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\begin{document}

\title{Inducible transgene expression in CHO cells using an artificial
transcriptional activator with estrogen-binding domain}



\author[1]{Md. Rashidur Rahman}%
\author[1]{Yoshinori Kawabe}%
\author[1]{Kozumi Suzuki}%
\author[1]{Satoshi Chen}%
\author[1]{Yuki Amamoto}%
\author[2]{Masamichi Kamihira}%
\affil[1]{Affiliation not available}%
\affil[2]{Kyushu University}%


\vspace{-1em}



  
  \date{May 27, 2024}


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\selectlanguage{english}
\begin{abstract}
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 in conjunction with cell
growth. In this study, we developed a technology that controls cell
growth and recombinant protein production to induce recombinant protein
production with arbitrary 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 \selectlanguage{greek}β-\selectlanguage{english}estradiol. When scFv-Fc was continuously produced under
inducing conditions, stable production was possible for more than 2
weeks while maintaining high specific productivity (57 pg
cell\textsuperscript{-1} day\textsuperscript{-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.%
\end{abstract}%



\sloppy


\textbf{1 INTRODUCTION}

Biopharmaceuticals are high-molecular-weight biological drugs
manufactured using biological processes.\textsuperscript{{[}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.\textsuperscript{{[}2{]}} The biopharmaceutical market is rapidly
expanding and has promising prospects due to enormous
demand.\textsuperscript{{[}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.\textsuperscript{{[}4{]}}

CHO cells can produce properly folded proteins with post-translational
modifications similar to those in
humans.\textsuperscript{{[}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.\textsuperscript{{[}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.\textsuperscript{{[}8,9{]}} Cell line
development, media composition, and culture optimization have increased
due to the demand for and regulatory requirements of
biopharmaceuticals.\textsuperscript{{[}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.\textsuperscript{{[}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.\textsuperscript{{[}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\textsuperscript{{[}13{]}} and CRISPR transcriptional activation
system.\textsuperscript{{[}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.\textsuperscript{{[}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.\textsuperscript{{[}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, \selectlanguage{greek}β-\selectlanguage{english}estradiol (E2), or the E2
antagonist 4-hydroxytamoxifen (4-OHT) to the culture
medium.\textsuperscript{{[}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.

\textbf{2 MATERIALS AND METHODS}

\textbf{2.1 Cells and media}

CHO-K1 (RIKEN Cell Bank, Tsukuba, Japan) and recombinant CHO cells were
cultured in F12 medium (\#N6760; Sigma-Aldrich, St Louis, MO, USA)
supplemented with 10\% fetal calf serum (BioWest, Nuaill\selectlanguage{ngerman}é, France) and
antibiotics (Penicillin-Streptomycin, \#15140122; Invitrogen, Waltham,
MA, USA). Cells were cultured in a 5\% CO\textsubscript{2} incubator at
37°C. For serum-free cultures, CD CHO AGT Medium (\#12490025,
Invitrogen) containing 4 mM L-glutamine (\#074-00522; Fujifilm Wako Pure
Chemical Corporation, Osaka, Japan), 0.2\% anti-clumping reagent
(\#0010057AE, Invitrogen), and antibiotics (penicillin-streptomycin) was
used. When cells were cultured in serum-containing medium, 100-mm cell
culture dishes (BioLite \#130182; Thermo Scientific, Waltham, MA, USA)
or 24-well tissue culture plates (BioLite \#130186, Thermo Scientific)
were used. For serum-free suspension culture, bioreactor tubes (\#87050;
TPP Techno Plastic Products AG, Trasadingen, Switzerland) placed in a
shaker (Model \#0081704-000; Taitec, Koshigaya, Japan) with a 45° angle
were incubated at 37°C in a 5\% CO\textsubscript{2} incubator.

