Manufacturing processes for autologous cell therapy need to reproducibly generate in specification (quality and quantity) clinical product. However, patient variability prevents the level of control of cell input material that could be achieved in a cell line or allogeneic-based process. We have applied literature data on bone marrow-derived mesenchymal stromal cells variability to estimate probability distributions for stem cell yields given underlying truncated normal distributions in total nucleated cell concentration, stem cell percentage and plausible aspirate volumes. Monte Carlo simulation identified potential variability in harvested stem cell number in excess of an order of magnitude. The source material variability was used to identify the proportion of donor manufacturing runs that would achieve a target yield specification of 2E7 cells in a fixed time window with given proliferative rates and different aspirate volumes. A rapid, screening, development approach was undertaken to assess culture materials and process parameters (T-flask surface, medium, feed schedule) to specify a protocol with identified proliferative rate and a consequent model-based target aspirate volume. Finally, four engineering runs of the candidate process were conducted and a range of relevant quality parameters measured including expression of markers CD105, CD73, CD44, CD45, CD34, CD11b, CD19, HLA-DR, CD146 (melanoma cell adhesion molecule), CD106 (vascular cell adhesion molecule) and SSEA-4, specific metabolic activity and vascular endothelial growth factor secretion, and osteogenic differentiation potential. Our approach of using estimated distributions from publicly available information provides a route for data-poor earl- stage developers to plan manufacture with defined risk based on rational assumptions; furthermore, the models produced by such assumptions can be used to evaluate candidate processes, and can be incrementally improved with accumulating distribution understanding or subdivision by new process variables.

中文翻译：

---

用于管理间充质基质细胞疗法制造自体细胞疗法的生物变异性的蒙特卡罗框架

自体细胞疗法的制造过程需要可重复地生成规范（质量和数量）的临床产品。然而，患者的变异性阻碍了在细胞系或基于同种异体的过程中可以实现的细胞输入材料的控制水平。我们已经应用有关骨髓来源间充质基质细胞变异性的文献数据来估计干细胞产量的概率分布，因为总有核细胞浓度、干细胞百分比和合理的抽吸体积的潜在截断正态分布。蒙特卡罗模拟确定了收获的干细胞数量超过一个数量级的潜在变异性。源材料变异性用于确定在给定增殖率和不同抽吸体积的固定时间窗口内实现 2E7 细胞目标产量规格的供体制造运行的比例。采取快速、筛选、开发方法来评估培养材料和工艺参数（T 瓶表面、培养基、进料计划），以指定具有确定的增殖率和随后基于模型的目标抽吸量的方案。最后，对候选工艺进行了四次工程运行，并测量了一系列相关质量参数，包括标记物 CD105、CD73、CD44、CD45、CD34、CD11b、CD19、HLA-DR、CD146（黑色素瘤细胞粘附分子）、CD106 的表达（血管细胞粘附分子）和 SSEA-4，特异性代谢活性和血管内皮生长因子分泌，以及成骨分化潜能。我们使用来自公开可用信息的估计分布的方法为缺乏数据的早期开发人员提供了一种途径，可以根据合理假设以定义的风险计划制造；此外，由这些假设产生的模型可用于评估候选过程，并且可以通过累积分布理解或按新过程变量细分来逐步改进。