Development of predictable and high-yield CHO cell line by site-specific integration.

Chinese hamster ovary (CHO) cell is a widely used cell line for the production of therapeutic proteins. Customarily, CHO host cell line is established through random integration, which requires multiple rounds of screening to identify the optimal producer. In contrast, site-specific integration (SSI) technology can boost cell line development efficiency by directing gene of interest (GOI) to certain genomic loci that supports sustained and stable expression. In this study, by utilizing AI algorithms and robotic systems, we have identified several CHO host cell lines carrying marker gene by Bxb1-mediated SSI technology. After the substitution of marker gene with different types of GOI at specific sites, some of cases delivered a titer exceeding 15 g/L. Thanks to the uniformity of SSI cell lines, protein titer and quality of monoclonal cells can be predicted based on the performance of corresponding minipools. Different monoclonal cells originated from the same minipool also showed consistent protein quality. Furthermore, it was observed that the monoclonal cells obtained by SSI demonstrated consistency in protein titer, quality and genetic stability after 26 consecutive passages (approximately 90 days). In summary, we have successfully developed an effective platform for the construction of SSI cell lines, which allows the rapid and efficient expression of various proteins while maintaining consistency in stability and product quality. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13036-025-00610-z.