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

单克隆抗体（mAbs）和其他蛋白质治疗药物是制造成本最高的药物之一。让这些疗法变得更便宜，更容易为公众所接受，将改善美国和全球数百万患者的健康和生活质量。本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抗产量的策略。在美国，CHO细胞用于生产所有蛋白质治疗剂的60-70%。这些研究将使用杨森研发公司提供的CHO细胞系，该细胞系具有高单克隆抗体生产能力。这种高产细胞系通常无法在学术实验室中获得，因此这次合作提供了一个独特的机会，可以在工业相关的宿主细胞系中测试所提出的代谢工程策略。这项工作意义重大，因为它将为提高哺乳动物细胞生物过程的生产力和一致性提供新方法，从而降低药物开发和治疗性抗体的制造成本。该项目还将为研究生和博士后研究人员提供独特的教育机会，与行业科学家进行合作研究，最终研究生将在杨森研发中心进行为期3个月的实习。这样的经历将为这些受训者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）治疗的加速趋势导致了对提高哺乳动物细胞生物过程生产力的迫切需求。先前nsf赞助的研究发现，与低或不产生的宿主相比，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尚不清楚，也不清楚是否可以合理地设计CAC通量以促进增加单克隆抗体的产生。本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。由于这种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设计CHO细胞以增强CAC通量，同时评估对单克隆抗体滴度、细胞特异性产率（CSPR）和聚糖谱的影响。首先，由杨森公司提供的工业CHO主机系列将被设计成组成性上调氧化CAC代谢。特定的线粒体调控蛋白会过表达，13C代谢通量分析（MFA）将指导局部途径工程，进一步增强CAC通量。其次，利用诱导表达载体在固定阶段将碳通量动态重定向到CAC。工作假设是，在固定期开始时诱导线粒体代谢将提高CSPR，同时使培养物在指数期达到峰值细胞密度，从而最大化最终的单抗滴度。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。这项拟议研究的基本原理是，它将确定是否可以设计CHO细胞的中心碳代谢，以提高单克隆抗体的产量，同时保持一致的产品质量。该项目由CBET部门的生物技术和生化工程项目、工业创新和伙伴关系部门的GOALI项目以及分子和细胞生物科学部的系统和合成生物学项目共同资助。

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.