Design and Development of Model-based Selection Systems in Mammalian Cell Lines

哺乳动物细胞系中基于模型的选择系统的设计和开发

• 批准号: 7746786
• 负责人: Renata Usaite Black
• 金额: $ 22.27万
• 依托单位: GT LIFE SCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7746786
• 关键词: Alleles; Ammonia; Antibodies; Antibody Formation; Biological Products; Biological Sciences; Cell Culture System; Cell Culture Techniques; Cell Line; Cell physiology; Cells; Characteristics; Chinese Hamster; Chinese Hamster Ovary Cell; Client; Cloning; Computer Simulation; Data; Development; Dihydrofolate Reductase; Disadvantaged; Engineering; Ensure; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Experimental Models; FDA approved; Gene Amplification; Gene Targeting; Genes; Genome; Glutamate-Ammonia Ligase; Goals; Growth; Hand functions; Healthcare; Housing; Human; Hybridomas; Knowledge; Letters; Licensing; Mammalian Cell; Marketing; Measures; Medicine; Metabolic; Metabolism; Methods; Methotrexate; Modeling; Mus; Nucleic Acids; Ovary; Patients; Pattern; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phase I Clinical Trials; Phenotype; Physiology; Population; Population Heterogeneity; Post-Translational Protein Processing; Price; Process; Production; Productivity; Proteins; Provider; Public Health; Reaction; Recombinant Proteins; Recombinants; Research; Screening procedure; Series; Services; Simulate; Site; Small Business Innovation Research Grant; Specificity; Staging; System; Systems Biology; Techniques; Technology; Testing; Therapeutic; Time; TimeLine; Transfection; Zinc Fingers; auxotrophy; base; bioprocess; candidate selection; cellular engineering; computerized tools; cost; deletion analysis; design; feeding; flasks; implantation; improved; meetings; model development; models and simulation; next generation; novel; novel therapeutics; nuclease; product development; programs; protein folding; simulation; success; therapeutic protein; uptake; validation studies; vector

DESCRIPTION (provided by applicant): The overall goal of this SBIR Phase I study is utilize our integrated modeling and experimental approach to computationally identify and experimentally develop a novel selection system in Chinese Hamster Ovary (CHO) cell line. The successful completion of this proof-of-concept study will demonstrate the utility of a systems biology based approach in rational design of novel selection systems in mammalian cell line protein production. Selection of high-producing mammalian cell lines represents a bottleneck in process development for the production of biopharmaceuticals. Production of therapeutic proteins in mammalian cell lines has been dominated by the use of selection markers that have metabolic origin. However, the current selection methods are hampered by a number of disadvantages, including extensive development timelines and cost. Our systems biology approach utilizes the knowledge of a whole cell metabolism and is capable to provide rational designs for identifying new selection systems. We aim to utilize our integrated computational and experimental approach to identify novel selection systems in CHO cell line and experimentally implement the most promising and advantageous candidate to validate our approach. We will perform this proof-of-concept study in three stages. In Aim I, we will identify essential metabolic reactions that are candidate targets for designing novel and superior selection systems using a computational model of CHO metabolism. We will rank-order and prioritize the candidate targets based on a number of criteria, including the predicted stringent specificity of the new selection system and improved cell physiology using our in-house computational tools. In Aim II, we will experimentally implement the top candidate selection system in the CHO-S cell line by: (a) creating an auxotrophic clone, (b) transiently transfecting cells with a selection vector that includes an antibody-expressing gene, and (c) selecting protein producing cell lines based on their auxotrophy. In Aim III, we will evaluate the development and implementation of a model-based selection system in CHO cells by comparing experimentally generated cell culture data with those calculated by the reconstructed model. Successful completion of this validation study will provide a demonstration of the scientific and technical feasibility of our integrated platform for design of new and superior metabolic selection systems in mammalian based protein production. PUBLIC HEALTH REVELANCE: Protein-based therapeutic products have contributed immensely to health care and constitute a large and growing percentage of the total pharmaceutical market. The majority of these FDA approved products are currently manufactured using mammalian cell culture systems. Mammalian cell line development for therapeutic protein production is an expensive and lengthy process. The need to engineer better performing selection systems to select stable protein producer cell lines calls for the use of a rational approach that combines modeling technologies with established experimental techniques to fundamentally change the way selection systems are developed. Reducing the cost and timelines of high producing cell lines using a superior selection system would ensure that the manufacturing of the next generation of medicines can be created in amounts large enough to meet patients' needs and at a price low enough that patients can afford them.

描述（由申请方提供）：本SBIR I期研究的总体目标是利用我们的综合建模和实验方法，在中国人卵巢（CHO）细胞系中通过计算鉴定和实验开发一种新型选择系统。这一概念验证研究的成功完成将证明基于系统生物学的方法在哺乳动物细胞系蛋白质生产中合理设计新型选择系统中的实用性。高产哺乳动物细胞系的选择代表了生物药物生产工艺开发中的瓶颈。哺乳动物细胞系中治疗性蛋白质的生产一直由具有代谢来源的选择标记的使用主导。然而，目前的选择方法受到许多缺点的阻碍，包括广泛的开发时间表和成本。我们的系统生物学方法利用了全细胞代谢的知识，能够为识别新的选择系统提供合理的设计。我们的目标是利用我们的综合计算和实验方法来确定新的选择系统在CHO细胞系和实验实施最有前途的和有利的候选人，以验证我们的方法。我们将分三个阶段进行这项概念验证研究。在目的I中，我们将确定基本的代谢反应，这些反应是使用CHO代谢的计算模型设计新颖和上级选择系统的候选目标。我们将根据一系列标准对候选靶点进行排序和优先排序，包括新选择系统的预测严格特异性和使用我们内部计算工具改进的细胞生理学。在目的II中，我们将通过以下方式在CHO-S细胞系中实验性地实施最佳候选选择系统：（a）创建营养缺陷型克隆，（B）用包含抗体表达基因的选择载体瞬时转染细胞，以及（c）基于其营养缺陷型选择产生蛋白质的细胞系。在目标III中，我们将通过比较实验生成的细胞培养数据与重建模型计算的数据，评估CHO细胞中基于模型的选择系统的开发和实施。本验证研究的成功完成将证明我们的集成平台在哺乳动物蛋白质生产中设计新型和上级代谢选择系统的科学和技术可行性。 公共卫生部门：基于蛋白质的治疗产品对医疗保健做出了巨大贡献，并在整个医药市场中占很大比例，而且还在不断增长。这些FDA批准的产品中的大多数目前使用哺乳动物细胞培养系统生产。用于治疗性蛋白质生产的哺乳动物细胞系开发是昂贵且漫长的过程。需要设计更好的选择系统来选择稳定的蛋白质生产细胞系，需要使用合理的方法，将建模技术与已建立的实验技术相结合，从根本上改变选择系统的开发方式。使用上级选择系统降低高产细胞系的成本和时间表将确保下一代药物的制造可以以足够大的量产生以满足患者的需求，并且价格足够低以使患者能够负担得起。