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.