Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells

Abstract

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.

Complete Knockout of the Lactate Dehydrogenase A Gene is Lethal in Pyruvate Dehydrogenase Kinase 1, 2, 3 Down-Regulated CHO Cells

“…However, the rate of ZFN-induced mutations due to NHEJ in CHO cells was at *5 % in our study. This is comparable to the efficiency reported previously for ZFN-mediated knockout in CHO cells using a different ZFN pair [23]. Therefore, we decided to carry out the knockout attempt of the 2nd allele and screen all the resulting clones by sequencing.…”

Emerging synthetic biology tools for engineering mammalian cell systems and expediting cell line development

“…Overexpression of other anti-apoptotic genes resulted in shifted nutrient consumption profiles and decreased lactate and ammonia accumulation [69]. Improvements in efficiency and cellular metabolism have also been achieved by generating glutamine synthetase null strains using ZFNs [70].…”

Cell Culture Processes for Biopharmaceutical Manufacturing

“…CHO cells have an endogeneous functional GS gene. To improve GS selection in CHO, endogeneous GS gene was knocked-out which increased the productivity achieved in CHO from GS-MSX selection-amplification 41 . Improvements to the basic selection strategy have been reported, such as attenuation of selection marker to reduce its activity thus requiring greater expression of the marker, and by association with the transgene, to survive the selection pressure 42 .…”