Rapid Mycoplasma control in Bioreactors: moving from culture to PCR

2. Why mycoplasma still matters

Unlike typical bacteria, mycoplasmas lack a cell wall and can slip through certain sterilizing filters. They often propagate without visible turbidity, so low-level contamination may erode cell health, productivity, and product quality long before anyone notices [2]. Once established, contamination can move with media, feeds, or aerosols and cause recurring deviations – hence the shift toward pairing culture with rapid molecular checks in day-to-day decisions [3].

3. The direction of travel in guidance

European and US compendia continue to recognize culture as the reference method, yet increasingly allow risk-based implementation of nucleic acid tests when suitability is demonstrated for the intended matrices. In practice, manufacturers use PCR or dPCR to make timely continue/hold calls during upstream growth and at harvest, while maintaining bridging to compendial results for release [1,3].

4. Where PCR/dPCR adds value

Rapid methods shine in three places. First, in-process checks during expansion, N-1, and production confirm culture health before committing materials or time. Second, pre-harvest and harvest testing can prevent contaminated material from entering downstream operations. Third, environmental and raw-material monitoring helps close gaps that traditional programs may miss [3]. qPCR remains the workhorse for speed and scalability; dPCR is useful when absolute quantification near the limit of detection or added tolerance to inhibitors would change a decision. Recent method papers illustrate sensitive, internally controlled assays designed for routine cell-culture screening [4,5].

5. Sampling and matrices: design for the real world

Sample preparation – not the thermocycler – usually determines whether rapid testing works at the line. Clarified supernatants typically need minimal pre-treatment beyond clarification and appropriate biosafety handling. Crude harvests and protein-rich intermediates benefit from magnetic-bead or high-stringency spin-column chemistries that remove surfactants and host proteins that suppress amplification. Media, feeds, and filtrates may require volume reduction by membrane capture or centrifugation before lysis, especially when expected bioburden is low. Environmental swabs introduce their own inhibitors; swab-compatible lysis and extraction controls reduce the risk of false negatives [3].

6. Designing the method

Start with the species you intend to detect. A practical panel covers the usual cell-culture contaminants – common Mycoplasma species alongside Acholeplasma – and excludes near neighbors. Establish the limit of detection in each matrix you plan to test, not just in buffer; protein-rich materials behave differently from clarified supernatants. Build inhibition checks (internal controls or spike-recoveries) into every run, and define clear acceptance criteria so analysts aren’t left interpreting marginal traces. If the rapid method will inform release decisions before compendial results are available, perform a structured bridging study and capture the logic for discordant results in the deviation process [1,3].

7. A workable at-line workflow

Define the sampling scheme by risk: which stages to test, how often, and what volume to collect. Clarify the sample and reserve parallel aliquots for molecular testing and culture as required. Extract nucleic acids using a matrix-appropriate protocol – rapid lysis for clarified supernatants; higher-stringency bead or column workflows for harvests and intermediates – and include extraction blanks. Run qPCR or dPCR with positive, negative, and inhibition controls. Interpret results against predefined thresholds and codified actions; trend Ct or copies over time to spot weak signals before they become batch-threatening [3–5].

8. Implementation essentials

Before rollout, ensure the risk assessment is complete per unit operation; the target panel is defined; matrix-specific limits of detection are verified; inhibition controls are routine; bridging to the compendial method is documented where required; and sampling frequencies, acceptance criteria, and escalation paths are captured in SOPs. With these pieces in place, rapid mycoplasma control becomes a routine part of process monitoring rather than an emergency measure [1,3].

9. Conclusion

Rapid mycoplasma testing is most effective when it is designed around real matrices and decisions, not around instruments. By pairing matrix-appropriate extraction with qPCR or dPCR, teams can replace weeks-long uncertainty with actionable results in hours - tightening reactor control and accelerating, better-documented decisions [4,5].

10. What Invitek offers

While our Mycoplasma spp. qPCR test kit is in development, the upstream foundation is already available: automation-ready nucleic acid extraction for complex bioprocess matrices. For example, researchers have extracted Mycoplasma agassizii DNA from swabs with the Invisorb® Spin Universal Kit in wildlife health studies [6]. Avian mycoplasmology groups routinely purified M. synoviae and M. gallisepticum genomic DNA using the RTP® Pathogen Kit  to support strain characterization, cloning, and PCR assay development [7,8,9]. Together, these independent studies underscore kit robustness across challenging sample types – from culture isolates to field swabs – and give us a strong foundation to pair with our in-house method adaptations for bioprocess matrices. Rapid lysis options support at-line testing of clarified supernatants, while magnetic-bead and spin-column chemistries with robust wash capacity address protein-rich harvests and intermediates. Flexible volumes and formats fit both manual spot checks and higher-throughput screening. Once the qPCR kit launches, we’ll add matrix-specific performance data and method cards here for a drop-in, pharmacopoeia-aligned workflow.

Bibliography

1. European Pharmacopoeia 2.6.7, “Mycoplasmas.” European Directorate for the Quality of Medicines & HealthCare (EDQM).

2. Drexler HG, Uphoff CC. Mycoplasma contamination of cell cultures: incidence, sources, effects, detection, elimination, prevention. Cytotechnology. 2002;39(2):75–90. doi: 10.1023/A:1022913015916.
3. Angart P, Kohnhorst C, Chiang M-J, Arden NS. Considerations for risk and control of mycoplasma in bioprocessing. Curr Opin Chem Eng. 2018;22:161–166. doi: 10.1016/j.coche.2018.09.012.
4. Sung J, Hawkins C. A highly sensitive, internally controlled real-time PCR assay for mycoplasma detection in cell cultures. Biologicals. 2020;63:1–6. doi: 10.1016/j.biologicals.2019.12.007.
5. Siegl D, et al. A PCR protocol to establish standards for routine mycoplasma testing. iScience. 2023;26(7):106724. doi: 10.1016/j.isci.2023.106724.

6. Louro M, et al. First molecular detection of Mycoplasma agassizii in captive tortoises in Portugal. Frontiers in Veterinary Science. 2025;12:1652362. doi: 10.3389/fvets.2025.1652362.

7. Berčič RL, et al. Neuraminidase of Mycoplasma synoviae desialylates heavy chain of the chicken immunoglobulin G and glycoproteins of chicken tracheal mucus. Avian Pathology. 2011;40(3):299–308. doi: 10.1080/03079457.2011.565311.

8. Slavec B, et al. Variation of vlhA gene in Mycoplasma synoviae clones isolated from chickens. Avian Pathology. 2011;40(5):481–489. doi: 10.1080/03079457.2011.604840.

9. Cizelj I, et al. Mycoplasma gallisepticum and Mycoplasma synoviae express a cysteine protease CysP, which can cleave chicken IgG into Fab and Fc. Microbiology. 2011;157(2):362–372. doi: 10.1099/mic.0.045641-0.