Why Mycoplasma testing is required
Mycoplasmas, the smallest known free living organisms, are relatively common bacterial contaminants of mammalian cell cultures. Regulatory guidance requires that all products derived from mammalian cell culture undergo mycoplasma testing. Mycoplasma detection is difficult, sometimes impossible, when using a traditional 28-day culture-based test. Regulatory agencies have expanded acceptance of nucleic acid tests, such as real-time PCR, as an alternative method to traditional Mycoplasma detection after appropriate validation and regulatory review. Over 40 major global biotechnology companies have implemented, validated, and received regulatory approval to use the MycoSEQ system as their mycoplasma lot release test.
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. The MycoSEQ Mycoplasma Detection System integrates real-time PCR assays, instruments, and software with optimized PrepSEQ sample preparation reagents—delivering results in hours, not days. This enables you to test at multiple points in cell line and cell bank production and media harvest, to help increase your productivity and reduce the risk of downstream contamination.
Following validation, regulatory review, and acceptance, the MycoSEQ assay can be used as a preferred same-day mycoplasma lot release test, replacing the 28-day culture-based test.
Detection of more than 90 Mycoplasma, Acholeplasma, and Spiroplasma species, including commonly known contaminants within bioprocessing facilities with no cross reactivity with non-mycoplasma DNA
Streamlined workflow with results in less than 5 hours
The MycoSEQ Mycoplasma Detection System’s easy workflow delivers actionable results in less than 5 hours—enabling you to detect contamination quickly in process development, equipment, raw materials, harvest, helping to expedite the release of your product.
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.
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
Sensitivity
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.
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.
Specificity
Broad detection of Mycoplasmas
Below you can see a partial listing of the >90 species inclusion panel*, designed to detect the most common isolated species recommended for testing and validation of mammalian cell cultured biologics.
Partial inclusion panel | |||
Acholeplasma granularum | Mycoplasma genitalium | Mycoplasma testudinis | |
Acholeplasma laidlawii | Mycoplasma gypis | Mycoplasma timone | |
Acholeplasma pleciae | Mycoplasma hominis | Spiroplasma citri | |
Mycoplasma alkalescens | Mycoplasma hyorhinis | Spiroplasma endosymbionts | |
Mycoplasma alvi | Mycoplasma imitans | Spiroplasma insolitum | |
Mycoplasma anseris | Mycoplasma indiense | Spiroplasma kunkelii | |
Mycoplasma arginini | Mycoplasma lagogenitalium | Spiroplasma melliferum | |
Mycoplasma auris | Mycoplasma lipofaciens | Spiroplasma mirum | |
Mycoplasma buccale | Mycoplasma mobile | Spiroplasma phoeniceum | |
Mycoplasma californicum | Mycoplasma molare | Spiroplasma poulsonii | |
Mycoplasma canadense | Mycoplasma mycoides | Mycoplasma bovirhinis | |
Mycoplasma capricolum | Mycoplasma neurolyticum | Mycoplasma bovis | |
Mycoplasma caviae | Mycoplasma orale | Mycoplasma bovigenitalium | |
Mycoplasma collis | Mycoplasma phocidae | Mycoplasma canis | |
Mycoplasma cricetuli | Mycoplasma pirum | Mycoplasma felis | |
Mycoplasma equirhinis | Mycoplasma pneumoniae | Mycoplasma fastidiosum | |
Mycoplasma fermentans | Mycoplasma salivarium | Mycoplasma muris | |
Mycoplasma gallinaceum | Mycoplasma simbae | Mycoplasma pulmonis | |
Mycoplasma gallisepticum | Mycoplasma spumans | ||
Mycoplasma gateae | Mycoplasma synoviae | ||
*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 | |||
Species | Positive/negative | Tm | Derivative |
Human | Negative | n/a | n/a |
CHO | Negative | n/a | n/a |
Mouse | Negative | n/a | n/a |
Bacillus cereus | Negative | n/a | n/a |
Bacillus subtilis | Negative | n/a | n/a |
Candida albicans | Negative | n/a | n/a |
Clostridium perfringens | Negative | n/a | n/a |
Clostridium sporogenes | Negative | n/a | n/a |
Escherichia coli | Negative | n/a | n/a |
Lactobacillus delbrueckii | Negative | n/a | n/a |
Micrococcus luteus | Negative | n/a | n/a |
Staphylococcus aureus | Negative | n/a | n/a |
Staphylococcus epidermidis | Negative | n/a | n/a |
Steptococcus faecalis | Negative | n/a | n/a |
Support throughout the validation process
Implementations and technical support
- Our mission is to support your MycoSEQ 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, including instrument installation with IQ/OQ and CSV services, 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.
Validation guidance
- 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
Regulatory support
- 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
Regulatory documentation
- 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)
Webinar spotlight: Making the Switch–The Evaluation, Implementation, and Validation of Bringing Rapid Mycoplasma Testing In-House to Expedite Lot Release of Recombinant Protein Product
Octapharma AB in Stockholm, Sweden is the manufacturing site for NUWIQ®, a recombinant human FVIII produced in human cells. Regulatory guidelines state that recombinant bulk harvest must be tested for mycoplasma prior to further processing. This presentation will give an overview of Octapharma’s journey on replacing an out-sourced 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 as an alternative to the traditional growth-based mycoplasma testing and the process of laboratory set-up and instrument qualifications and software requirements. This webinar also presents a summary of the method development and validation, as well as how to help provide assay sensitivity meets regulatory requirements.
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 5 minutes of hands on time.
Powerful platform
The Applied Biosystems QuantStudio 5 (QS5) system is designed for both new and experienced users who need simple and affordable real-time PCR systems without compromising performance and quality. The QuantStudio 5 Real-Time PCR 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 3rd generation platform features an innovative optical system that enhances sensitivity.
Dedicated software for easy interpretation
The Applied Biosystem AccuSEQ Real-Time Detection Software is designed to work with the Applied Biosystems QuantStudio 5 Real-Time PCR instrument or the Applied Biosystems 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.
Highlights
- 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 AccuSEQ software demonstrates one of the key parameters (Ct value) to show the amplification which indicates the presence of DNA.
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.
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Take advantage of knowledge sharing sessions with industry experts
Educational programs | Description |
A journey to cell therapy manufacturing: From R&D to lot-release | 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. |
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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. | |
Our panel of experts answered questions regarding implementation and validation of a rapid mycoplasma system, covering topics including regulatory requirements, lot release, in-process testing, risk mitigation, live mycoplasma requirements, sensitivity, specificity, validation considerations, 28-day vs. same day, and compendial vs. rapid microbial method. | |
Regulatory FAQs & common concerns for cell & gene therapy raw & starting materials | 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. |
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Setting up for Success: Raw Materials Roundtable Discussion & Webinar | Cell and gene therapy industry leaders discuss the critical issues to consider when developing your raw material sourcing strategy. They share their thoughts on sourcing, supply, and regulatory documentation that should be taken into consideration at such a key step in the manufacturing of therapies. |
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