PDF(Pubmed)
Abstract：
Secretion levels required of industrial Chinese hamster ovary (CHO) cell lines can challenge endoplasmic reticulum (ER) homeostasis, and ER stress caused by accumulation of misfolded proteins can be a bottleneck in biomanufacturing. The unfolded protein response (UPR) is initiated to restore homeostasis in response to ER stress, and optimization of the UPR can improve CHO cell production of therapeutic proteins. We compared the fed-batch growth, production characteristics, and transcriptomic response of an immunoglobulin G1 (IgG1) producer to its parental, non-producing host cell line. We conducted differential gene expression analysis using high throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) to study the ER stress response of each cell line during fed-batch culture. The UPR was activated in the IgG1 producer compared to the host cell line and our analysis of differential expression profiles indicated transient upregulation of ATF6α target mRNAs in the IgG1 producer, suggesting two upstream regulators of the ATF6 arm of the UPR, ATF6β and WFS1, are rational engineering targets. Although both ATF6β and WFS1 have been reported to negatively regulate ATF6α, this study shows knockdown of either target elicits different effects in an IgG1-producing CHO cell line. Stable knockdown of ATF6β decreased cell growth without decreasing titer; however, knockdown of WFS1 decreased titer without affecting growth. Relative expression measured by qPCR indicated no direct relationship between ATF6β and WFS1 expression, but upregulation of WFS1 in one pool was correlated with decreased growth and upregulation of ER chaperone mRNAs. While knockdown of WFS1 had negative impacts on UPR activation and product mRNA expression, knockdown of ATF6β improved the UPR specifically later in fed-batch leading to increased overall productivity.
摘要：
工业中国仓鼠卵巢（CHO）细胞系所需的分泌水平可以挑战内质网（ER）稳态，错误折叠蛋白的积累引起的内质网压力可能是生物制造的瓶颈。未折叠的蛋白质反应（UPR）启动以恢复内质网应激的稳态，优化UPR可以提高CHO细胞产生治疗性蛋白。我们比较了分批补料生长，生产特点,和免疫球蛋白G1（IgG1）生产者对其亲本的转录组反应，非生产宿主细胞系。我们使用高通量RNA测序（RNASeq）和定量聚合酶链反应（qPCR）进行了差异基因表达分析，以研究补料分批培养过程中每个细胞系的ER应激反应。与宿主细胞系相比，IgG1生产者中的UPR被激活，我们对差异表达谱的分析表明，IgG1生产者中ATF6α靶mRNA的瞬时上调，建议UPRATF6部门的两个上游监管机构，ATF6β和WFS1是合理的工程目标。虽然ATF6β和WFS1都有负调控ATF6α的报道，这项研究表明，任一靶标的敲除在产生IgG1的CHO细胞系中引起不同的作用。ATF6β的稳定敲低降低了细胞生长而不降低滴度;然而，WFS1的敲低降低了滴度而不影响生长。通过qPCR测量的相对表达表明ATF6β和WFS1表达之间没有直接关系，但是一个池中WFS1的上调与ER伴侣mRNA的生长减少和上调相关。WFS1敲低对UPR激活和产物mRNA表达有负面影响，ATF6β的敲低特别是在补料分批中提高了UPR，从而提高了整体生产率。
