Why is mycoplasma testing necessary for GMP manufacturing?
Mycoplasmas, the smallest known free living organisms, are relatively common and difficult to detect bacterial contaminants of mammalian cell cultures. In therapeutic applications, an undetected mycoplasma contamination can negatively impact product quality and patient safety. GMP manufacturers develop stringent risk mitigation plans to ensure early detection of mycoplasmas to avoid costly manufacturing delay or remediation activities. For lot-release testing, regulatory guidance requires that all products derived from cell culture be tested for the presence of mycoplasma.
Proven, regulatory-accepted method for mycoplasma detection
The Applied Biosystems MycoSEQ Mycoplasma Detection Assay has been accepted world-wide to meet existing regulation and guidance for the detection of mycoplasmas across multiple therapeutic modalities.
Regulatory acceptance track-record for the MycoSEQ Detection System used for lot-release testing as of 2021
Therapeutic drug category
Number of commercially released drugs
Number of drugs pending approval
Regulatory agencies granting approval
*EMA - European Medicines Agency; FDA - United States Food and Drug Administration; PMDA - Pharmaceuticals and Medical Devices Agency, Japan; MFDS - Ministry of Food and Drug Safety, Korea)
Requirements for regulatory acceptance
For mycoplasma detection assays, regulatory agencies require validation studies to confirm the sensitivity, specificity, and robustness of the method to meet their standards. The MycoSEQ detection assay has achieved such success through demonstrating these parameters:
Proven specificity of the MycoSEQ Detection Assay:
The MycoSEQ kit has been validated in numerous independent studies to specifically detect a wide variety of Mycoplasma, Acholeplasma, and Spiroplasma species with no known cross-reactivity with closely related bacterial species. Specificity testing results and bioinformatics analysis are detailed in the Drug Master File (DMF) which is filed with the United States Food and Drug Administration (FDA) and Health Canada.
High sensitivity of the MycoSEQ Detection Assay:
Internal and external validation methods have demonstrated limit of detection (LOD) to meet or exceed the recommended regulatory guidance of 10 CFU/mL, or the genomic equivalent of 10 genome copies (GC)/mL for a vast panel of mycoplasma species including those known to contaminant bioprocessing cultures.
Robustness of the MycoSEQ Detection Assay:
Deliberate variations in the method parameters have been extensively evaluated by Thermo Fisher Scientific as part of the assay development process and validated by independent studies.
Press release: Rapid Mycoplasma Testing Method Now Accepted by Regulators
Learn the history of how The Applied Biosystems MycoSEQ Mycoplasma Detection Kit was accepted by regulators for QA/QC and lot release.
E-book: Rapid Mycoplasma Testing Method for Lot-Release
Learn how the MycoSEQ Drug Master File with the US FDA and Health Canada can assist in regulatory acceptance of the method.
Webinar: Mycoplasma detection in cell therapy products: GMP-compliant implementation and validation
Explore a case study of the implementation and validation of the MycoSEQ mycoplasma detection system for routine quality control and lot release testing for a mesenchymal cell therapy ATMP.
MycoSEQ Mycoplasma Detection System
The MycoSEQ Mycoplasma Detection System is a real-time PCR-based mycoplasma detection assay, providing sensitivity that meets or exceeds the regulatory guidance for mycoplasma detection and same-day actionable results to enable early detection of mycoplasma contamination.
- Method accepted by regulatory agencies for Mycoplasma detection in a cGMP environment
- Proven sensitivity to less than 10 copies/reaction with no known cross-reactivity to closely related non-mycoplasma species
- Total workflow solution, from sample prep to analysis with SAE compatible software
- Detection of more than >90 Mycoplasma species
- Supported throughout the validation process with our regulatory specialists and our Field Application Specialists
Streamlined workflow with results in less than five hours
The MycoSEQ Mycoplasma Detection System’s easy workflow delivers actionable results in less than five hours—enabling you to detect contamination quickly in process development, equipment, raw materials, harvest, helping to expedite the release of your product.
Automated sample preparation
Superior sample prep for consistent detection across multiple matrices
Efficient sample preparation and DNA extraction are the base for a high-sensitivity assay. Our Applied Biosystems PrepSEQ nucleic acid extraction kits utilize magnetic bead-based technology to provide quantitative recovery of nucleic acids from a variety of complex sample matrices. Our MycoSEQ system is optimized with sample preparation protocols for use with either automated or manual workflows.
