An increasing number of engineered therapeutic recombinant proteins with unpredictable manufacturability are currently filling industrial cell line development pipelines. These proteins can be "difficult-to-express" (DTE) in that production of a sufficient quantity of correctly processed recombinant product by engineered mammalian cells is difficult to achieve. In these circumstances, identification of appropriate cell engineering strategies to increase yield is difficult as constraints are cell line and product-specific. Here we describe and validate the development of a high-throughput microscale platform for multiparallel testing of multiple functional genetic components at varying stoichiometry followed by assessment of their effect on cell functional performance. The platform was used to compare and identify optimal cell engineering solutions for both transient and stable production of a model DTE IgG1 monoclonal antibody. We simultaneously tested the functional effect of 32 genes encoding discrete ER or secretory pathway components, each at varying levels of expression and utilized in different combinations. We show that optimization of functional gene load and relative stoichiometry is critical and optimal cell engineering solutions for stable and transient production contexts are significantly different. Our analysis indicates that cell engineering workflows should be cell line, protein product and production-process specific; and that next-generation cell engineering technology that enables precise control of the relative expression of multiple functional genetic components is necessary to achieve this.

中文翻译：

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一个用于CHO细胞生产重组蛋白的针对特定环境的基因工程的平台。

目前，越来越多的具有不可预测的可加工性的工程治疗性重组蛋白正在填补工业细胞系的开发流程。这些蛋白质可以是“难于表达的”（DTE），因为工程化的哺乳动物细胞难以产生足够量的正确加工的重组产物。在这些情况下，很难确定适当的细胞工程策略以提高产量，因为限制条件取决于细胞系和特定产品。在这里，我们描述并验证了高通量微型平台的开发，该平台可用于在不同化学计量比下对多个功能遗传组件进行多重平行测试，然后评估它们对细胞功能性能的影响。该平台用于比较和识别用于模型DTE IgG1单克隆抗体的瞬时和稳定生产的最佳细胞工程解决方案。我们同时测试了32个编码离散ER或分泌途径成分的基因的功能效果，每个基因处于不同的表达水平并以不同的组合使用。我们表明功能基因负载和相对化学计量的优化是至关重要的，并且稳定和瞬时生产环境的最佳细胞工程解决方案存在显着差异。我们的分析表明，细胞工程工作流程应针对细胞系，蛋白质产品和生产过程。