A systematic approach combining process optimization and clinically-relevant quality attributes to manufacture high potency mesenchymal stromal cells for clinical and veterinary applications

问题-尽管生物反应器技术的进步允许可规模制造间充质干细胞(MSCs)，但这些方法中的许多都是借鉴疫苗和抗体行业的，并不优先考虑细胞功能。MSCs的商业规模扩张导致多家公司在后期试验中表现不佳。需要改进的制造方法来生产可扩展和功能性的MSCs，以利用对MSCs的大量投资。*解决方案-我的实验室已经申请了一项专利，可以制造具有增强抗炎特性的可伸缩3D MSCs。使用基于统计学的方法，我们将系统地优化生物工艺参数，以在不增加成本的情况下生产足够数量和质量的具有增强抗炎特性的治疗相关的MSCs。首先，使用迭代实验设计(DoE)和高通量读出，将对生物工艺参数组合进行建模和测试，以确定它们对MSC产量、抗炎因子和成本的相对影响。将调查和查询影响MSC培养代谢谱的其他工艺参数，以进一步优化生物工艺参数的组合。人和狗的MSCs将被大规模制造，并在患有炎症性疾病的家犬身上进行测试，以验证这种优化生物过程参数的方法。*O1-统计建模，以优化同时提高MSC产量和抗炎属性的工艺参数-在此，我们使用确定性筛选设计(DSD)来模拟生物工艺参数的各种排列对MSC产量、效力和计算成本的影响。响应曲面图允许生物过程参数组合的分级聚类，从而以低成本最大化细胞产量和效力。*O2-评估代谢参数读数，以进一步优化MSC生物过程参数-在这个目标中，将使用拉曼光谱来优化代谢参数以及MSC大小和细胞结构的变化。这些参数随后可以在MSC制造容器中进行在线监测，为就地快速“纠正”工艺参数提供反馈。*O3-验证MSCs在兽医应用中的可扩展性和治疗效力-将使用优化的生物工艺参数组合以1L规模制造人和犬MSCs。犬AT-MSCs将在患有关节疾病的家犬身上进行测试。*意义--工艺优化将导致大规模生产具有治疗功能的MSCs，用于炎症性疾病的有效临床和兽医应用。该项目提供HQP的统计建模、生物过程优化、细胞制造和兽医应用方面的培训机会。

Problem – Although advances in bioreactor technology allow for scalable manufacturing of mesenchymal stromal cells (MSCs), many of these methods are borrowed from the vaccine and antibody industry, and do not prioritize cell functionality. Commercial-scale expansion of MSCs contributed to sub-optimal performance in late-phase trials for multiple companies. Enhanced manufacturing methods that produce scaleable and fuctional MSCs are needed to capitalize on the substantial investment in MSCs. ***Solution – My lab has filed a patent to manufacture scalable 3D MSCs with enhanced anti-inflammatory properties. Using a statistically-based approach, we will systematically optimize bioprocess parameters to manufacture sufficient quantities and quality of therapeutically-relevant MSCs with enhanced anti-inflammatory properties, without increasing costs. First, using iterative Design of Experiment (DoE) and high throughput read-outs, combinations of bioprocess parameters will be modeled and tested for their relative impact on MSC yield, anti-inflammatory factors and cost. Additional processing parameters that affect the metabolic profile of the MSC cultures will be investigated and queried to further optimize combination of bioprocess parameters. Human and canine MSCs will be manufactured in large scales, and tested in domestic dogs with inflammatory disease to validate this approach to optimize bioprocess parameters. ***O1 – Statistical modeling to optimize process parameters that simultaneously improve MSC yield and anti-inflammatory attributes – Herein, we use Definitive Screening Designs (DSD) to model the effects of various permutations of bioprocess parameters on MSC yield, potency and calculated cost. A response surface map allows hierarchical clustering of combination of bioprocess parameters that maximize cell yield and potency at low costs. ***O2 – Assess metabolic parameters read-outs to further optimize MSC bioprocess parameters – Optimizing metabolic parameters and changes in MSC size and cellular structures will be conducted in this objective using Raman spectroscopy. These parameters can subsequently be monitored on-line in MSC manufacturing vessels providing feedback to rapidly “correct” process parameters in situ.***O3 – Validate scalability and therapeutic potency of MSCs in veterinary applications – Human and canine MSCs will be manufactured in 1L scales using the optimized combination of bioprocess parameters. Canine AT-MSCs will be tested in domestic dogs with joint diseases.***Significance – The process optimization will result in large scale manufacturing of therapeutically-potent MSCs for effective clinical and veterinary applications in inflammatory diseases. This program offers training opportunities in statistical modeling, bioprocess optimization, cell manufacturing, and veterinary application for HQPs.