Traits like robust growth, easy scaling of suspension cultures, and reliable cloning frequency have crowned Chinese hamster ovary (CHO) cells as an ideal mammalian cell line for bioprocessing. However, optimizing recombinant CHO cell development to produce biologics like antibodies can still be quite the challenge.
Dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection systems are common but require multiple rounds of selection, which can result in longer timelines and genetic instability.
Glutamine synthetase (GS) based selection utilizes GS gene co-expression with the gene of interest as a selectable marker for high-expressing GS CHO clones. Because CHO cells naturally express glutamine, these cells must be under selection of the GS inhibitor MSX. GS CHO selection can be considered an improvement over DHFR as the reduced need for selection entails shorter timelines. 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 that cells with low endogenous glutamine levels often survive the MSX selection. This decreases the GS CHO system selection stringency. Therefore, creating GS CHO knockout cells as an 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) technology. The knockout efficiency using ZFN in CHO cells was 2%.
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).
ZFNs have obvious advantages when it comes to improving biopharma production capabilities. Moreover, other tools like CRISPR have also been used to make GS knockout CHO cells. Using CRISPR and ZFNs to improve CHO cells through GS knockout is just one example of how the production process in biopharma is changing.
More recent tools like Cas-CLOVER are taking these advancements one step further by building on the CRISPR-Cas model. Cas-CLOVER utilizes Clo51, a dimeric endonuclease that is only active upon dimerization. This requires two guide RNAs (gRNA) to bring nuclease inactivated Cas9 proteins to the site where cutting is desired, thereby bringing the dimers together. This dimerization requirement results in higher accuracy for Cas-CLOVER than regular CRISPR-Cas9, meaning little to no off-target effects.
Demeetra AgBio, Inc. holds exclusive licenses to Cas-CLOVER for organizations looking to use the technology for therapeutic bioprocessing. We have distinct patents than those covering CRISPR-Cas9 and, unlike our competitors, our licensing process is straightforward no matter how far along you are to commercialization. If you’re interested in learning more about our product and licensing structure schedule a call to talk to one of our representatives.