{"title":"Sar1A overexpression in Chinese hamster ovary cells and its effects on antibody productivity and secretion","authors":"","doi":"10.1016/j.jbiosc.2024.05.003","DOIUrl":null,"url":null,"abstract":"<div><p>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.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 2","pages":"Pages 171-180"},"PeriodicalIF":2.3000,"publicationDate":"2024-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of bioscience and bioengineering","FirstCategoryId":"5","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1389172324001324","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
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.
中国仓鼠卵巢(CHO)细胞是用于生产治疗性抗体最广泛的细胞。在细胞系开发过程中,设计分泌过程(如折叠相关蛋白上调)是构建高重组蛋白生产率细胞系的有效方法。然而,有关重组蛋白在内质网(ER)和高尔基体之间转运的研究却很少。在本研究中,Sar1A 是一种参与 COPII 囊泡形成的蛋白质,研究的重点是通过增强 COPII 囊泡介导的抗体从 ER 到高尔基体的转运来提高抗体的生产率,并阐明其对分泌过程的影响。对构建的过表达 Sar1A 的 CHO 细胞系进行了批量培养,结果表明它们的特异性抗体生产率有所提高。使用翻译抑制剂进行追逐实验,并通过免疫荧光成像分析观察了细胞内抗体的积累和特异性定位。结果表明,Sar1A的过表达减少了细胞内抗体的积累,尤其是在ER中。液相色谱-质谱法和 UPR 相关基因表达评估分别分析了工程化抗体转运对抗体糖基化特征和未折叠蛋白反应(UPR)途径的影响。Sar1A 的过表达降低了糖半乳糖基化,并在批次培养结束时诱导了更强的 UPR。Sar1A 过表达通过改变 CHO 细胞的分泌过程提高了其抗体生产率。这种方法不仅有助于生产单克隆抗体,还有助于生产其他需要 COPII 囊泡运输的治疗蛋白。
期刊介绍:
The Journal of Bioscience and Bioengineering is a research journal publishing original full-length research papers, reviews, and Letters to the Editor. The Journal is devoted to the advancement and dissemination of knowledge concerning fermentation technology, biochemical engineering, food technology and microbiology.