1 INTRODUCTION
Biopharmaceuticals are high-molecular-weight biological drugs manufactured using biological processes.[1] Many biopharmaceuticals include proteins or nucleotides consisting of hundreds or thousands of amino acids or nucleosides, such as monoclonal antibodies, cytokines, enzymes, viruses, and nucleic acid-based products. Among them, monoclonal antibodies have received the most approvals from regulatory agencies and entered the market.[2] The biopharmaceutical market is rapidly expanding and has promising prospects due to enormous demand.[3] In the biopharmaceutical market, mammalian cell lines are preferred for producing recombinant therapeutic proteins. Chinese hamster ovary (CHO) cell lines are particularly notable. The use of CHO cells as hosts in production systems has become an established method for producing therapeutic proteins, primarily due to their ability to proliferate in high-cell-density serum-free cultures.[4]
CHO cells can produce properly folded proteins with post-translational modifications similar to those in humans.[5,6]Additionally, CHO cells are recognized as safe hosts with resistance to human viral infections because they do not express genes for viral entry.[4,7] There are several challenges to improve the specific productivity of target proteins. The main barriers include vector construction, transfection efficiency, and integration into specific loci on the transgenic chromosome. Another limitation is the availability of high-producing cell lines. Establishing high-producing cell lines is time-consuming and labor-intensive, so targeted integration of the desired transgene into specific genomic loci may be a potential solution to streamline the complex screening process.[8,9] Cell line development, media composition, and culture optimization have increased due to the demand for and regulatory requirements of biopharmaceuticals.[10] However, novel research is needed to develop high-producing cell lines that meet market requirements.
In the current mainstream production method, fed-batch cultivation, the desired substance is produced simultaneously with cell proliferation. For biotechnology applications, it is expected that the target gene will be expressed in a controllable manner.[11] This can be achieved with the help of inducible systems, which allow for specific control of transgene expression in certain cells during specific periods.[12] By switching between growth and production phases, it may be possible to reduce the burden on cells during proliferation while maintaining a high proliferation capacity and achieving high productivity by inducing production after reaching high cell density in a short period of time.
In recent years, gene expression control systems have become important techniques for analyzing various biological functions of both prokaryotes and eukaryotes. Various gene expression control systems based on artificial transcription activators have been developed, such as the tetracycline repressor-based transcriptional activation system[13] and CRISPR transcriptional activation system.[14] We previously developed an artificial gene expression system that responds to external environments such as heat treatment and hypoxia in mammalian cells derived from liver and muscle.[15–24] Using these systems, we succeeded in inducing artificial gene expression and making it functional by autonomously responding to the environment.
In this study, we aimed to construct a biopharmaceutical production system that utilizes the estrogen response of estrogen receptors to control target gene expression. Estrogen receptors translocate to the nucleus upon binding to their ligand estrogen and function as transcriptional activators.[25,26] Therefore, inducible expression systems using estrogen as a switch can be used to construct biopharmaceutical production systems that can switch between cell growth and cell production modes. We employed an artificial gene expression system using the chimeric protein Gal4-ERT2-VP16 (GEV), which is activated by the addition of estrogen receptor ligands. GEV is a transcriptional activator that can induce target gene expression in response to the addition of estrogen, β-estradiol (E2), or the E2 antagonist 4-hydroxytamoxifen (4-OHT) to the culture medium.[27,28] We generated CHO cells constitutively expressing GEV in which gene expression can be controlled by estrogen receptor ligands. First, a reporter gene expression system capable of responding to GEV activated in the presence of a ligand was introduced into cells and the induced expression behavior was analyzed. Subsequently, a gene expression cassette for producing an anti-prion single-chain antibody fused with the Fc-region of human IgG1 (scFv-Fc), as a model antibody, was incorporated into cells and the induced antibody production in response to ligand drugs was evaluated using serum-free medium. Finally, continuous production of scFv-Fc using this artificial gene expression control system was attempted by semi-continuous suspension culture with high cell density, and antibody production stability was investigated.