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

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 accept- able productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and labo- rious. Currently, there are two main CHO expression sys- tems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based me- thionine 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 pro- cess. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) tech- nology designed to specifically target the endogenous CHO GS gene. The high efficiency (� 2%) of bi-allelic modifica- tion 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 reduc- tion of non-producing and low-producing cells after 25 mM 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: 1007-1015. 2011 Wiley Periodicals, Inc.