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

2015年，美国至少有50万人死于癌症，150万新发病例。在临床试验中，大量肿瘤浸润性T细胞或具有癌症靶向受体的基因工程T细胞的过继转移在根除肿瘤方面显示出巨大的希望。然而，现有的大量制造这种人类T细胞的方法存在效率低、不一致和缺乏足够的质量控制的严重局限性。这项EAGER提案旨在开发一种新的人类T细胞生物制造平台，用于大规模、稳健和高质量的细胞生产。这项研究的完成不仅将提供概念验证，还将为临床免疫癌症治疗的T细胞扩增新手段提供现成的生物生产平台。在合理的生产过程工程中采用的新技术也将能够为其他治疗细胞的制造提供指导并易于应用。虽然本研究获得的结果和知识将对生物制药工业和学术研究有用，但所有癌症患者都可能从本研究项目的产品中受益。本提案的主要目标是开发一种全新的、基于代谢细胞过程工程（MCPE）的细胞生物制造平台，利用搅拌槽生物反应器生产可靠且可重复的大量用于免疫癌症治疗的人类T细胞，旨在有效生产超过20亿的高质量T细胞。传统的T细胞生物制造存在以下几个缺点：1)传质效率低，经常导致异种细胞代谢、细胞活力和产品质量下降；2)过程参数控制无效，鲁棒性、可靠性和可扩展性较低；3)在工艺开发的早期和中期缺乏关键的质量属性，限制了质量设计的应用。该项目的重点是开发一种创新的基于搅拌槽的细胞生物制造平台，以生产可靠且可重复的大量人类T细胞（或CAR - T细胞），用于免疫癌症治疗。在实验设计（Design of Experiment, DoE）的支持下，将应用蛋白质组学和代谢组学来评估和确定控制T细胞代谢和细胞生长的关键生物生产工艺参数（如搅拌池参数、培养基、添加物等）。将开发基于氧传递系数的放大策略，以指导T细胞的大规模制造，该策略将使用放大系数为10的中小型罐式生物反应器进行验证。此外，在细胞生物生产的多个关键步骤中，将通过监测和评估细胞密度、活力、T细胞表面标记物和功能来建立T细胞质量控制。现有的烧瓶、LifeCell袋或Wave袋中的T细胞生物制造存在批量差异、放大时产品质量异源、重复性低等缺点。所提出的方法，即在搅拌槽生物反应器中以mcpe为基础的进料批量T细胞生产，能够在大规模T细胞制造中实现均匀的细胞扩增，高细胞密度，高活力和良好的产品质量，将是该领域的主要方法进步。此外，系统生物学方法将有助于提高宿主细胞蛋白表达和人类T细胞在各种培养条件下的细胞内代谢物谱的知识。此外，该策略中的液体活化剂可以避免异种悬浮培养，提高细胞生长效率，简化制造操作，降低生产成本。从这一应用中学到的关键放大因素将指导未来大规模的T细胞生物制造。最后，工艺开发多个阶段的质量控制将有助于识别T细胞生物生产过程中潜在的产品质量和工艺放大痛点。据PI所知，这是通过了解细胞代谢和工艺参数之间的相互作用来合理开发T细胞生物生产过程的第一次努力。

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.