Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells
Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells
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DOI：
10.1002/bit.24365
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发表时间：
2012-04-01
影响因子：
3.8
通讯作者：
Frye, Christopher C.
中科院分区：
文献类型：
--
作者：
Fan, Lianchun;Kadura, Ibrahim;Frye, Christopher C.
尽管中国仓鼠卵巢（CHO）细胞以其独特的特性已成为生产治疗性重组蛋白的主要主力，但CHO细胞系产生（CLG）的主要挑战之一是如何有效地从大量低生产性和非生产性克隆中鉴定那些稀有的高产克隆。通常需要筛选几百个单独的克隆以鉴定具有可接受的生产力和生长特征的商业克隆细胞系，从而使细胞系适合于商业应用。这种低效率使得CLG的过程既耗时又费力。目前，有两种主要的CHO表达系统，基于二氢叶酸还原酶（DHFR）的甲氨蝶呤（MTX）选择和基于谷氨酰胺合成酶（GS）的甲硫氨酸亚砜亚胺（MSX）选择，其已在工业上广泛使用。由于GS-CHO系统中重组细胞系的选择是基于表达质粒引入的GS基因的表达与添加GS抑制剂L-MSX之间的平衡，因此亲本CHOK 1 SV细胞中内源GS基因的GS表达可能会干扰选择过程。为了研究内源GS表达对选择效率的潜在影响，使用设计用于特异性靶向内源CHO GS基因的锌指核酸酶（ZFN）技术产生GS敲除CHOK 1 SV细胞系。CHO GS基因上双等位基因修饰的高效率（类似于2%）支持ZFN技术的独特优势，尤其是在CHO细胞中。通过观察所有GS敲除细胞系的谷氨酰胺依赖性生长证实GS酶功能破坏。在标准工业细胞培养过程中对GS敲除细胞系进行了全面评价。通过使用GS敲除细胞作为亲本细胞，批量培养生产率提高了2至3倍。的选择严格性显着增加，所示的大量减少非生产和低生产细胞后25？μ M L-MSX选择，并导致在鉴定给定重组单克隆抗体的类似数目的高产细胞系方面的六倍效率提高。GS敲除细胞对重组蛋白质质量的潜在影响也进行了讨论。Biotechnol. Bioeng. 2012; 109：10071015. (c)2011 Wiley Periodicals，Inc.
Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (similar to 2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was significantly increased, as indicated by the large reduction of non-producing and low-producing cells after 25?mu M L-MSX selection, and resulted in a six-fold efficiency improvement in identifying similar numbers of high-productive cell lines for a given recombinant monoclonal antibody. The potential impact of GS-knockout cells on recombinant protein quality is also discussed. Biotechnol. Bioeng. 2012; 109:10071015. (c) 2011 Wiley Periodicals, Inc.