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，MichaelMonoclonal抗体（mAB）和其他蛋白质疗法是制造所有药物中最昂贵的。使这些疗法更加负担得起，公众可用，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工程化中国仓鼠卵巢（CHO）细胞的代谢来确定改善MAB生产的策略。 CHO细胞用于在美国产生60-70％的蛋白质治疗剂。这些研究将使用Janssen R＆D提供的CHO细胞系，该细胞系能够具有很高的MAB生产率。这种类型的高产生细胞系通常不适用于学术实验室，因此，这种合作提供了一个独特的机会，可以在工业相关的宿主线上测试拟议的代谢工程策略。这项工作很重要，因为它将实现新的方法来提高哺乳动物细胞生物处理的生产率和一致性，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，以与行业科学家进行合作研究，最终在3个月的实习中进行，研究生将在Janssen R＆D设施中进行实验。这样的经验将为这些受训者提供理想的准备工作，以为生物技术行业或政府或学术实验室的职业做准备。高度靶向单克隆抗体（MAB）疗法的加速趋势导致了哺乳动物细胞生物过程提高生产率的迫切需求。以前的NSF赞助的研究发现，与低生产或非生产宿主相比，高生产的CHO细胞系始终表现出增强的柠檬酸周期（CAC）活性。但是，这种代谢表型在驱动高收益蛋白表达所需的程度尚不清楚，并且尚不清楚CAC通量是否可以合理地设计以促进MAB的产生增加。这项研究的长期目标是确定代谢工程策略，以促进哺乳动物宿主的高生产率代谢表型，从而提高产品产量和质量。 因为预计该表型将涉及线粒体氧化代谢的上调，所以当前应用的总体目标是工程师CHO细胞增强CAC通量，同时评估对MAB滴度，细胞特异性生产率（CSPR）和Glycan概况的影响。首先，由Janssen研发提供的工业CHO宿主系列将经过设计，以构成上调氧化CAC代谢。特定的线粒体调节蛋白将过表达，并将13C代谢通量分析（MFA）应用于指导局部途径工程以进一步增强CAC通量。其次，在固定阶段，将使用诱导表达载体将碳通量动态重定向到CAC。工作假设是，在固定相开始时，线粒体代谢的诱导将增强CSPR，同时使培养物在指数阶段达到峰值细胞密度，从而最大程度地提高最终mAb滴度。第三，将评估IgG聚糖曲线，以确定操纵中央代谢如何影响糖基化。拟议的研究的基本原理是，它将确定CHO细胞中的中央碳代谢是否可以设计以推动MAB的生产增加，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物化学工程计划共同资助，该项目是由工业创新和群落计划和群群体群体和群落计划的靶向群体计划和群落计划的群体计划以及合成的群落计划和cy;生物科学。