通过实现从体外模型系统到治疗性生物工厂的广泛应用,工程化哺乳动物细胞是生物技术的关键。工程化细胞系作为含有细胞克隆的亚谱系的群体存在,所述细胞克隆表现出显著的遗传和表型异质性。对这种克隆间异质性的来源及其对生物技术应用的影响的理解仍然有限。这里,我们为基于靶向整合(TI)的CHO抗体生产细胞系开发过程开发了基因组条形码策略。这项技术提供了关于在池水平上稳定细胞系选择过程中克隆多样性的新见解,在单细胞克隆后实现了独立于成像的单克隆性评估,并最终通过在细胞系发育(CLD)过程中监测细胞系来改善抗体生产者克隆的命中选择。具体来说,我们观察到,由两个质粒在单个基因组位点的TI产生的CHO生产者池显示出低多样性(<0.1%RMCE效率),这进一步取决于表达的分子,在常规培养过程中,种群迅速向优势克隆倾斜。与来自不同TI事件的细胞系相比,来自一个单独TI事件的克隆细胞系在生产相关和表型参数方面表现出显著较低的差异。这意味着观察到的细胞多样性位于预先存在的细胞内在因素中,并且大多数克隆变异在CLD过程中没有发展。特别是在单细胞克隆期间。使用细胞条形码作为细胞多样性的代理,我们改进了我们的CLD筛选工作流程,并显著丰富了生产相关参数的多样性.这项工作,通过实现克隆多样性监测和控制,为具有经济价值和数据驱动的CLD流程铺平了道路。
Engineered mammalian cells are key for biotechnology by enabling broad applications ranging from in vitro model systems to therapeutic biofactories. Engineered cell lines exist as a population containing sub-lineages of cell clones that exhibit substantial genetic and phenotypic heterogeneity. There is still a limited understanding of the source of this inter-clonal heterogeneity as well as its implications for biotechnological applications. Here, we developed a genomic
barcoding strategy for a targeted integration (TI)-based CHO antibody producer cell line development process. This technology provided novel insights about clone diversity during stable cell line selection on pool level, enabled an imaging-independent monoclonality assessment after single cell cloning, and eventually improved hit-picking of antibody producer clones by monitoring of cellular lineages during the cell line development (CLD) process. Specifically, we observed that CHO producer pools generated by TI of two plasmids at a single genomic site displayed a low diversity (< 0.1% RMCE efficiency), which further depends on the expressed molecules, and underwent rapid population skewing towards dominant clones during routine cultivation. Clonal cell lines from one individual TI event demonstrated a significantly lower variance regarding production-relevant and phenotypic parameters as compared to cell lines from distinct TI events. This implies that the observed cellular diversity lies within pre-existing cell-intrinsic factors and that the majority of clonal variation did not develop during the CLD process, especially during single cell cloning. Using cellular barcodes as a proxy for cellular diversity, we improved our CLD screening workflow and enriched diversity of production-relevant parameters substantially. This work, by enabling clonal diversity monitoring and control, paves the way for an economically valuable and data-driven CLD process.