Strategies Of CHO Cell Line Development And Toolbox Implementation For Improving Cell Line Productivity And Stability

By AnQiang (Andy) Zhou, Michael Brem, April Stanley, Jon Mitschelen, Jesse McCool, R&D Services, Cytovance Biologics Inc.

It is well known that for better expressing protein of interest (POI), the codon usage has to be compatible to host cells, but when we ordered synthetic genes optimized for expressing a monoclonal antibody (mAb) in CHO-S cells, that pair of synthetic genes expressed the target Ab very poorly. After thorough investigation, we concluded that current codon optimization cannot guarantee good POI expression. To get good pair of synthetic genes for expressing Ab in CHO cells, multiple pairs of synthetic genes need to be evaluated. The selection of good pair of synthetic genes becomes a process that is not only labor intense, time consuming, but also costly. To simplify this unappealing process, we use a scaffold that contains both our proven compatible secretory signal peptide and hIgG1 Constant region DNA sequences, where we simply incorporated a single optimization at the variable region. Our data demonstrate that this strategy is successful. Using commercially available dicistronic mammalian expression vector for antibody cell line development, we encountered two challenges: 1) higher selection stringency cannot guarantee higher productivity, and 2) extremely low recovery rate of stable high producer clones. To overcome these issues, Cytovance􀂣 built its own dicistronic mammalian expression vectors. These vectors link the genes of interest (GOI) and the genes for selection (GFS) together via Internal Ribosome Entry Site (IRES). As a result, the GOI and GFS are on a same mRNA where we use a higher selection stringency that leads to a higher GOI expression. Moreover, the vectors also contain CHO genomic DNA targeting elements that direct the transgenes integration into specific sites of CHO genome to enhance the transgene stability. Consequently, the recovery rate of stable high producer clones should be much higher. To date, our preliminary data prove that it is the case.

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