Understanding Mycoplasma and Advances in Detection: The Enrichment MycoSEQ™ Plus Approach

Mycoplasma is a genus of bacteria distinguished by its lack of a cell wall, a feature that gives it a high degree of flexibility but also makes it resistant to many antibiotics that target cell wall synthesis. With more than 200 known species, mycoplasmas are widespread across the globe and found in diverse environments. Considered auxotrophs, lacking many essential biochemical pathways and depending heavily on external nutrients for survival and replication, they are adaptable and persistent contaminants in both laboratory and manufacturing environments. Testing for mycoplasma contamination is critical because several species are pathogenic, capable of causing pneumonia and inflammatory diseases. Their small size and resistance to filtration make them difficult to eliminate once contamination occurs. Moreover, antibiotic resistance has been documented in some species, heightening the need for reliable detection methods. It is important to conduct mycoplasma testing to ensure biopharmaceutical products are free of contaminants, promoting therapeutic efficacy and reliable testing results. While traditional culture methods remain the gold standard for Mycoplasma detection, they are slow and time-consuming, posing challenges for products with short shelf lives such as cell and gene therapy (CGT) products. This article explores how molecular testing can provide sensitive and specific results more rapidly.  

Specific challenges for advanced therapies

Mycoplasma testing typically occurs at multiple stages — from raw drug material to final product release — and includes routine checks on cell banks such as the Master Cell Bank (MCB) and Working Cell Bank (WCB). These assays ensure that analytical and manufacturing cell lines remain uncontaminated. Environmental monitoring also plays a role, assessing facility cleanliness, staff technique, and airflow control to prevent recurring contamination. 

CGT products present unique testing challenges. Since these therapies often involve patient-derived materials with limited batch volumes and short shelf lives, standard mycoplasma testing — which can take up to 28 days — is impractical. Additionally, the small sample volumes available for testing complicate method validation and performance assessment. 

Traditional and alternative testing methods

Mycoplasma testing has been a regulatory requirement since the 1990s, governed by compendial standards from the United States Pharmacopeia (USP), European Pharmacopoeia (EP), and Japanese Pharmacopoeia (JP), as well as US Food and Drug Administration (FDA) guidance. These established methods include: 

• Culture method: Involves inoculating samples into liquid media and monitoring colony formation on agar plates over 28 days. While reliable, this process is labor-intensive and slow. 

• Indicator cell culture method: Samples are co-cultured with Vero cells, then stained with a fluorescent dye that binds DNA to detect mycoplasma presence. The assay typically requires one week but has lower sensitivity. 

• Nucleic Acid Amplification Technique (NAT): Recognized by the EP and JP, this PCR-based approach isolates nucleic acids from samples and can deliver results within a day. However, due to the need for extensive product-specific validation to demonstrate equivalence to culture methods under the FDA/EMA/JP regulations, it remains an alternative option. 

Each method poses trade-offs between time, sensitivity, and practicality. The lengthy timelines and high material requirements of compendial methods contrast with the limited sampling capacity and validation demands of nucleic acid techniques. 

Developing a more sensitive and efficient assay

To overcome these challenges, researchers at the PPD™ clinical research business of Thermo Fisher Scientific sought to design a more sensitive, faster, and scalable mycoplasma detection method. Their goal was to create an assay that could detect multiple mycoplasma species with reduced sample volumes and shorter turnaround times, while maintaining high accuracy. 

Using four mycoplasma species — including both aerobic and anaerobic types as well as Mycoplasma pneumoniae, a leading human pathogen — the team evaluated culture conditions, enrichment durations, and sensitivity across a range of colony-forming unit (CFU) concentrations. They also optimized the nucleic acid extraction process, improving cycle threshold (CT) values for negative controls, thereby enhancing detection precision. 

The Enrichment MycoSEQ Plus assay

These efforts culminated in the development of Enrichment MycoSEQ Plus, an assay combining enrichment step compliant with USP/EP/JP mycoplasma culture and indicator cell culture methods with qPCR detection using the MycoSEQ Plus Mycoplasma Detection Kit. This method involves culturing samples in both Vero E6 cells and broth media for three to six days, followed by qPCR analysis. The MycoSEQ Plus Mycoplasma Detection Kit employs TaqMan-based chemistry for increased specificity and can detect over 200 mycoplasma species. 

Advantages of the Enrichment MycoSEQ Plus approach

Compared to established techniques, Enrichment MycoSEQ Plus offers several significant advantages: 

1. Enhanced sensitivity: Detects as low as 0.6 CFU/mL in just 6 days, outperforming both the gold standard (10 CFU/mL after 28 days) and standard MycoSEQ™ (10 CFU/mL in one day). 

2. Improved specificity: Detects only viable mycoplasma instead of both viable and non-viable.  

3. Reduced testing time: Shortens turnaround from weeks to days, critical for CGT products. 

4. Improved sampling volume: Uses up to 1.5mL per test, reducing false negatives common in small-volume PCR assays. 

Incorporating automation

The assay’s nucleic acid extraction is automated using the KingFisher Flex™ platform, which employs magnetic bead-based technology to process entire plates simultaneously. This approach minimizes variability, enhances consistency, minimizes potential for contamination, and reduces labor time. Extraction is completed in under 90 minutes, and the system’s flexible scripting allows for method customization based on product formulation. 

Impact and future directions

The Enrichment MycoSEQ Plus assay represents a significant advance in mycoplasma detection — offering greater sensitivity, faster turnaround, and scalability compared to legacy methods. This improvement not only boosts confidence in product sterility but also supports the rapid release of time-sensitive therapies such as CGTs. 

Final thoughts

Mycoplasma testing remains a cornerstone of biopharmaceutical quality assurance, ensuring the safety and purity of drug products and cell banks. While traditional methods are thorough, they are long and resource-intensive. The Enrichment MycoSEQ Plus assay bridges the gap, offering a faster, more sensitive, and scalable alternative that aligns with modern biomanufacturing needs — paving the way for next-generation analytical platforms that could redefine microbial testing altogether.