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

Becherucci, Valentina; Curini, L.; Ceccantini, Riccardo; Bisin, S.; Gori, V.; Gentile, Francesca Romana; Rienzo, Elena De; Piccini, L.; Bindi, B.; Pavan, Paola; Cunial, V.; Allegro, Elisa; Ermini, Stefano; Brugnolo, Francesca; Bambi, Franco

doi:10.1016/j.biologicals.2021.07.006

Cited by 3 publications

(3 citation statements)

References 35 publications

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“…We intentionally used M. orale DNA for sensitivity testing because this mycoplasma strain has a very low percentage of genomic copies per colony-forming unit (GC/CFU), thus representing a detection target with an overall low genomic mycoplasma DNA content. 31 Although our detection limits of 6.3 pg for the standard PCR and 0.8 pg mycoplasma DNA for the qPCR protocol are above those reported by some commercial kits or other protocols, 43 , 44 , 45 the here presented method will be more than sufficient for reliable routine laboratory testing, as shown in Figures 5 and S4 . This result is not surprising, considering that a single mycoplasma bacterium forms about 10 6 CFU/mL within 3–5 days 6 and taking into account that contaminated cell cultures usually contain about 10 6 -10 8 CFU/mL.…”

Section: Discussionmentioning

confidence: 55%

“…A good example, where a very high PCR sensitivity is required, is the screening of cell therapy products for medical use. For reaching the minimum LOD of 10 CFU in such applications, specialized commercial kits 43 , 44 or TaqMan-based multi-primer qPCRs 45 are needed. However, the here presented mycoplasma detection system is not intended for specialized applications but designed as a generally applicable and highly reliable standard for routine mycoplasma testing.…”

Section: Discussionmentioning

confidence: 99%

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A PCR protocol to establish standards for routine mycoplasma testing that by design detects over ninety percent of all known mycoplasma species

Siegl¹,

Kruchem²,

Jansky³

et al. 2023

iScience

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Section: Discussionmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 99%

A PCR protocol to establish standards for routine mycoplasma testing that by design detects over ninety percent of all known mycoplasma species

Siegl¹,

Kruchem²,

Jansky³

et al. 2023

iScience

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“…Swine trypsin solutions are a major source of M. hyorhinis [30]. One of the tiniest free-living bacterium species, Mycoplasma can be found in sizes between 0.15 and 0.3 µm [31]. Special tip organelles, containing high concentrations of adhesins, allow mycoplasmas to attach to and penetrate their host cells (Figure 1a).…”

Section: Bacteriamentioning

confidence: 99%

Methods and Analysis of Biological Contaminants in the Biomanufacturing Industry

et al. 2023

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The advent of bioprocessing has revolutionized the biomanufacturing industry, leading to the rise of biotherapeutics derived from biologic products such as chimeric antigen receptor (CAR) T-cells used for targeted cancer treatment and the Vero cell line for the production of viral vectors and vaccines. Despite these promising developments, most biologic products are characterized by fragile macromolecular structures that are heterogenous with a purity profile that varies with each batch making them susceptible to microorganism contamination. Regulatory oversight of biologic products is imperative to ensure adherence to good manufacturing practices and compliance with quality management systems. Current quality assurance protocols during production include monoclonality during cell line development, real-time monitoring of process parameters, flow cytometry for microbial monitoring, polymerase chain reaction, and immunoassay techniques to amplify DNA sequences related to bacterial or biological contaminants. FDA guidance recommends the implementation of process analytical technology within biomanufacturing production to measure critical quality parameters, which includes screening for potential biological contamination. Future advancements in bioprocess monitoring and control should capitalize on providing cheap, real-time, and sensitive detection. Biosensors, mass spectrometry, and polymerase chain reaction present robust, rapid, and real-time capabilities for multiplexed detection of contaminant analytes and have shown promise in meeting these needs. This review discusses the main biological contaminants of bioprocesses, European Union and FDA regulatory guidelines for monitoring and control within biologics production, existing methods and their limitations, and future advancements for biological contamination detection.

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Analysis of GMP for Marketing Authorization of ATMPs: Comparison in the US, the EU, Japan and South Korea

Shin

Kim

2022

Regen. Med.

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Aim: This study compared regulatory systems of competent authorities related to GMP for marketing authorization of advanced therapy medicinal products (ATMPs). Methods: Dossiers for GMP and regulations and guidelines for facilities and equipment were analyzed using gap analysis. The risk-based approach (RBA) and GMP inspection were evaluated with regulations and guidelines. Results: The dossier was similar for the competent authorities. However, whereas a site master file is required in the EU, Japan and South Korea, the US requires only a biologics license application. The regulations and guidelines of facilities and equipment emphasized preventing contamination. There are differences among the competent authorities in GMP inspection and RBAs. Conclusion: Differences among the competent authorities in the marketing authorization process related to GMP for ATMPs should be considered.

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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

Cited by 3 publications

References 35 publications

A PCR protocol to establish standards for routine mycoplasma testing that by design detects over ninety percent of all known mycoplasma species

A PCR protocol to establish standards for routine mycoplasma testing that by design detects over ninety percent of all known mycoplasma species

Methods and Analysis of Biological Contaminants in the Biomanufacturing Industry

Analysis of GMP for Marketing Authorization of ATMPs: Comparison in the US, the EU, Japan and South Korea

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