Collaborative Research: GOALI: Exploiting metabolism-apoptosis interactions to enhance mammalian cell culture

包括单克隆抗体（mab）和其他蛋白质疗法在内的生物制药是所有药物中制造成本最高的。哺乳动物细胞培养过程产生了这些化合物的绝大部分，它们代表着每年超过900亿美元的总市场。对单克隆抗体治疗药物的加速需求导致了对提高哺乳动物细胞培养生物过程生产率的迫切需要。然而，向更高的细胞和产物浓度的推进伴随着抑制性代谢物的积累和凋亡细胞死亡的增加，这两者都限制了产物的产量。最近的研究表明，在中国仓鼠卵巢（CHO）细胞中表达抗凋亡基因会导致代谢变化，包括在指数生长后期快速消耗乳酸。这开启了以前无法预见的策略的可能性，可以利用代谢-细胞凋亡相互作用来限制有毒副产物如乳酸和氨的积累。因此，该项目的长期目标是了解代谢和凋亡途径之间的调节联系，以便调节这些过程以增强哺乳动物细胞培养。总体目标是应用系统方法来识别受抗凋亡工程强烈影响的关键代谢节点，并可以靶向增强抗凋亡（ApoR） CHO细胞的单克隆抗体生产。该方法依赖于代谢通量分析（MFA）和定量细胞成像来绘制通过关键细胞内途径的营养物质和信号分子的动态流动。该项目的总体目标将通过追求以下两个具体目标来实现。首先，将确定抗凋亡蛋白过度表达导致CHO细胞乳酸代谢重编程的机制。其次，这种理解将应用于优化培养基和补料批培养条件，以最大限度地提高ApoR克隆的细胞活力和单克隆抗体的产量。拟议的研究具有创新性，因为它旨在制定提高细胞活力和抗体产生的综合策略，同时减少副产物积累，而不是试图单独解决这些问题。这项工作有望通过对代谢-凋亡相互作用的定量理解来填补一个关键的知识空白，从而可以利用它们来提高生物反应器的性能。这项研究具有重要意义，因为它将为提高哺乳动物细胞生物过程的生产力提供新的策略，从而降低药物开发和治疗性抗体的生产成本。该项目还将为PI的研究生提供独特的教育机会。美国实验室与业界科学家进行合作研究。在为期3个月的实习中，学生将在Centocor的生物加工设施中进行实验，这将为学生在生物技术行业的职业生涯或在政府或学术实验室进行行业相关研究提供理想的准备。

Biopharmaceuticals encompassing monoclonal antibodies (mAbs) and other protein therapeutics are among the most expensive of all drugs to manufacture. Mammalian cell culture processes are responsible for producing the vast majority of these compounds, which represent a total market of more than 90 billion dollars annually. The accelerating demand for mAb therapeutics has led to a critical need for enhanced productivity in mammalian cell culture bioprocesses. However, the push toward higher cell and product concentrations has been accompanied by the accumulation of inhibitory metabolites and increased apoptotic cell death, both of which limit product yields. It has recently been shown that expressing anti-apoptosis genes in Chinese hamster ovary (CHO) cells causes a metabolic shift involving rapid lactate consumption during late exponential-phase growth. This opens the possibility of previously unforeseen strategies that can harness metabolism-apoptosis interactions to limit the accumulation of toxic by-products such as lactate and ammonia. The long-term goal of this project is therefore to understand the regulatory connections between metabolic and apoptotic pathways so that these processes can be modulated to enhance mammalian cell culture. The overall objective is to apply systems approaches to identify critical metabolic nodes that are strongly impacted by anti-apoptosis engineering and can be targeted to enhance mAb production in apoptotic-resistant (ApoR) CHO cells. The approach relies upon metabolic flux analysis (MFA) and quantitative cell imaging to map the dynamic flow of nutrients and signaling molecules through key intracellular pathways. The overall objective of this project will be accomplished by pursuing the following two specific aims. First, the mechanism by which overexpression of anti-apoptotic proteins causes reprogramming of lactate metabolism in CHO cells will be determined. Second, this understanding will be applied to optimize media and fed-batch culture conditions to maximize cell viability and mAb production of ApoR clones. The proposed research is innovative because it aims to develop integrated strategies for improving cell viability and antibody production while reducing by-product accumulation, rather than attempting to address these problems individually. This work is expected to fill a critical knowledge gap by contributing a quantitative understanding of metabolism-apoptosis interactions so that they can be exploited to enhance bioreactor performance. The research is significant because it will enable novel strategies for increasing productivity of mammalian cell bioprocesses, thus lowering drug development and production costs of therapeutic antibodies. This project will also provide the unique educational opportunity for a graduate student from the PI?s lab to engage in collaborative research with industry scientists. This will culminate in a 3-month internship in which the student will perform experiments in a Centocor bioprocessing facility, an experience that will provide ideal preparation for a career in the biotech industry, or alternatively to pursue industry-relevant research in a government or academic lab.