Cell Culture Technology, Technical Department, Bielefeld University, Bielefeld, Germany.
Center for Biotechnology, CeBiTec, Bioinformatics Resource Facility, Bielefeld University, Bielefeld, Germany.
Appl Microbiol Biotechnol. 2019 Oct;103(19):8127-8143. doi: 10.1007/s00253-019-10020-z. Epub 2019 Aug 16.
Chinese hamster ovary (CHO) cells are commonly used for the production of monoclonal antibodies. Omics technologies have been used to elucidate cellular switch points which result in higher monoclonal antibody (mAb) productivity and process yields in CHO and other biopharmaceutical production cell lines such as human or mouse. Currently, investigations of the phosphoproteome in CHO cell lines are rare yet could provide further insights into cellular mechanisms related to target product expression. Therefore, we investigated CHO IGF-signaling events using a comparative expression and phosphoproteomic approach in recombinant mAb-producing XL99 cell lines and corresponding parental strain. Differences were found on the level of protein expression between producer and parental cells in the exponential growth phase, mainly in proteins related to the lysosome, oligosaccharide metabolic processes, stress response, and cellular homeostasis. Within a stable isotope labeling by amino acids in cell culture (SILAC)-based phosphoproteomic investigation of IGF signaling, expected general regulation of phosphorylation sites and cell line-specific responses were observed. Detected early phosphorylation events can be associated to observed effects of IGF on cellular growth, metabolism, and cell cycle distribution. Producer cell line-specific signaling exhibited differences to parental cells in intracellular trafficking and transcriptional processes, along with an overall lower amount of observable cross talk to other signaling pathways. By combining label-free and SILAC-based expression for phosphoproteomic analyses, cellular differences in the highly interactive levels of signaling and protein expression were detected, indicating alterations in metabolism and growth following treatment with an exogenous growth factor. The characterization of cell lines and effects of IGF addition resulted in identification of metabolic switch points. With this data, it will be possible to modulate pathways towards increased CHO process yield by targeted application of small-molecule inhibitors.
中国仓鼠卵巢（CHO）细胞常用于生产单克隆抗体。组学技术已被用于阐明细胞转换点，这导致 CHO 和其他生物制药生产细胞系（如人或鼠）中的单克隆抗体（mAb）生产力和过程产量更高。目前，对 CHO 细胞系磷酸蛋白质组的研究很少，但可能进一步深入了解与靶标产品表达相关的细胞机制。因此，我们使用比较表达和磷酸蛋白质组学方法在重组 mAb 产生的 XL99 细胞系及其相应的亲本株中研究 CHO IGF 信号事件。在指数生长期，在产生细胞和亲本细胞之间发现了蛋白质表达水平的差异，主要在与溶酶体、寡糖代谢过程、应激反应和细胞内稳态相关的蛋白质中。在基于稳定同位素标记的细胞培养物中的氨基酸（SILAC）的 IGF 信号磷酸蛋白质组学研究中，观察到预期的磷酸化位点的一般调节和细胞系特异性反应。检测到的早期磷酸化事件可以与 IGF 对细胞生长、代谢和细胞周期分布的观察到的影响相关联。与亲本细胞相比，产生细胞系特异性信号显示在细胞内运输和转录过程中有差异，并且与其他信号通路的可观察到的交叉对话总体上较少。通过结合无标记和 SILAC 基于表达的磷酸蛋白质组学分析，检测到信号和蛋白质表达的高度交互水平的细胞差异，表明在添加外源性生长因子后代谢和生长发生改变。细胞系的特征和 IGF 添加的影响导致了代谢转换点的鉴定。有了这些数据，通过靶向应用小分子抑制剂，可以调节途径以提高 CHO 过程产量。