\textbf{2.2 Plasmid construction}

To construct a transposon-based GEV expression vector, the GEV gene was
amplified by PCR using pME-GAL4-ERT2-VP16 (\#82588, Addgene, Watertown,
MA, USA)\textsuperscript{{[}28{]}} as a template. A DNA fragment of the
Chinese hamster-derived EF1\selectlanguage{greek}α \selectlanguage{english}promoter\textsuperscript{{[}8{]}} was
chemically synthesized. A hygromycin-resistant gene expression unit
(selection marker) was obtained by PCR using pCEP4 (\#V04450,
Invitrogen) as a template. These DNA fragments were inserted into a
PiggyBac transposon vector plasmid (\#PB513B-1; System Biosciences, Palo
Alto, CA, USA) to generate PB/chEF1\selectlanguage{greek}α/\selectlanguage{english}GEV\_Hyg (Figure 1A).

Next, to generate the UAS/scFv-Fc expression vector, a DNA fragment of
scFv-Fc gene was obtained by cutting plasmid
R2\textsuperscript{{[}8{]}}with \emph{Eco} RI and \emph{Spe} I. A DNA
fragment of the UAS response element and minimal region of the
adenovirus major late promoter were chemically synthesized. A
zeocin-resistant gene expression unit was amplified by PCR using
pcDNA4/GP\textsuperscript{{[}29{]}} as a template. By inserting these
DNA fragments into the PiggyBac transposon vector plasmid,
PB/UAS/scFv-Fc\_Zeo was constructed (Figure 1A). To construct the
UAS/GFP expression vector, a \emph{GFP} gene was amplified by PCR using
pGreenFire1-RARE (\#TR037PA-P, System Biosciences) as a template.
PB/UAS/GFP\_Zeo was constructed by replacing the scFv-Fc gene region in
PB/UAS/scFv-Fc\_Zeo with the resulting fragment (Figure 1A).

For PCR, KOD plus Neo DNA polymerase (\#KOD-401; Toyobo, Tsuruga, Japan)
and the primers shown in Supplementary Table S1 were used for
amplification. All PCR products were subjected to gene sequence analysis
by Sanger sequencing to confirm correct sequences.

\textbf{2.3 Generation of recombinant CHO cells}

To generate CHO cells expressing the GEV gene (CHO/GEV), CHO-K1 cells
were seeded in a 24-well plate at 1.0 x 10\textsuperscript{5}cells/well.
The next day, PB/chEF1\selectlanguage{greek}α/\selectlanguage{english}GEV\_Hyg and PiggyBac transposase expression
vector plasmids (pPBase; \#PB210PA-1, System Biosciences) were
transiently transfected into CHO-K1 cells using Lipofectamine 2000
(\#11668019, Invitrogen) according to the manufacturer's procedure.
After 48 h, cells were plated in six-well plates (BioLite \#130184,
Thermo Scientific), Hygromycin B (\#008-07683, Wako) was added at a
concentration of 400 \selectlanguage{greek}µ\selectlanguage{english}g/mL, and drug selection was performed for 14
days. Stable transformed cells expressing GEV were obtained. Cell clones
(CHO/GEV) were established by limiting dilution.

Next, PB/UAS/GFP\_Zeo was transiently introduced into CHO/GEV together
with pPBase using Lipofectamine 2000 as described above. Drug selection
was performed for 14 days using 400 \selectlanguage{greek}µ\selectlanguage{english}g/mL Zeocin (\#R25001, Invitrogen).
Next, 1 \selectlanguage{greek}µ\selectlanguage{english}M 4-OHT (\#H6278, Sigma-Aldrich) was added to the cells. The
day after drug addition, single-cell cloning of GFP-positive cells was
performed using a cell sorter (SH800; Sony, Tokyo, Japan) to establish
drug-inducible GFP-expressing cell clones (CHO/GEV\_GFP).

To generate drug-inducible scFv-Fc-producing cells, PB/UAS/scFv-Fc\_Zeo
linearized with \emph{Fsp} I was introduced into CHO/GEV using
Lipofectamine 2000. Selection with Zeocin was performed in the same
manner as described above to establish stable transformed cells, and
cell clones (CHO/GEV\_scFv-Fc) were obtained by limiting dilution.

