Productivity and stability are critical for the producing cell lines for manufacturing protein drug. Given that the integration sites of gene of interest (GOI) could contribute remarkable effect on the productivity and stability of GOI expression, we intended to develop a time-saving and less labor-intensive strategy to generate the high-producing cell lines through targeting the active and stable regions of CHO genome. To identify the active and stable regions located in CHO genome, we generated high producing stable cell line, and employed PCR-based or NGS-based method to analyze the integration sites of the high producing stable cell line. The pair-end reads with one read mapped to the vector and the other read mapped to the CHO reference genome are extracted to identify the integration sites. In order to verify the expression activity and stability of the integration sites, we employed CRISPR/Cas9 to specifically knock-in the expression vector with antibody gene into CHO genome. Our data showed the cell pool generated by knock-in of expression vector into the IS1 integration site present higher expression titer than cell pools generated by random integration or integrating into other sites . We further cultured the single cell clones derived from this cell pool through screening and selection by Clonepix and limiting dilution. These single cell clones have high expression titer ranging from 254 to 804 mg/L in batch culture of after 6 Days. A single cell clone(376 mg/L in batch culture) can reached 2 g/L in fed-batch culture. The stability analysis showed this clone maintain stable expression of GOI after 60 generation. Here, we demonstrated the generation of stable cell line with high protein expression by CRISPR/Cas9 mediated target integration. The target designed strategy will serve as a platform for therapeutic antibody production.

Development of high-producing CHO cell lines through target-designed strategy

255th National Meeting (2018)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.