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 Q₂.

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 specific PCR-based Nucleic Acid Test assay that can be used as an alternative to official mycoplasma test methods for lot release of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP). Moreover, our study underlines the presence of interference on real-time PCR reaction due to matrix composition, pointing out a practical approach for method validation (i.e interference removal).

中文翻译：

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一种符合 gmp 标准验证实时 PCR 方法在人类间充质基质细胞中检测支原体作为先进治疗药物的实用方法

背景

将人类间充质基质细胞作为用于临床使用的高级治疗药物 (ATMP) 的制造涉及体外扩增，这会导致微生物制剂污染的风险。即使在良好生产规范 (GMP) 许可下进行生产将这种风险降到最低，支原体对细胞培养物的污染仍然是一个普遍存在的问题。此外，没有支原体污染是 ATMP 释放标准之一。自 2007 年 7 月起，欧洲药典 (EuPh) 提供了在适当验证后用核酸扩增技术取代官方支原体检测方法的可能性。作为意大利授权的细胞工厂，我们开发了符合 GMP 的内部实时 PCR 方法验证，用于检测人类间充质基质细胞中的支原体，根据 EuPh 秒。2.6.7 和国际协调会议 Q₂ .

材料和方法

这项研究是顺从与MycoSEQ™支原体检测测定（GMP的国际要求进行默飞世尔）上QuantStudio5实时PCR（Applied Biosystems公司）。开发了测定验证以评估灵敏度、与基质相关的干扰、特异性和稳健性。

结果

MycoSEQ™ 支原体检测分析已在人类间充质基质细胞上成功验证，结果符合验证方案验收标准。

结论

MycoSEQ™ 支原体检测分析是一种快速、灵敏和特异的基于 PCR 的核酸测试分析，可用作官方支原体测试方法的替代方法，用于批量发布人类间充质基质细胞作为高级治疗药物 (ATMP)。此外，我们的研究强调了由于基质组成对实时 PCR 反应的干扰，指出了方法验证（即干扰去除）的实用方法。