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

描述（由申请人提供）：这项SBIR一期研究的总体目标是利用我们的综合建模和实验方法来计算鉴定和实验开发一种新的中国仓鼠卵巢（CHO）细胞系选择系统。这项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合理设计哺乳动物细胞系蛋白质生产中的新型选择系统中的实用性。选择高产的哺乳动物细胞系是生物制药生产过程发展的瓶颈。在哺乳动物细胞系中，治疗性蛋白的生产一直以使用具有代谢起源的选择标记为主导。然而，目前的选择方法受到一些缺点的阻碍，包括冗长的开发时间和成本。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识别CHO细胞系的新选择系统，并通过实验实现最有希望和最有利的候选系统来验证我们的方法。我们将分三个阶段进行这个概念验证研究。在Aim I中，我们将使用CHO代谢的计算模型来确定必要的代谢反应，这些代谢反应是设计新颖和优越的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，包括新选择系统预测的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Aim II中，我们将通过实验在CHO-S细胞系中实现最佳候选选择系统：(a)创建一个缺陷营养克隆，(b)用包含抗体表达基因的选择载体瞬时转染细胞，以及(c)根据缺陷营养选择产生蛋白质的细胞系。在Aim 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来评估CHO细胞中基于模型的选择系统的开发和实施。这项验证研究的成功完成将为我们的集成平台在哺乳动物蛋白质生产中设计新的和优越的代谢选择系统提供科学和技术可行性的证明。

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.