleic acid amplification methods in China. Specificity, Detection Limit, and Robustness are essential for validating mycoplasma NAT methods. If commercial kits are utilized for mycoplasma detection, method suitability validation can be conducted based on the supplier's comprehensive kit performance report, combined with the laboratory environment and sample type.
Specificity:
Specificity refers to the ability of an analytical method to accurately determine the analyte in the presence of other components (such as impurities, degradation products, excipients, etc.). The EP emphasizes the need to focus on cross-reactions between other bacterial species closely related to the phylogenetic tree, such as Clostridium, Lactobacillus, and Streptococcus within the Gram-positive bacteria. Attention should also be given to common contaminant types like human DNA contamination in host cells and experimental environments.
Detection Limit:
The detection limit is the lowest amount of analyte that can be detected in a sample. It serves as a qualitative identification basis and does not need to be quantified as an exact value. The EP requires 24 detection data points for each mycoplasma species at each dilution concentration. This can be achieved by performing three independent 10-fold serial dilutions on different days, with eight repeated detections for each dilution gradient, or by conducting four independent 10-fold serial dilutions on different days, with six repeated detections for each dilution gradient. A detection positivity rate of over 95% can be considered as the detection limit. For mycoplasma types that require confirmation of the detection limit, kit manufacturers need to cover as many mycoplasma types as possible as required by regulatory pharmacopoeias and explore the detection limits for each mycoplasma type. For biopharmaceutical companies, it is advisable to consider human mycoplasma types as well.
- If avian cells or materials are used or encountered during the production process, the detection limit verification for Mycoplasma synoviae should be conducted.
- If insect or plant materials are used or encountered during the production process, the detection limit verification for Acholeplasma should be conducted.
- Porcine nasal mycoplasma is highly prevalent in mycoplasma-contaminated samples and has attracted attention from the China Institute for Food and Drug Control.
In comparison to the European Pharmacopoeia, the Japanese Pharmacopoeia does not mention avian mycoplasma for which detection limit verification is required but includes salivary mycoplasma.
Robustness:
Robustness refers to the ability of a method to withstand small variations in measurement conditions without being affected, providing a basis for the established method's use in routine testing. Evaluation of robustness should focus on the tolerance of mycoplasma detection methods to variations in the concentration of MgCl2, primers, and dNTPs in detection reagents, changes in nucleic acid extraction kits or extraction steps, and the use of different nucleic acid amplifiers.
Comparability Study
If NAT is to be used as a substitute for pharmacopeial methods, it is necessary to confirm through comparison that NAT can replace culture-based methods or indicator cell culture methods. This typically involves considering the detection limit of the method, as well as specificity (such as the range of mycoplasma coverage and possible false positives). For comparing detection limits, it should meet the criteria that "a detection limit of 10 CFU/mL can replace culture-based methods, and a detection limit of 100 CFU/mL can replace indicator cell culture methods."
There are two optional approaches for conducting comparability studies:
- Conducting synchronous experiments using the same validated mycoplasma strains for both NAT and pharmacopeial methods to confirm the detection limit.
- Comparing the results of NAT validation with previous pharmacopeial method validation results, but careful documentation of the confirmation files for the mycoplasma standards used in both methods is required.
NAT Mycoplasma qPCR Detection - Comprehensive Solution
Related Products
Yeasen MycAway™ Mycoplasma qPCR Test Kit (probe method) is a rapid qualitative detection product based on NAT (nucleic acid amplification techniques) for potential mycoplasma contamination in raw materials, cell banks, viral seeds, virus or cell harvest fluids, and therapeutic cells. This kit, based on quantitative PCR technology, uses Taqman fluorescence probes to qualitatively detect mycoplasma DNA in the test sample, covering over 160 mycoplasma DNA sequences. It strictly follows the guidelines and requirements of EP 2.6.7 and JP G3 for mycoplasma detection, demonstrating high sensitivity, good specificity, and safety. It can be used in conjunction with the MolPure® Magnetic Bead Residual DNA Sample Pretreatment Kit to manually extract samples or automatically extract nucleic acids using the Auto-Pure 32A Fully Automatic Nucleic Acid Extractor. After sample pretreatment to remove interfering impurities and obtain purified mycoplasma DNA, the fluorescence signal of the probe is collected through qPCR to determine the detection result.
Technical Services
- Providing comprehensive validation reports for the MycAway™ Mycoplasma qPCR Test Kit.
- Providing technical services for client sample applicability validation.
- Providing audit support.
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Description |
Part Number |
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18461ES |
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| MycAway™ Mycoplasma qPCR Detection Kit (2G) | 40619ES |