A Highly Stringent Split Intein-Mediated DHFR Selectable Marker Enables Efficient Development of High-Producing CHO Cells for Therapeutic Proteins

The dihydrofolate reductase (DHFR)-based selection system widely used to develop Chinese hamster ovary (CHO) cell lines for the production of therapeutic proteins is limited by low selection stringency, reliance on stepwise gene amplification, and restricted capacity for efficient multigene selection. In this study, we developed a highly stringent split DHFR selectable marker by integrating a computationally guided split-site design with split intein-mediated protein ligation (SiMPl) in CHO cells. Using the synthetic SiMPl-DHFR selection system, the proportion of Fc-fusion glycoprotein-producing cells in stable cell pools reached 98.5%, and specific protein productivity increased 22.5-fold compared with the wild-type DHFR (WT-DHFR) selection system. When combined with dhfr /methotrexate-mediated gene amplification, the SiMPl-DHFR system further increased mRNA expression levels and Fc-fusion glycoprotein production. Notably, dhfr -nonamplified cell pools generated using the SiMPl-DHFR selection system exhibited higher expression levels than all dhfr -amplified cell pools established using the WT-DHFR selection system. Furthermore, the SiMPl-DHFR selection system permitted the efficient generation of stable monoclonal antibody- and bispecific antibody-producing CHO cell pools that require coordinated expression of two and four genes, respectively. Overall, this study establishes a synthetic selectable marker architecture based on intein-mediated protein splicing that rewires DHFR selection logic and provides a highly stringent and extensible platform for mammalian cell line development.