Reduce hands-on time with automation
The Applied Biosystems AutoMate Express Nucleic Acid Extraction System delivers the power of efficient extraction in a fully automated system. Implementing the same PrepSEQ chemistry in preloaded cartridges, the AutoMate Express provides complete extraction of up to 13 samples in under two hours with only five minutes of hands-on time.
The Applied Biosystems Pharmaceutical Analytics QuantStudio 5 Real-Time PCR System is designed for both new and experienced users who need a simple and affordable real-time PCR system without compromising performance and quality. The QuantStudio 5 System detects differences in target quantity as small as 1.5-fold in singleplex reactions and provides 10 logs of linear dynamic range.
The Applied Biosystems 7500 Real-Time PCR System is a powerful platform for labs requiring superior performance and maximum dye versatility. It is a sophisticated platform for users who require extended capabilities and maximum versatility. The third generation platform features an innovative optical system that enhances sensitivity.
Software to help enable 21 CFR Part 11 compliance
The Applied Biosystem AccuSEQ Real-Time Detection Software is designed to work with the QuantStudio 5 Real-Time PCR instrument or the 7500 Fast Real-Time PCR instrument. Dedicated templates and data analysis algorithms allow for easy plate set up and automated mycoplasma presence-absence calling. Security, audit, and e-signature features enable 21 CFR Part 11 compliance.
- Optimized and validated for the MycoSEQ Mycoplasma Detection assay
- Easy visual result summary provides immediate understanding of presence or absence of mycoplasma in your samples
- Confidence in positive results with automated detection of discriminatory positive control (DPC)
- Robust data analysis parameters for the MycoSEQ Mycoplasma Detection assay (Ct, Tm, and DV values)
- Security, audit, and signature capabilities to enable 21 CFR Part 11 compliance
The AccuSEQ software features an intuitive interface that provides an easy way to identify presence or absence of mycoplasma. The software demonstrates one of the key parameters.
Figure 1. The AccuSEQ software demonstrates one of the key parameters (Ct value) to show the amplification which indicates the presence of DNA.
Figure 2. The specificity of the qPCR reaction can be confirmed using melting curve analysis. The melting curve shows what you amplified based on Tm, while the derivative shows how much of the product/target we have.
Designed for purpose using a multi-parameter analysis
Three distinct assay acceptance criteria provide confidence in the determination of the presence or absence of mycoplasma. The cycle threshold (Ct) value indicates that amplification has occurred, while melting temperature (Tm) and derivative value (DV) confirm that amplification was due to mycoplasma DNA. All three criteria must meet specific values established during validation for a test sample to be positive for the presence of mycoplasma DNA.
Assay sensitivity is critical to meet regulatory expectations and provide results for early detection of mycoplasma contamination. The MycoSEQ assay consistently detects between 1 to 10 genome copies of mycoplasma target DNA. This highly sensitive method meets the rigorous expectations of regulatory agencies.
A measure of target DNA level at the beginning of the PCR reaction
A measure of amplicon size and base composition (known for mycoplasma using this assay)
A measure of specific amplicon quantity generated during the PCR reaction
Figure 3. Amplification plot of a 10-fold dilution series of purified M. arginini DNA performed in triplicate. The MycoSEQ assay provides highly reproducible detection of mycoplasma DNA down to 1 genome copy per PCR reaction.
Positive means positive
Our proprietary Discriminatory Positive/Extraction Control gives you the confidence in your results.
- Our positive control DNA is made with mycoplasma amplicon modified to have a melting temperature (Tm) outside the range of mycoplasma amplicons, providing an additional level of confirmation of positive test results.
- Higher Tm allows discrimination between true mycoplasma and accidental contamination of test sample with positive control.
- This helps enable simple extraction control spiking of test samples.
- This can be used as a surrogate for mycoplasma DNA during method qualification.
Detection of more than 90 bacterial species and contaminants
Equally important is specificity to demonstrate that only the target species are detected. The MycoSEQ assay provides highly sensitive detection of more than 90 Mycoplasma, Sprioplasma, and Acholeplasma species with no detection of non-related bacteria. The assay detects all species required by European Pharmacoepia <2.6.7>.
Broad detection of Mycoplasmas
View the Partial Inclusion Panel based on 10 ng purified DNA/reaction
Partial inclusion panel
*Common isolated species recommended for use in qualification and validation are listed in bold.
