National Research Council Canada, 6100 Royalmount Avenue, Montréal, QC H4P 2R2, Canada; Département de microbiologie et immunologie, Faculté de médecine, Université de Montréal, QC, Canada.
National Research Council Canada, 6100 Royalmount Avenue, Montréal, QC H4P 2R2, Canada.
J Biotechnol. 2019 Apr 20;296:32-41. doi: 10.1016/j.jbiotec.2019.03.009. Epub 2019 Mar 15.
Chinese hamster ovary (CHO) cells are the most widely used mammalian host for industrial-scale production of monoclonal antibodies (mAbs) and other protein biologics. Isolation of rare high-producing CHO cell lines from heterogeneous populations of stable transfectants is a daunting task and delays the process of manufacturing of novel biologics. A variety of factors that contribute to the low frequency of high-producing clones have been described; however, the impact of metabolic burden and other stresses (eg. ER stress) associated with sustained high-level expression of recombinant protein (r-protein) during selection of stable transfectants has not been fully appreciated. CHO cell line development has not traditionally received much optimization in this area because the vast majority of platforms use constitutive expression systems to produce biologics. Previously, we developed a cell line (CHO) containing a robust inducible expression system, based on the cumate gene switch, that allows r-protein expression to be down-regulated during selection. Using this switch, we generated inducible CHO pools expressing an Fc-fusion protein within two weeks of transfection with volumetric productivity of up to 1.1 g/L at 17 days post-induction in a fed-batch culture process. Herein, we show that the ability to regulate r-protein expression during pool generation confers a substantial advantage for selecting high-producing stable clones. Reducing expression levels ("off-state") during pool selection dramatically enhances high-producer frequency compared to a pool in which expression was maintained at a high level during selection ("on-state", mimicking a constitutive expression system). Overexpression of the r-protein during the pool selection process negatively affects pool recovery and is associated with subtle but significant increases in BiP expression and cell death compared to pool selection in the "off-state". Our data shows that the cumate gene switch is a valuable platform for stable clone generation and supports the wider application of inducible systems for scalable production of biologics in CHO cells.
中国仓鼠卵巢（CHO）细胞是用于工业规模生产单克隆抗体（mAb）和其他蛋白生物制剂的最广泛使用的哺乳动物宿主。从稳定转染子的异质群体中分离出罕见的高产 CHO 细胞系是一项艰巨的任务，会延迟新型生物制剂的制造过程。已经描述了许多导致高产克隆低频率的因素；然而，在选择稳定转染子时，与重组蛋白（r-蛋白）持续高水平表达相关的代谢负担和其他应激（例如内质网应激）对高产克隆的影响尚未得到充分认识。CHO 细胞系的开发在这方面并没有得到太多优化，因为绝大多数平台都使用组成型表达系统来生产生物制剂。以前，我们开发了一种基于 cumate 基因开关的稳健诱导表达系统的细胞系（CHO），该系统允许在选择过程中下调 r-蛋白表达。使用该开关，我们在两周内生成了表达 Fc 融合蛋白的诱导型 CHO 池，在 fed-batch 培养过程中，诱导 17 天后的体积生产率高达 1.1 g/L。在此，我们表明，在池生成过程中调节 r-蛋白表达的能力为选择高产稳定克隆提供了很大的优势。与在选择过程中维持高水平表达的池（“开状态”，模拟组成型表达系统）相比，在池选择过程中降低表达水平（“关状态”）可显著提高高产率的频率。在池选择过程中 r-蛋白的过表达会对池的恢复产生负面影响，并与比池选择在“关状态”时的 BiP 表达和细胞死亡的细微但显著增加相关。我们的数据表明，cumate 基因开关是稳定克隆生成的有价值的平台，并支持在 CHO 细胞中广泛应用诱导型系统进行可扩展的生物制剂生产。
