Collaborative Research: GOALI: Metabolic Engineering of Next Generation CHO Hosts for Monoclonal Antibody Production

合作研究：GOALI：用于单克隆抗体生产的下一代 CHO 宿主的代谢工程

• 批准号: 1604527
• 负责人: Michael Betenbaugh
• 金额: $ 20万
• 依托单位: Johns Hopkins University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1604527&HistoricalAwards=false
• 关键词: Collaborative; Research; GOALI; Metabolic; Engineering

1604426/1604527Young, Jamey D./Betenbaugh, MichaelMonoclonal antibodies (mAbs) and other protein therapeutics are among the most expensive of all drugs to manufacture. Making these therapies more affordable and available to the public will improve both the health and quality of life of millions of patients in the U.S. and around the globe. The proposed research aims to identify strategies for improving mAb production by engineering the metabolism of Chinese hamster ovary (CHO) cells. CHO cells are used to produce 60-70% of all protein therapeutics in the US. The studies will use a CHO cell line provided by Janssen R&D that is capable of high mAb productivity. High-producing cell lines of this kind are not typically available to academic labs, and therefore this collaboration provides a unique opportunity to test the proposed metabolic engineering strategies in an industrially relevant host line. This work is significant because it will enable novel approaches for enhancing the productivity and consistency of mammalian cell bioprocesses, thus lowering drug development and manufacturing costs of therapeutic antibodies. This project will also provide the unique educational opportunity for a graduate student and post-doctoral researcher to engage in collaborative research with industry scientists, culminating in a 3-month internship in which the graduate student will perform experiments in a Janssen R&D facility. Such an experience will provide these trainees with ideal preparation for a career in the biotechnology industry or in a government or academic lab. The accelerating trend toward highly targeted monoclonal antibody (mAb) therapeutics has led to a critical need for enhanced productivity in mammalian cell bioprocesses. Previous NSF-sponsored research has found that high-producing CHO cell lines consistently exhibit enhanced citric acid cycle (CAC) activity compared to low- or non-producing hosts. However, the extent to which this metabolic phenotype is required to drive high-yield protein expression is still unclear, and it is unknown whether CAC flux can be rationally engineered to promote increased mAb production. The long-term goal of this research is to identify metabolic engineering strategies that promote a high-productivity metabolic phenotype in mammalian hosts leading to increased product yield and quality. Because this phenotype is expected to involve up-regulation of mitochondrial oxidative metabolism, the overall objective of the current application is to engineer CHO cells to enhance CAC flux while assessing the impacts on mAb titer, cell specific production rate (CSPR), and glycan profile. First, an industrial CHO host line provided by Janssen R&D will be engineered to constitutively up-regulate oxidative CAC metabolism. A specific mitochondrial regulatory protein will be overexpressed and 13C metabolic flux analysis (MFA) will be applied to guide local pathway engineering to further enhance CAC flux. Second, an inducible expression vector will be used to dynamically redirect carbon flux into CAC during stationary phase. The working hypothesis is that induction of mitochondrial metabolism at the onset of stationary phase will enhance CSPR while enabling the culture to reach peak cell density during exponential phase, thus maximizing final mAb titer. Third, IgG glycan profiles will be assessed to determine how manipulating CHO central metabolism impacts product glycosylation. The rationale for the proposed research is that it will determine whether central carbon metabolism of CHO cells can be engineered to drive increased mAb production while maintaining consistent product quality.This project is co-funded by the Biotechnology and Biochemical Engineering Program of the CBET Division, by the GOALI Program of the Division of Industrial Innovation and Partnerships and by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biosciences.

1604426/1604527Young、Jamey D./Betenbaugh、Michael 单克隆抗体 (mAb) 和其他蛋白质治疗药物是所有药物中制造成本最昂贵的药物。使这些疗法更加经济实惠并可供公众使用，将改善美国和全球数百万患者的健康和生活质量。拟议的研究旨在通过改造中国仓鼠卵巢（CHO）细胞的代谢来确定提高单克隆抗体产量的策略。在美国，60-70% 的蛋白质治疗药物均使用 CHO 细胞生产。这些研究将使用 Janssen R&D 提供的 CHO 细胞系，该细胞系具有高单克隆抗体生产力。学术实验室通常无法获得这种高产细胞系，因此这次合作提供了一个独特的机会，可以在工业相关宿主系中测试所提出的代谢工程策略。这项工作意义重大，因为它将提供提高哺乳动物细胞生物过程的生产率和一致性的新方法，从而降低治疗性抗体的药物开发和制造成本。该项目还将为研究生和博士后研究员提供独特的教育机会，让他们与行业科学家进行合作研究，最终获得为期 3 个月的实习，研究生将在杨森研发设施中进行实验。这样的经历将为这些学员在生物技术行业或政府或学术实验室的职业生涯提供理想的准备。高度靶向单克隆抗体 (mAb) 疗法的加速趋势导致迫切需要提高哺乳动物细胞生物过程的生产力。之前 NSF 资助的研究发现，与低产或不产宿主相比，高产 CHO 细胞系始终表现出增强的柠檬酸循环 (CAC) 活性。然而，这种代谢表型在多大程度上需要驱动高产蛋白表达仍不清楚，并且尚不清楚是否可以合理地设计CAC通量以促进单克隆抗体产量的增加。这项研究的长期目标是确定代谢工程策略，促进哺乳动物宿主的高生产力代谢表型，从而提高产品产量和质量。 由于该表型预计涉及线粒体氧化代谢的上调，因此当前应用的总体目标是改造 CHO 细胞以增强 CAC 通量，同时评估对 mAb 滴度、细胞比生产率 (CSPR) 和聚糖谱的影响。首先，杨森研发中心提供的工业 CHO 宿主系将被设计为组成性上调氧化 CAC 代谢。特定的线粒体调节蛋白将被过度表达，并应用 13C 代谢通量分析 (MFA) 来指导局部通路工程，以进一步增强 CAC 通量。其次，诱导型表达载体将用于在稳定期动态地将碳通量重定向到 CAC 中。工作假设是，在稳定期开始时诱导线粒体代谢将增强 CSPR，同时使培养物在指数期达到峰值细胞密度，从而最大化最终的 mAb 滴度。第三，将评估 IgG 聚糖谱，以确定操纵 CHO 中心代谢如何影响产物糖基化。拟议研究的基本原理是，它将确定是否可以对 CHO 细胞的中心碳代谢进行改造，以提高 mAb 产量，同时保持稳定的产品质量。该项目由 CBET 部门的生物技术和生化工程项目共同资助，由工业创新与合作司的 GOALI 计划以及分子和细胞生物科学司的系统和合成生物学计划提供。