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使间充质间质细胞（MSCs）的规模化生产成为可能，但其中许多方法借鉴于疫苗和抗体行业，并没有优先考虑细胞的功能。在多家公司的后期试验中，MSCs的商业规模扩张导致了不理想的性能。为了充分利用对间充质干细胞的大量投资，需要改进制造方法，生产可扩展和功能性的间充质干细胞。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扩展3D间充质干细胞。使用基于统计的方法，我们将系统地优化生物工艺参数，在不增加成本的情况下，制造足够数量和质量的具有增强抗炎特性的治疗相关MSCs。首先，使用迭代实验设计（DoE）和高通量读数，生物工艺参数组合将建模并测试它们对MSC产量、抗炎因子和成本的相对影响。影响MSC培养物代谢谱的其他工艺参数将被调查和查询，以进一步优化生物工艺参数的组合。人类和犬间充质干细胞将大规模生产，并在患有炎症疾病的家养狗身上进行测试，以验证这种优化生物过程参数的方法。*** 1 -统计建模优化工艺参数，同时提高MSC产量和抗炎属性-在这里，我们使用确定筛选设计（DSD）来模拟各种生物工艺参数排列对MSC产量，效力和计算成本的影响。响应面图允许以低成本最大化细胞产量和效力的生物过程参数组合的分层聚类。***O2 -评估代谢参数读数以进一步优化MSC生物过程参数-优化代谢参数和MSC大小和细胞结构的变化将在本目标中使用拉曼光谱进行。这些参数随后可以在MSC制造容器中进行在线监测，并提供反馈，以快速“纠正”现场的工艺参数。***O3 -验证MSCs在兽医应用中的可扩展性和治疗效力-人类和犬MSCs将使用优化的生物工艺参数组合以1L的规模生产。犬AT-MSCs将在患有关节疾病的家养狗身上进行试验。***意义-工艺优化将导致大规模生产具有治疗作用的MSCs，用于炎症疾病的有效临床和兽医应用。该项目为HQPs提供了统计建模、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.