A practical approach for gmp-compliant validation of real-time PCR method for mycoplasma detection in human mesenchymal stromal cells as advanced therapy medicinal product
Abstract
Background Manufacturing of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP) for clinical use involves an ex vivo expansion, which leads to a risk of contamination by microbiological agents. Even if manufacturing under Good Manufacturing Practice (GMP) license minimizes this risk, contamination of cell cultures by mycoplasmas still represents a widespread problem. Furthermore, the absence of mycoplasma contamination represents one of ATMPs release criteria. Since July 2007, European Pharmacopoeia (EuPh) offers the possibility to replace official mycoplasma detection methods with Nucleic Acid Amplification techniques, after suitable validation. As an Italian authorized Cell Factory, we developed an in-house GMP-compliant validation of real-time PCR method for mycoplasma detection in human Mesenchymal Stromal Cells, according to EuPh sec. 2.6.7 and International Conference on Harmonization Q 2 . Materials and methods The study was performed in compliance with GMP international requirements with MycoSEQ™ Mycoplasma Detection Assay (Thermofisher) on QuantStudio5 real-Time PCR (Applied Biosystems). Assay validation was developed to evaluate sensitivity, interferences matrix-related, specificity and robustness. Results MycoSEQ™ Mycoplasma Detection Assay has been successfully validated on human Mesenchymal Stromal Cells as results comply with validation protocol acceptance criteria. Conclusions MycoSEQ™ Mycoplasma Detection Assay is a fast, sensitive and…
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qPCR-based mycoplasma detection assays have largely replaced traditional culture methods due to their ability to deliver validated results within 1-4 h, supporting rapid batch release in POC settings.
“qPCR-based mycoplasma detection assays have largely replaced traditional culture methods due to their ability to deliver validated results within 1–4 h, supporting rapid batch release in POC settings (Becherucci et al., 2021).”
From Isolator-based point-of-care manufacturing: a practical solution for GMP-compliant cell and extracellular vesicles therapy production (2025) · cites this paper as Becherucci et al., 2021
Cites
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Chiu, Y.-S., Wang, K.-L., & Wu, K.-L. (2025) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2025.1644318 · Cited by 17 · see citing works
- Authors
- Becherucci, V., Curini, L., Ceccantini, R., Bisin, S., Gori, V., Gentile, F., De Rienzo, E., Piccini, L., Bindi, B., Pavan, P., Cunial, V., Allegro, E., Ermini, S., Brugnolo, F., & Bambi, F.
- Affiliations
- Meyer Children's Hospital (IT)
- Corresponding author
- Becherucci, V.
- Venue
- Biologicals
- Publisher
- Elsevier BV
- Published
- 2021-09
- Volume
- 73
- Pages
- 31-40
- ISSN
- 1045-1056 (ISSN-L)
- OpenAlex
- W3190721116
- Cited by
- 6
- References
- 3
- Citations by year
- 2025: 3 · 2023: 2 · 2022: 1
- FWCI
- 0.53
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biologicals (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Immunology and Microbiology › Microbiology › Microbial infections and disease research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Physical Sciences › Chemistry › Analytical Chemistry › Analytical Methods in Pharmaceuticals
- Keywords
- mycoplasma detection, human mesenchymal stem cells, advanced therapy medicinal products, GMP compliance, nucleic acid amplification tests, lot release testing, matrix effects, cell culture contamination, European Pharmacopoeia
- MeSH terms
- Humans, Mycoplasma, Cell Culture Techniques, Mesenchymal Stem Cells, Real-Time Polymerase Chain Reaction
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