Abstract：
Chinese hamster ovary (CHO) cells are the most widely used for therapeutic antibody production. In cell line development, engineering secretion processes such as folding-related protein upregulation is an effective way of constructing cell lines with high recombinant protein productivity. However, there have been few studies on the transport of recombinant proteins between the endoplasmic reticulum (ER) and the Golgi apparatus. In this study, Sar1A, a protein involved in COPII vesicle formation, was focused on to improve antibody productivity by enhancing COPII vesicle-mediated antibody transport from the ER to the Golgi apparatus, and to clarify its effect on the secretion process. The constructed Sar1A-overexpressing CHO cell lines were batch-cultured, in which they showed an increased specific antibody production rate. The intracellular antibody accumulation and the specific localization of the intracellular antibodies were investigated by chase assay using a translation inhibitor and observed by immunofluorescence-based imaging analysis. The results showed that Sar1A overexpression reduced intracellular antibody accumulation, especially in the ER. The effects of the engineered antibody transport on the antibody\'s glycosylation profile and the unfolded protein response (UPR) pathway were analyzed by liquid chromatography-mass spectrometry and UPR-related gene expression evaluation, respectively. Sar1A overexpression lowered glycan galactosylation and induced a stronger UPR at the end of the batch culture. Sar1A overexpression enhanced the antibody productivity of CHO cells by modifying their secretion process. This approach could also contribute to the production of not only monoclonal antibodies but also other therapeutic proteins that require transport by COPII vesicles.
摘要：
中国仓鼠卵巢(CHO)细胞最广泛地用于产生治疗性抗体。在细胞系发育中，工程分泌过程，如折叠相关蛋白上调是构建具有高重组蛋白生产率的细胞系的有效方法。然而,关于内质网（ER）和高尔基体之间的重组蛋白转运的研究很少。在这项研究中,Sar1A,一种参与COPII囊泡形成的蛋白质，致力于通过增强COPII囊泡介导的抗体从ER到高尔基体的转运来提高抗体生产率，并阐明其对分泌过程的影响。将构建的Sar1A过表达CHO细胞系分批培养，其中它们显示出增加的特异性抗体产生率。通过使用翻译抑制剂的追踪测定研究了细胞内抗体的积累和细胞内抗体的特异性定位，并通过基于免疫荧光的成像分析进行了观察。结果表明，Sar1A过表达减少了细胞内抗体的积累，尤其是在急诊室。通过液相色谱-质谱和UPR相关基因表达评估分析了工程化抗体转运对抗体糖基化谱和未折叠蛋白反应(UPR)途径的影响。分别。Sar1A过表达降低了聚糖半乳糖基化并在分批培养结束时诱导了更强的UPR。Sar1A过表达通过改变CHO细胞的分泌过程来增强其抗体生产力。这种方法不仅有助于单克隆抗体的产生，还有助于需要通过COPII囊泡转运的其他治疗性蛋白质的产生。
