Strategy and validation of a consistent and reproducible nucleic acid technique for mycoplasma detection in advanced therapy medicinal products
Related Experiment Video
Updated: Dec 30, 2025
Optimized PCR-based Detection of Mycoplasma
Published on: June 20, 2011
Danilo D'Apolito1, Lucia D'Aiello2, Salvatore Pasqua2
1Unità Prodotti Cellulari (GMP), Fondazione Ri.MED c/o IRCCS-ISMETT, Via E. Tricomi 5, 90127, Palermo, Italy; Unità di Medicina di Laboratorio e Biotecnologie Avanzate, IRCCS-ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione), Via E. Tricomi 5, 90127, Palermo, Italy.
Abstract:
Advanced therapy medicinal products (ATMP) are required to maintain their quality and safety throughout the production cycle, and they must be free of microbial contaminations. Among them, mycoplasma contaminations are difficult to detect and undesirable in ATMP, especially for immunosuppressed patients. Mycoplasma detection tests suggested by European Pharmacopoeia are the "culture method" and "indicator cell culture method" which, despite their effectiveness, are time consuming and laborious. Alternative methods are accepted, provided they are adequate and their results are comparable with those of the standard methods. To validate a novel in-house method, we performed and optimized, a real time PCR protocol, using a commercial kit and an automatic extraction system, in which we tested different volumes of matrix, maximizing the detection sensitivity. The results were compared with those obtained with the gold standard methods. From a volume of 10 ml, we were able to recognize all the mycoplasmas specified by the European Pharmacopoeia, defined as genomic copies per colony forming unit ratio (GC/CFU). Our strategy allows to achieve faster and reproducible results when compared with conventional methods and meets the sensitivity and robustness criteria required for an alternative approach to mycoplasmas detection for in-process and product-release testing of ATMP.
Insights
A novel real-time PCR method offers faster and more reliable detection of mycoplasma contamination in advanced therapy medicinal products (ATMP). This validated approach ensures product safety for patients, especially those who are immunosuppressed.
Area of Science:
- Microbiology
- Pharmaceutical Science
- Molecular Biology
Background:
- Advanced therapy medicinal products (ATMP) require stringent microbial control for patient safety.
- Mycoplasma contamination poses a significant risk in ATMPs, particularly for immunocompromised individuals.
- Current European Pharmacopoeia methods for mycoplasma detection are time-consuming and labor-intensive.
Purpose of the Study:
- To validate a novel, rapid, and sensitive in-house real-time PCR method for mycoplasma detection in ATMPs.
- To optimize the real-time PCR protocol for maximum detection sensitivity using an automated system.
- To compare the performance of the novel method against gold-standard detection techniques.
Main Methods:
- Development and optimization of a real-time PCR protocol using a commercial kit.
- Implementation of an automated nucleic acid extraction system.
- Testing of various matrix volumes to enhance detection sensitivity.
- Comparative analysis against European Pharmacopoeia-standard culture and indicator cell culture methods.
Main Results:
- The optimized real-time PCR method successfully detected all European Pharmacopoeia-specified mycoplasmas from a 10 ml sample volume.
- The assay demonstrated high sensitivity, defined by the genomic copies per colony forming unit ratio (GC/CFU).
- Results were reproducible and met the required sensitivity and robustness criteria for an alternative method.
Conclusions:
- The novel real-time PCR method provides a faster and more reproducible alternative to conventional mycoplasma testing for ATMPs.
- This validated method is suitable for in-process and product-release testing, enhancing ATMP quality and safety.
- The optimized protocol ensures reliable mycoplasma detection, crucial for protecting vulnerable patient populations.
