Bioprocess Research and Development, Eli Lilly and Company, LTC-North, 1200 Kentucky Avenue, Indianapolis, Indiana 46221, USA.
Biotechnol Bioeng. 2012 Apr;109(4):1007-15. doi: 10.1002/bit.24365. Epub 2011 Nov 21.
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 (∼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 µM 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.
尽管中国仓鼠卵巢（CHO）细胞因其独特的特性已成为生产治疗性重组蛋白的主要工具，但 CHO 细胞系生成（CLG）的主要挑战之一是如何在大量低产和无产克隆中高效鉴定那些罕见的高产克隆。通常需要筛选数百个单个克隆，才能鉴定出具有可接受生产力和生长特性的商业克隆细胞系，使其适合商业应用。这种低效率使得 CLG 过程既耗时又费力。目前，有两种主要的 CHO 表达系统，二氢叶酸还原酶（DHFR）-甲氨蝶呤（MTX）选择和谷氨酰胺合成酶（GS）-蛋氨酸亚砜imine（MSX）选择，已广泛应用于工业生产。由于 GS-CHO 系统中的重组细胞系的选择是基于引入表达质粒的 GS 基因的表达与 GS 抑制剂 L-MSX 的添加之间的平衡，因此亲本 CHOK1SV 细胞中的内源性 GS 基因的表达可能会干扰选择过程。为了研究内源性 GS 表达对选择效率的潜在影响，使用锌指核酸酶（ZFN）技术生成了 GS 敲除 CHOK1SV 细胞系，该技术旨在专门针对内源性 CHO GS 基因。CHO GS 基因的双等位基因修饰效率高达约 2%，支持了 ZFN 技术的独特优势，尤其是在 CHO 细胞中。所有 GS 敲除细胞系均观察到谷氨酰胺依赖性生长，证实了 GS 酶功能被破坏。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估。使用 GS 敲除细胞作为亲本细胞，使批量培养生产力提高了 2 到 3 倍。通过 25 μM L-MSX 选择后，非生产性和低生产性细胞大量减少，选择严格性显著提高，从而在鉴定相同数量的高产细胞系时，效率提高了 6 倍，用于给定的重组单克隆抗体。还讨论了 GS 敲除细胞对重组蛋白质量的潜在影响。
