Knockout GS CHO cells in biologics manufacturing
Specific traits ranging from hearty growth & suspension culture scale up to cloning frequency have crowned CHO as the top bioprocessing host. However, due to low specific productivity during the cell line development and selection process, optimization of recombinant Chinese hamster ovary (CHO) cell line development for commercial bioprocess of therapeutic biologics such as antibodies, is an ever-evolving landscape.
Dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection systems are common and effective but requires multiple rounds of selection resulting in longer time-lines and the possibility of genetic instability.
Glutamine synthetase “GS CHO” based selection utilizes GS gene co-expression with the gene of interest (i.e. antibody production) as a selectable marker for high expressing GS CHO pools and clones. Since CHO cells express suitable amounts of GS, these cells must be under selection of the GS inhibitor MSX, which is an improvement over DHFR as it requires shorter timelines due to a reduced need for selection. Figure 1 demonstrates monoclonal antibody production is increased with MSX selection in this characterization study (1).
One key flaw to MSX selection in GS CHO is low expressing cells often survive the MSX selection decreasing the GS CHO system selection stringency. Therefore creating GS CHO knockout cells as a expression system would be ideal. One publication by the same group at Eli Lilly who utilized piggyBac on CHO engineering, demonstrated GS CHO knockout with zinc finger nuclease (ZFN). The knockout efficiency using ZFN in CHO cells was 2% (a comparison to Cas-CLOVER will be the subject of a separate post).
GS CHO knockout cells exhibit more stringent selection, with bulk culture productivity double that of CHOK1 control cells under the same conditions and about 4x higher than previously shown (2).
1. Noh et al. (2018) Scientific Reports
2. Fan et al. (2011) Biotechnology & Bioengineering