SYSTEMATIC STEM CELL BIOPROCESS ENGINEERING

生物过程工程在分子生物学和重组DNA技术的发现中发挥了变革性作用，最终产生了新的治疗方法-单克隆抗体和疫苗。基于加拿大在干细胞生物学方面的优势，细胞疗法的生物过程工程代表了治疗或治愈疾病的生物制剂发展的下一波浪潮。这些产品能否到达患者手中，取决于以稳健和经济有效的方式制造细胞的技术。干细胞作为制造细胞疗法的“原材料”发挥着重要作用，类似于大肠杆菌和杂交瘤细胞作为生物制药工业的主力。事实上，该产品是活细胞，而不是它们分离和富集的蛋白质产品，这就提出了一套不同的制造和控制挑战。生物工程基础包括生物反应器设计、代谢建模和过程控制需要与系统生物学原理相结合，以指导制造基于细胞的产品的创新技术的发展。本研究项目的总体目标是开发一个基于机制的细胞治疗制造平台，重点关注两个新颖且相互关联的项目：** a)将代谢建模和控制集成到多能干细胞生物过程中。**理解和控制细胞代谢在生物制药生产中发挥着重要作用，有证据表明，细胞疗法将同样受益于对细胞输出优化中代谢网络的更多理解。诱导多能干细胞（iPSC）是通过所谓的重编程从任何有核细胞类型生成的细胞，是研究代谢在细胞产生中的作用的强大模型。在本项目中，我们特别致力于建立iPSC重编程过程中代谢网络的计算模型，测试关键模型控制点对重编程频率的影响，并优化iPSC生物过程中的代谢控制参数以提高细胞产量。**B)用于增强干细胞繁殖的自动分泌因子反馈控制生物反应器。**生物制药生产中的过程控制策略通常依赖于转化细胞或微生物对培养设定点的相对稳定和均匀的反应，以优化重组蛋白的产量。相反，细胞治疗生产涉及异质细胞群，其中竞争的生长速率和培养要求产生不断变化且通常不稳定的培养物。我们的NSERC研究表明，内源性可溶性因子介导的反馈抑制是原代造血干细胞培养输出的主要控制点。在这个项目中，我们将开发和验证基于实时和动态控制内源性产生的信号因子的生物反应器系统。该项目不仅将产生新的策略来实现细胞治疗制造中的鲁棒控制，而且还将提供关于原代细胞系统反馈控制的基本信息。**总的来说，通过利用基于工程的方法，如数学建模和生物反应器设计，拟议的研究计划应该允许技术的发展，使新兴的加拿大细胞治疗行业。拟议的工作还将支持继续培训适合为高价值下一代工业生物技术制造工艺做出贡献的人员。

Bioprocess engineering has played a transformative role in enabling discoveries in molecular biology and recombinant DNA technologies, ultimately yielding new therapeutics - monoclonal antibodies and vaccines. Building on Canadian strengths in stem cell biology, bioprocess engineering of cell-based therapies represents a next wave in the development of biologics to treat or cure disease. Whether or not these products ever reach patients depends upon technologies to manufacture cells in a robust and cost effective manner. Stem cells play an important role as the "raw material" in manufacturing cell therapies, analogous to E. coli and hybridoma cells as the workhorses of the biopharmaceutical industry. The fact that the product is living cells, not their isolated and enriched protein products, brings forward a different set of manufacturing and control challenges. Bioengineering fundamentals including bioreactor design, metabolic modeling and process control need to be combined with systems biology principles to guide the development of innovative technologies for manufacturing cell based products. The overall goal of this research program is to develop a mechanism-based platform for cell therapy manufacturing, focusing on two novel and interrelated projects: **A) Integrating metabolic modeling and control into pluripotent stem cell bioprocesses.**Understanding and controlling cell metabolism has played an important role in biopharmaceutical production and evidence suggests that cell therapies will similarly benefit from an increased understanding of metabolic networks in cell output optimization. Induced pluripotent stem cells (iPSC), cells generatable from any nucleated cell type via so-called reprogramming, are a powerful model with which to study the role of metabolism in cell production. In this project we specifically aim to develop computational models of the iPSC metabolic network during reprogramming, test the influence of key model control points on reprograming frequencies, and optimize metabolic control parameters in iPSC bioprocesses to increase cell yields. **B) Automated secreted-factor feedback-controlled bioreactors for enhanced stem cell propagation.**Process control strategies in biopharmaceutical production typically rely on the relatively stable and homogenous responses of transformed cells or microbes to culture set-points to optimize recombinant protein yield. In contrast, cell therapy production involves heterogeneous cell populations where competing growth rates and culture requirements yield continuously changing and often unstable cultures. Our NSERC research has demonstrated that endogenous soluble factor-mediated feedback inhibition represents a dominant control point in primary blood stem cell culture outputs. In this project we will undertake the development and validation of bioreactor systems based on real-time and dynamic control of endogenously produced signaling factors. This project will not only yield new strategies to achieve robust control in cell therapy manufacturing, but also fundamental information on feedback control of primary cell systems. **Overall, by utilizing engineering-based approaches such mathematical modeling and bioreactor design, the proposed research program should allow the development of technologies that enable the emerging Canadian cell therapy industry. The proposed work will also support the continued training of personnel ideally suited to contribute to high value next-generation industrial biotechnology manufacturing processes.