4 DISCUSSION
Recombinant protein production in Escherichia coli cells has been carried out using lac -based inducible promoters.[33] Although gene expression control systems have been extensively reported in animal cells, inducible expression systems are rarely employed for industrial-scale production of recombinant proteins. Cell growth inhibition is often observed in cell lines that produce recombinant proteins at extremely high levels. In such cases, a production system that switches between cell growth and protein production phases may effectively improve productivity per unit time. In this study, to demonstrate the concept of separating growth and production phases, we constructed a biopharmaceutical production system with inducible target gene expression using the estrogen-binding domain of an estrogen receptor. As a gene expression inducer, 4-OHT can bind to the estrogen receptor with high affinity compared with the natural ligand E2,[32] enabling expression induction at low concentrations. Using 4-OHT as an induction switch, scFv-Fc protein production could be induced with 4.4-times higher concentration and 5.5-times higher specific productivity compared with E2, under lower induction concentrations. The model antibody scFv-Fc was produced by inducing expression in CHO cells using a serum-free medium commonly used for biopharmaceutical production. In serum-free medium, sensitivity to the inducer and responsiveness to gene expression induction are increased. After achieving the required cell number in 4-OHT-free medium, production culture was performed under the condition of 0.1 µM 4-OHT. Within 3 days, cells were induced to a maximum production level. scFv-Fc production in CHO/GEV_scFv-Fc cells with high cell density was stably maintained during semi-continuous culture for over 2 weeks, with specific productivity ranging from 37 to 57 pg cell–1day–1 and secretion levels of 0.31–0.44 g/L. Interestingly, the specific productivity of cells at high density was 2.8 to 4.4-fold higher compared with the growth state at low cell density (13 pg cell–1 day–1).
In this gene induction system, GFP-positive cells with over 95% efficiency were observed from day 1 after 0.5 µM 4-OHT addition. Maximum GFP expression intensity or maximum scFv-Fc production was generally observed 2–3 days after adding the inducer drug. In ERT2 fusion proteins, including ERT2-Cre, the mutant ERT2[32]derived from the ligand-binding domain of human ERalpha (amino acids 282–595; Accession No. NP_000116)[34] binds to the molecular chaperone HSP90 in the cytoplasm. In the presence of 4-OHT, HSP90 is replaced by 4-OHT and undergoes nuclear translocation.[25,26] GEV utilizes the same region as the ligand-binding domain of Cre-ERT2 but lacks the nuclear localization signal region (243-RKC YEV GMM KGG IRK DRR GGR MLK HKR QRD-272)[35] of human ER⍺ in ERT2. Nuclear translocation of proteins can be controlled by the type of nuclear localization signal, mutations, or their placement.[36,37] Thus, screening for an optimal nuclear translocation signal for GEV may enhance nuclear translocation and rapid induction of transgene expression following addition of the inducer drug.
When using a seeding cell density of 2.0 × 107cells/mL in suspension culture under high-cell density conditions, a significant decrease in cell viability was observed from day 2 of culture (data not shown). Therefore, the initial seeding cell density was set to 0.5 or 1.0 × 107 cells/mL, and daily medium changes were performed. This strategy allowed for stable induction and production while maintaining constant cell viability. Glucose in the medium was consumed at a rate of 86%–93% and the specific consumption rate was calculated as 0.4–1.0 ng cell–1day–1. With a seeding cell density of 1.0 × 107 cells/mL, a lactate concentration of 66 mM was measured, corresponding to consumption of approximately 33.5 mM glucose per day. This suggests that almost all of the consumed glucose was converted to lactate. Cell viability was around 80%, indicating that oxygen depletion occurred in this culture. With a seeding cell density of 0.5 × 107 cells/mL, the glucose consumption rate was 31.3 mM and lactate accumulation was approximately 34 mM, almost half the amount of the consumed glucose. This suggests that although most glucose was consumed, lactate accumulation was suppressed, resulting in cell viability being maintained at nearly 100% and a high specific production rate (57 pg cell–1day–1). In this experiment, a commonly used serum-free medium was employed for evaluation; however, in recent years, data-driven medium development and feed development in CHO cell metabolism have been actively pursued.[38,39]Accordingly, improving the oxygen supply and developing medium suitable for this inducible gene expression system may further enhance productivity.
A drug-inducible nuclear translocation system based on estrogen receptors enables spatiotemporal control of gene function. Numerous applications have been reported using recombinase,[32]transposase,[40] transcription factors,[41] and other approaches. In this study, we constructed a gene expression system incorporating an estrogen-responsive domain into an artificial transcription factor designed for target gene expression and applied it to the inducible production of scFv-Fc antibody. The system demonstrated high-level induction of expression and long-term stable production, highlighting its effectiveness as a production system that switches between cell proliferation and production modes. Due to its anticipated versatility, this gene induction system is considered applicable to CHO cells as well as HEK293 cells, which are commonly used for viral production, and newly developed Chinese hamster-derived cells[42,43] for the production of valuable substances.
In conclusion, we developed an inducible gene expression system for biopharmaceutical production utilizing the estrogen-responsive property of the estrogen receptor. Using this system, we demonstrated an efficient recombinant protein production system in CHO cells that effectively separates the cell proliferation phase from the production phase. The production system, built upon a synthetic biology-based approach for an artificial gene expression, is expected to become an important method in biopharmaceutical production.