No cross reactivity with non-mycoplasma DNA
Multiple non-mycoplasma species have been tested and confirmed to not be detected by the assay, including but not limited to Bacillus spp., Enterobacter spp., E. coli, Pseudomonas spp., Staphylococcus spp., and Streptococcus spp. Additionally, the assay has no cross-reactivity with chicken, chimpanzee, CHO, HeLa, mouse, rat, and many others.
|Partial exclusion panel based on 10 ng purified DNA/reaction|
Implementation and technical support
Our mission is to support your MycoSEQ Detection System implementation—from the decision stage through validation and routine use. Our experienced Field Application Scientists are dedicated to facilitating seamless implementation into your process and will train the lab on the appropriate protocols. We know it’s not easy. We’ve helped many companies and will collaborate with your team to develop a comprehensive implementation plan, which could include instrument installation with IQ/OQ and Computer System Validation, team training, method/protocol optimization, and regulatory support.
Regulatory consulting support
Our team of regulatory consultants provide support to address your bioprocessing and regulatory compliance challenges. We can:
- Advise customers on the features of our products that enable alignment with regulatory guidance
- Support evaluation, implementation, and validation of bioproduction solutions
- Provide guidance on regulatory strategy during implementation and qualification of bioproduction solutions
- Provide consultation to support deviation or nonconformance investigations, including guidance on development of CAPA
For decades, Thermo Fisher Scientific’s extensive network of Field Application Specialists around the world have helped customers establish analytical assays that have satisfied regulatory guidelines, and our Regulatory Consulting Services specialists have offered customers needed support when working with regulatory agencies.
- Consultation on validation, strategy, and approach
- Assist in alignment with current industry guidance documents: International Committee for Harmonization (ICH), regional pharmacopeias (USP, EP, JP, CP), FDA Guidance for Industry, World Health Organization (WHO)
- Review validation protocols, acceptance criteria, and reports
- Validation support at Customer Experience Centers
- Consult on regulatory strategy: Prior Approval Supplement (PAS), Type C, Chemistry, Manufacturing, and Controls (CMC) update
- Review briefing documents and presentations
- Organization of informational meetings with key regulatory thought leaders
- National Institute of Standards and Technology (NIST), Standards Coordinating Body (SCB)
- Regulatory submissions support documentation is available to support regulatory filings in multiple jurisdictions
- MycoSEQ Drug Master File (DMF) on file with FDA and Canada
- Validation and comparability reports
Industry organizations: active engagement
- Parenteral Drug Association (PDA)
- BioPhorum Operations Group (BPOG)
- Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB)
- Advanced Virus Detection Technologies Interest Group (AVDTIG)
- NSF Center for Cell Manufacturing Technologies (CMaT)
- National Institute of Standards and Technology (NIST), Standards Coordinating Body (SCB)
Bringing Rapid Mycoplasma Testing In-House to Expedite Lot Release of Recombinant Protein Product
This presentation will give an overview of one customer’s journey to replace an out-sourced mycoplasma test with an in-house mycoplasma qPCR assay. Octapharma received regulatory approval for the assay in October 2019 and now use in-house mycoplasma testing for lot release, saving lead time for their recombinant production.
Take advantage of knowledge sharing sessions with industry thought leaders
This webinar discusses three key areas to consider when commercializing a cell therapy—starting/raw materials, controlled manufacturing processes, and testing safety and quality. The starting and raw materials you choose at discovery can ultimately define the success of your development and GMP manufacturing process. We highlight some key watch outs and review essential tests that are required for lot release and regulatory filing and present solutions that save time and are accepted by regulators for lot release testing.
This webinar addresses regulatory requirements for safety and purity testing during the vaccine production process and highlights the benefits of implementing automated systems to save time and demonstrate product quality and safety.
Every IND or BLA is different, as is every therapy being considered for regulatory approval. The CMC validation is a significant part of the regulatory filing, and interpretation of regulatory guidance is not always easy, with variability depending on the product. Thermo Fisher’s team takes a retrospective look at some of the commonly asked regulator’s questions regarding mycoplasma testing and validation.
This presentation provides an overview of Octapharma’s journey on replacing an outsourced mycoplasma test with an in-house mycoplasma qPCR assay. Octapharma received regulatory approval for the assay in October 2019 and plan to run in-house mycoplasma testing for lot release, saving lead time for their recombinant production.
In cell and gene therapy, materials matter. However, misconceptions abound, exacerbating a lack of harmonization and standardization in key areas. This presentation aims to debunk some popular myths, provide practical guidance based upon long experience in the field, and clarify key regulatory considerations and requirements across the cell and gene therapy raw and starting materials area.
This webinar addresses regulatory requirements for safety and purity testing during the AAV production process and discusses the benefits of implementing automated systems to save time and demonstrate product quality and safety.