EAGER: Biomanufacturing: Metabolic cell process engineering (MCPE)-based stirred-tank bioproduction of large quantities of human T cells

EAGER：生物制造：基于代谢细胞过程工程 (MCPE) 的大量人类 T 细胞的搅拌罐生物生产

• 批准号: 1645031
• 负责人: Xiaoguang Liu
• 金额: $ 29.99万
• 依托单位: University of Alabama Tuscaloosa
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1645031&HistoricalAwards=false
• 关键词: EAGER; Biomanufacturing; Metabolic; cell; process

1645031-LiuIn 2015, cancer caused at least 0.5 million deaths and 1.5 million new cases were diagnosed in the US. The adoptive transfer of large numbers of tumor-infiltrating T cells or genetically engineered T cells with cancer-targeting receptors has shown tremendous promise for eradicating tumors in clinical trials. The existing methods to manufacture large quantities of such human T cells, however, have severe limitations of low efficiency, inconsistency and lack of sufficient quality control. This EAGER proposal aims to develop a novel human T cell biomanufacturing platform for large-scale, robust, and high-quality cellular production. The accomplishment of this study will provide not only the proof-of-concept but also the ready-to-use bioproduction platform for new means of T cell expansion for clinical immune cancer therapy. The novel technology employed in the rational production process engineering will also be able to provide guidelines and apply easily to the manufacturing of other therapeutic cells. Whereas the results and knowledge obtained in this study will be useful for both the biopharmaceutical industry and academic research, all cancer patients may benefit from the products of this research project. The primary goal of this proposal is to develop an entirely new, metabolic cell process engineering (MCPE)-based, cellular biomanufacturing platform using stirred-tank bioreactor to produce reliable and reproducible large quantities of human T cells for immune cancer therapy, aiming to effectively produce more than 2,000 million T cells with high quality. The traditional T cell biomanufacturing presents several weaknesses: 1) low efficiency of mass transfer that often results in heterologous cellular metabolism, cell viability and product quality; 2) ineffective process parameter control that causes low robustness, reliability and scalability; and 3) lack of critical quality attributes in the early and middle stages of process development, limiting the application of quality by design. This project focuses on developing an innovative stirred-tank-based cellular biomanufacturing platform to produce reliable and reproducible large quantities of human T cells (or CAR T cells) for immune cancer therapy. Supported by Design of Experiment (DoE), proteomics and metabolomics will be applied to evaluate and determine the key bioproduction process parameters (such as stirred-tank parameters, media, supplements, etc.) to control T cell metabolism and cell growth. The oxygen transfer coefficient-based scale-up strategy will be developed to guide large-scale manufacturing of T cells, which will be validated using small- and medium- size tank bioreactors with scale-up factor of 10. In addition, at multiple key steps of the cellular bioproduction, the T cell quality control will be established via monitoring and evaluating cellular density, viability, T cell surface markers and functions. The existing T cell biomanufacturing in flask, LifeCell bag or Wave bag is limited by the weaknesses of lot-to-lot variation, heterologous product quality during scale-up, and low reproducibility. The proposed approach, i.e. MCPE-based fed-batch T cell production in stirred-tank bioreactor, that enables homogenous cell expansion, high cell density, high viability and good product quality in large-scale T cell manufacturing would be a major methodological advance for the field. Moreover, the systems biology approach will help advance the knowledge of host cell protein expression and intracellular metabolite profiling of human T cells under various culture conditions. In addition, the liquid activators in this proposed strategy will avoid heterologous suspension culture, improve cell growth efficiency, simplify manufacturing operation and reduce production cost. The critical scale-up factors learned from this application will guide future large-scale T cell biomanufacturing. Finally, the quality control at multiple stages of the process development will help identify potential product quality and process scale-up pain points during T cell bioproduction. To the PI's best knowledge, this is the first effort to rationally develop T cell bioproduction process via understanding the interaction between cellular metabolism and process parameters.

1645031-Liuin 2015，癌症在美国诊断出了至少50万例死亡和150万例新病例。在临床试验中，具有癌症受体的大量肿瘤浸润T细胞或具有癌症靶向受体的基因工程T细胞的过继转移对消除肿瘤的巨大有望。但是，现有的生产大量这种人类T细胞的方法严重限制了低效率，不一致和缺乏足够的质量控制。这项渴望的建议旨在开发一个新型的人类T细胞生物制造平台，用于大规模，健壮和高质量的细胞生产。这项研究的完成不仅将提供概念证明，而且还提供了用于临床免疫癌症T细胞扩展新手段的现成生物生产平台。合理生产过程工程中采用的新技术也将能够提供指南，并轻松地申请其他治疗细胞的生产。尽管本研究中获得的结果和知识将对生物制药行业和学术研究都有用，但所有癌症患者都可以从该研究项目的产物中受益。该提案的主要目的是开发一种全新的，代谢细胞工艺工程（MCPE），基于搅拌坦克生物反应器的基于基于的细胞生物制造平台，以生产可靠且可再现的大量人类T细胞，以进行免疫癌症治疗，旨在有效地生产超过2000万个具有高质量的T细胞。传统的T细胞生物制造提出了几个弱点：1）低效率的传质通常会导致异源细胞代谢，细胞活力和产品质量； 2）无效的过程参数控制会导致较低的鲁棒性，可靠性和可伸缩性； 3）在过程开发的早期和中间阶段缺乏关键质量属性，从而限制了质量的应用。该项目着重于开发一种创新的基于坦克的细胞生物制造平台，以生产可靠且可重复的大量人T细胞（或CAR T细胞）进行免疫癌症治疗。在实验设计（DOE）的支持下，蛋白质组学和代谢组学将用于评估和确定关键的生物生产过程参数（例如搅拌坦克参数，培养基，补充剂等），以控制T细胞代谢和细胞生长。将制定基于氧气系数的扩大策略来指导T细胞的大规模制造，该策略将使用中小型储罐生物反应器进行验证，其扩展系数为10。此外，在细胞生物生物生产的多个关键步骤中，T细胞质量控制将通过监测和评估细胞密度，Vialular密度，Vibribility，Tybibility，t Cellbility，t Cells Surfactions和功能来建立。现有的T细胞生物制造在烧瓶，Lifecell Bag或Wave Bag中受到限制，在缩放期间较低的差异，异源产品质量以及较低的可重复性。提出的方法，即搅拌坦克生物反应器中基于MCPE的Fed批量T细胞产生，可​​以使大规模T细胞生产中的同质细胞扩张，高细胞密度，高生存能力和良好的产品质量成为该领域的重大方法论进步。此外，系统生物学方法将有助于促进人类T细胞在各种培养条件下的宿主细胞蛋白表达和细胞内代谢产物分析的知识。此外，该提出的策略中的液体激活剂将避免异源悬浮培养物，提高细胞生长效率，简化制造运营并降低生产成本。从该应用中汲取的关键扩展因素将指导未来的大规模T细胞生物制造。最后，过程开发的多个阶段的质量控制将有助于确定T细胞生物生产过程中潜在的产品质量和过程缩放疼痛点。据PI的最佳知识，这是通过了解细胞代谢和过程参数之间的相互作用来合理发展T细胞生物生产过程的第一个努力。