Inducible transgene expression in CHO cells using an artificial transcriptional activator with estrogen-binding domain.

Biopharmaceuticals, including therapeutic antibodies, are rapidly growing products in the pharmaceutical market. Mammalian cells, such as Chinese hamster ovary (CHO) cells, are widely used as production hosts because recombinant antibodies require complex three-dimensional structures modified with sugar chains. Recombinant protein production using mammalian cells is generally performed with cell growth. In this study, we developed a technology that controls cell growth and recombinant protein production to induce recombinant protein production with predetermined timing. Expression of green fluorescent protein (GFP) gene and a single-chain antibody fused with the Fc-region of the human IgG1 (scFv-Fc) gene can be induced and mediated by the estrogen receptor-based artificial transcription factor Gal4-ERT2-VP16 and corresponding inducer drugs. We generated CHO cells using an artificial gene expression system. The addition of various concentrations of inducer drugs to the culture medium allowed control of proliferation and transgene expression of the engineered CHO cells. Use of 4-hydroxytamoxifen, an antagonist of estrogen, as an inducing agent yielded high gene expression at a concentration more than 10-fold lower than that of ß-estradiol. When scFv-Fc was produced under inducing conditions, continuous production was possible for more than 2 weeks while maintaining high specific productivity (57 pg cell-1 day-1 ). This artificial gene expression control system that utilizes the estrogen response of estrogen receptors can be an effective method for inducible production of biopharmaceuticals.

High-Level Production of scFv-Fc Antibody Using an Artificial Promoter System with Transcriptional Positive Feedback Loop of Transactivator in CHO Cells.

With the increasing demand for therapeutic antibodies, CHO cells have become the de facto standard as producer host cells for biopharmaceutical production. High production yields are required for antibody production, and developing a high-titer production system is increasingly crucial. This study was established to develop a high-production system using a synthetic biology approach by designing a gene expression system based on an artificial transcription factor that can strongly induce the high expression of target genes in CHO cells. To demonstrate the functionality of this artificial gene expression system and its ability to induce the high expression of target genes in CHO cells, a model antibody (scFv-Fc) was produced using this system. Excellent results were obtained with the plate scale, and when attempting continuous production in semi-continuous cultures using bioreactor tubes with high-cell-density suspension culture using a serum-free medium, high-titer antibody production at the gram-per-liter level was achieved. Shifting the culture temperature to a low temperature of 33 °C achieved scFv-Fc concentrations of up to 5.5 g/L with a specific production rate of 262 pg/(cell∙day). This artificial gene expression system should be a powerful tool for CHO cell engineering aimed at constructing high-yield production systems.