Leveraging Transfection Dose Streamlines the Establishment of a Chinese Hamster Ovary Cell Targeted Integration System

Targeted integration (TI) expression system has emerged as an alternative to random integration in cell line development (CLD) involved in biologics development and manufacturing. A key element of a robust TI CLD system is the construction of a high-performance TI host cell line, which significantly influences the productivity, product quality, and cell line stability of expressed biologics. In this study, we implemented a low-copy transfection strategy by optimizing transfection dosages to efficiently achieve over 30% single-copy integrants in a single transfection. This approach dramatically reduces TI host cell screening efforts, enabling rapid isolation of functional TI host cells. In addition, using mRNA transfection of Cre recombinase enabled faster pool recovery and eliminated concerns related to residual Cre coding sequence integration. Under non-optimized fed-batch conditions, this resulting TI system supported monoclonal antibody (mAb) titers of up to 8.03 g/L in 15-L bioreactors.