Orellana Camila A, Marcellin Esteban, Gray Peter P, Nielsen Lars K
Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD 4072, Australia.
Biotechnol Bioeng. 2017 Aug;114(8):1825-1836. doi: 10.1002/bit.26316. Epub 2017 May 8.
For decades, Chinese hamster ovary (CHO) cells have been the preferred host for therapeutic monoclonal antibody (mAb) production; however, increasing mAb titer by rational engineering remains a challenge. Our previous proteomic analysis in CHO cells suggested that a higher content of glutathione (GSH) might be related to higher productivity. GSH is an important antioxidant, cell detoxifier, and is required to ensure the formation of native disulfide bonds in proteins. To investigate the involvement of GSH in mAb production, we generated stable CHO cell lines overexpressing genes involved in the first step of GSH synthesis; namely the glutamate-cysteine ligase catalytic subunit (Gclc) and the glutamate-cysteine ligase modifier subunit (Gclm). The two genes were reconstructed from our RNA-Seq de novo assembly and then were functionally annotated. Once the sequences of the genes were confirmed using proteogenomics, a transiently expressed mAb was introduced into cell lines overexpressing either Gclc or Gclm. The new cell lines were compared for mAb production to the parental cell line and changes at the proteome level were measured using SWATH. As per our previous proteomics observations, overexpressing Gclm improved productivity, titer, and the frequency of high producer clones by 70%. In contrast, overexpressing Gclc, which produced a higher amount of GSH, did not increase mAb production. We show that GSH cannot be linked to higher productivity and that Gclm may be controlling other cellular processes involved in mAb production yet to be elucidated. Biotechnol. Bioeng. 2017;114: 1825-1836. © 2017 Wiley Periodicals, Inc.
几十年来，中国仓鼠卵巢（CHO）细胞一直是生产治疗性单克隆抗体（mAb）的首选宿主；然而，通过合理工程提高mAb滴度仍然是一项挑战。我们之前对CHO细胞的蛋白质组学分析表明，较高的谷胱甘肽（GSH）含量可能与更高的生产力有关。GSH是一种重要的抗氧化剂、细胞解毒剂，是确保蛋白质中天然二硫键形成所必需的。为了研究GSH在mAb生产中的作用，我们构建了稳定的CHO细胞系，这些细胞系过表达参与GSH合成第一步的基因，即谷氨酸-半胱氨酸连接酶催化亚基（Gclc）和谷氨酸-半胱氨酸连接酶调节亚基（Gclm）。这两个基因是从我们的RNA-Seq从头组装中重建的，然后进行了功能注释。一旦使用蛋白质基因组学确认了基因序列，就将瞬时表达的mAb引入过表达Gclc或Gclm的细胞系中。将新的细胞系与亲本细胞系进行mAb生产比较，并使用SWATH测量蛋白质组水平的变化。根据我们之前的蛋白质组学观察，过表达Gclm可使生产力、滴度和高产克隆频率提高70%。相比之下，过表达产生更高量GSH的Gclc并没有增加mAb的产量。我们表明，GSH与更高的生产力无关，并且Gclm可能在控制mAb生产中尚未阐明的其他细胞过程。生物技术与生物工程。2017年；114：1825 - 1836。©2017威利期刊公司。
