BACKGROUND The recent concept of secretome-based tissue regeneration has profoundly altered the field of regenerative medicine and offers promising novel therapeutic options. In contrast to medicinal products with a single active substance, cell-derived secretomes comprise pleiotropic bioactive ingredients, representing a major obstacle for reproducible drug product efficacy and warranting patient safety. Good manufacturing practice (GMP)-compliant production guarantees high batch-to-batch consistency and reproducible efficacy of biological medicinal products, but different batches of cellular secretomes produced under GMP have not been compared yet, and suitable quality control parameters have not been established. To this end, we analyzed diverse biological and functional parameters of different batches produced under GMP of the secretome obtained from γ-irradiated peripheral blood mononuclear cells with proven tissue regenerative properties in infarcted myocardium, stroke, spinal cord injury, and skin wounds. METHODS We quantified key secretome ingredients, including cytokines, lipids, and extracellular vesicles, and functionally assessed potency in tube formation assay, ex vivo aortic ring sprouting assay, and cell-based protein and reporter gene assays. Furthermore, we determined secretome stability in different batches after 6 months of storage at various ambient temperatures. RESULTS We observed that inter-batch differences in the bioactive components and secretome properties were small despite considerable differences in protein concentrations and potencies between individual donor secretomes. Stability tests showed that the analytical and functional properties of the secretomes remained stable when lyophilisates were stored at temperatures up to + 5 °C for 6 months. CONCLUSIONS We are the first to demonstrate the consistent production of cell-derived, yet cell-free secretome as a biological medicinal product. The results from this study provide the basis for selecting appropriate quality control parameters for GMP-compliant production of therapeutic cell secretomes and pave the way for future clinical trials employing secretomes in tissue regenerative medicine.

中文翻译：

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治疗应激外周血单核细胞衍生分泌蛋白（一类新型生物医药产品）符合 GMP 生产的可重复性。

背景基于分泌组的组织再生的最新概念已经深刻地改变了再生医学领域，并提供了有前途的新型治疗选择。与具有单一活性物质的药品相比，细胞源性分泌蛋白组包含多效性生物活性成分，这是可重复的药品功效和保证患者安全的主要障碍。符合良好生产规范（GMP）的生产保证了生物医药产品批次间的高一致性和可重复的功效，但尚未对根据 GMP 生产的不同批次的细胞分泌蛋白组进行比较，也尚未建立合适的质量控制参数。为此，我们分析了在 GMP 下生产的不同批次的分泌蛋白组的多种生物学和功能参数，该分泌蛋白组从经 γ 照射的外周血单核细胞获得，在梗塞心肌、中风、脊髓损伤和皮肤伤口中具有经过验证的组织再生特性。方法我们量化了关键的分泌组成分，包括细胞因子、脂质和细胞外囊泡，并在管形成测定、离体主动脉环萌芽测定以及基于细胞的蛋白质和报告基因测定中功能评估效力。此外，我们还确定了在不同环境温度下储存 6 个月后不同批次的分泌蛋白组稳定性。结果我们观察到，尽管各个供体分泌蛋白组之间的蛋白质浓度和效力存在相当大的差异，但生物活性成分和分泌蛋白组特性的批次间差异很小。稳定性测试表明，当冻干物在高达 + 5 °C 的温度下储存 6 个月时，分泌蛋白组的分析和功能特性保持稳定。结论 我们是第一个证明细胞衍生的无细胞分泌蛋白组作为生物医药产品的一致生产的人。这项研究的结果为选择适当的质量控制参数以符合 GMP 的治疗性细胞分泌蛋白组生产提供了基础，并为未来在组织再生医学中使用分泌蛋白组的临床试验铺平了道路。