Transforming mycoplasma testing for faster biotherapeutic quality control

Mycoplasma contamination presents an unusual challenge for biotherapeutics manufacturers. These organisms lack a cell wall, can pass through 0.22μm filters and may proliferate without producing the turbidity or visible changes associated with many bacterial contaminants. Yet their presence can alter cell growth, metabolism and gene expression, potentially compromising process performance, product quality and, ultimately, patient safety.¹

The risk is particularly acute in cell-based manufacturing. Mammalian cell cultures provide favourable conditions for mycoplasma growth, while raw materials, personnel and contaminated cell banks can all provide routes of entry. For autologous cell therapies, the problem is compounded by small sample volumes, compressed manufacturing schedules and products whose shelf life may be shorter than the time required for a conventional microbiological test. Consequently, mycoplasma control is not just an end-product release exercise; it requires prevention, appropriately placed in-process monitoring and a validated method capable of supporting timely decisions.

Why conventional testing is under pressure

Culture-based testing has long been regarded as the reference approach because it demonstrates the presence of viable organisms and can achieve high sensitivity. Its principal weakness is speed, with incubation taking up to 28 days. Indicator cell culture can detect noncultivable organisms, but also requires skilled interpretation. Neither sits comfortably with intensified bioprocessing, real-time release ambitions or short-lived advanced therapies.¹

Nucleic acid amplification techniques (NAT), particularly quantitative PCR (qPCR), have therefore become the preferred rapid alternative for many manufacturers. By targeting conserved mycoplasma DNA sequences, qPCR can deliver sensitive, specific results within hours rather than weeks. Automation can further reduce manual handling, standardise extraction and amplification, increase throughput and strengthen data integrity. This results in faster laboratory testing and opens opportunity for moving quality control closer to the process.

Regulation is evolving in the same direction. Revised European Pharmacopoeia general chapter 2.6.7, which entered into force on 1 April, adopts a less prescriptive, risk-based approach. Sanctioning NAT as an alternative to conventional methods, it also includes updated expectations for reference preparations, inhibitory substances and method comparability. However, it does not make PCR an automatic substitute for culture. Unless otherwise justified and authorised, manufacturers must still ensure that their strategy detects both cultivable and non-cultivable mycoplasmas.²

Validation determines value

The speed of PCR is only useful when the result is reliable in the specific product matrix. Validation must demonstrate specificity across pharmacopoeia-relevant species, sensitivity at the required limit of detection, robustness, precision and comparability with compendial methods. Sample preparation is critical: media components, high cell concentrations or product-related substances may inhibit amplification and create false-negative results. Internal amplification controls are therefore essential, as are representative samples containing both cells and supernatant where appropriate.²

False-positive results present a different problem. NAT may detect residual DNA from non-viable organisms, so a positive signal does not necessarily demonstrate an active contamination event. Laboratories need predefined procedures for repeat testing, orthogonal investigation and root-cause analysis. Closed, automated workflows can reduce the risk of amplicon contamination, but technology cannot replace a scientifically justified contamination-control strategy.

Recent research illustrates how assay design is addressing these challenges. A Sanofi R&D team developed a qPCR method using two primers and two hydrolysis probes to detect 11 pharmacopoeia-relevant mycoplasma species. The approach was designed to combine broad coverage with specific detection while remaining compatible with pharmacopoeial expectations.³ A separate 2025 study compared universal PCR, enzymatic recombinase amplification and qPCR assays targeting the 16S–23S rRNA intergenic spacer region, highlighting how target selection and multimethod validation can improve species coverage and analytical sensitivity.⁴

Beyond qPCR

The next stage may be to integrate rapid detection more directly into process analytical technology (PAT). Zhang et al. combined recombinase polymerase amplification with CRISPR-Cas12a, reporting detection limits of 10 to 0.1 copies/μL in tested organisms and a total workflow of under one hour. The method was evaluated in antibody-production cell culture and downstream samples, indicating the potential for rapid monitoring beyond the final release point.¹

The next stage may be to integrate rapid detection more directly into process analytical technology (PAT)”

Other formats are also emerging. A closed ‘lab-in-a-pouch’ NAT workflow has demonstrated how simplified sample-to-answer systems could support decentralised testing with results in approximately one hour.⁵ A separate study has reported a NAT method for qualitative detection in biological products, adding to the evidence that rapid molecular methods can be designed around current pharmacopoeial requirements.⁶ These approaches remain subject to product-specific validation and regulatory acceptance, but they point towards testing that is faster, more automated and easier to position within manufacturing.

Other formats are also emerging…they point towards testing that is faster, more automated and easier to position within manufacturing”

PCR-based testing should therefore be viewed as one component of mycoplasma control – not its entirety. Supplier qualification, raw-material controls, closed processing, environmental monitoring, operator practices and segregation remain the first line of defence. Rapid detection adds most value when embedded within that broader system: placed at risk-based control points, protected by appropriate controls and linked to clear investigation procedures.

For manufacturers, the next challenge will be how to validate and integrate molecular methods without sacrificing the assurance provided by conventional microbiology. As regulation becomes more risk based and manufacturing timelines contract, well-designed PCR workflows can turn mycoplasma testing from a delayed confirmation into an active tool for protecting the process, the product and the patient.

References

1. Zhang D, et al. Rapid and Sensitive Mycoplasma Detection In Antibody Bioprocessing Via RPA-CRISPR/Cas12a’, Journal of Pharmaceutical and Biomedical Analysis. 2025; 263, 116904.

2. EPC Adopts Mycoplasmas General Chapter and Monographs, Updated To Incorporate Latest Analytical Developments. [Internet] European Directorate for the Quality of Medicines & HealthCare (EDQM). 2025. Available from: https://www.edqm.eu/en/-/epc-adopts-mycoplasmas-general-chapter-and-monographs-updated-to-incorporate-latest-analytical-developments

3. Dos Santos S, Lespinasse E, Bonnet B. Basmaciogullari S. Simple, Specific, Rapid, and Pharmacopoeia-Compliant qPCR Approach For The Detection Of Mycoplasma in Biopharmaceuticals. Molecular Therapy – Methods & Clinical Development, 2025;33(3), 101572.

4. Yin R, et al. Development and Validation Of Universal PCR, Basic ERA, and qPCR Assays Targeting the 16S–23S rRNA Intergenic Spacer Region for Mycoplasma detection. Microbial Pathogenesis. 2025; 205, 107669.

5. Houssenaly CK, et al. Strategy for Validation Of New Mycoplasma Nucleic Acid Detection Closed System And It Use In Routine Biopharmaceutical Manufacturing. PDA Journal of Pharmaceutical Science and Technology, 2025; 79(6), 686–693.

6. Guo Y, et al. Establishment of Nucleic Acid Amplification Technology For The Detection of Mycoplasma in Biological Products. Molecules, 2026; 31(11), 1794.