\textbf{2.4 Induction of target genes}

Cells were seeded with 1 mL per well in 24-well plates at a seeding
density of 1.0 \selectlanguage{ngerman}× 10\textsuperscript{5} cells/mL in serum-containing F12
medium or 1.0 × 10\textsuperscript{6} cells/mL in serum-free medium, and
cultured for 4 days. During culture, the medium was changed daily to
medium containing E2 (\#E2758, Sigma-Aldrich) or 4-OHT at various
concentrations. Cell numbers were counted using the trypan blue dye
exclusion method.

High-cell-density serum-free culture was performed as follows. After
culturing CHO/GEV\_scFv-Fc for 3 days in serum-free medium containing
0.1 \selectlanguage{greek}µ\selectlanguage{english}M 4-OHT, cells were adjusted to a density of 0.5 or 1.0 \selectlanguage{ngerman}×
10\textsuperscript{7} cells/mL. Thereafter, 10 mL of cell suspension was
seeded in a bioreactor tube and cultured for 15 days. The culture medium
was replaced with fresh medium every day, and the number of cells was
counted at that time.

Drug-induced GFP expression in CHO/GEV\_GFP was analyzed using a flow
cytometer (SH800). scFv-Fc secreted into the culture medium was
quantified using an enzyme-linked immunosorbent assay
method\textsuperscript{{[}30{]}}. The scFv-Fc specific production rate
(pg cell\textsuperscript{-1} day\textsuperscript{-1}) was calculated
from scFv-Fc concentrations in the spent medium and numbers of viable
cells. Glucose and lactate concentrations were measured using
commercially available kits (Glucose Assay Kit-WST, \#G264, Dojindo,
Kumamoto, Japan; Lactate Assay Kit-WST, \#L256, Dojindo).

\textbf{2.5 Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) analysis}

Medium samples (10 \selectlanguage{greek}µ\selectlanguage{english}L) harvested from semi-continuous cultures were
mixed with SDS sample buffer (5 \selectlanguage{greek}µ\selectlanguage{english}L) with or without 2-melcaptoethanol
and boiled for 5 min. Sample solutions were electrophoresed on 4\%--20\%
precast polyacrylamide gels (\#4561094; BioRad, Hercules, CA, USA).
Protein bands were visualized by incubating gels in protein staining
solution (\#LC6060, SimpleBlue SafeStain, Invitrogen) for 1 h. Next,
gels were immersed in distilled water for 2 days to remove excess dye.
Purified scFv-Fc was prepared from culture medium using a Protein A
Sepharose column (rProtein A Sepharose Fast Flow, \#17127901; Cytiva,
Marlborough, MA, USA).\textsuperscript{{[}31{]}}

\textbf{3 RESULTS}

\textbf{3.1 Establishment of founder CHO cells expressing GEV
transactivator}

The GEV, designed as an artificial transcription factor, is a fusion
protein consisting of the DNA-binding domain Gal4, ligand-binding domain
ERT2 of the estrogen receptor, and transcription activation domain VP16
derived from herpes simplex virus. In the absence of E2 or 4-OHT, GEV
remains in the cytoplasm; however, upon the addition of E2 or 4-OHT, GEV
translocates to the nucleus through ligand binding to the ERT2 domain.
Subsequently, GEV binds to the Gal4-binding sequence (UAS) of an
artificial promoter (UAS-P\textsubscript{min}) and induces expression of
the target gene under control of the artificial promoter using the
activity of VP16 (Figure 1B). Initially, to establish CHO cells
constitutively expressing GEV, the GEV gene was incorporated into a
PiggyBac transposon vector and introduced into CHO cells along with a
transposase expression vector. Stable expression cell lines were
obtained by selection with the corresponding drug. As a preliminary
investigation, a GFP reporter vector plasmid (PB/UAS/GFP) was
transiently transfected into stable GEV-expressing cell lines, and
drug-dependent GFP expression was confirmed for cells cultured in the
presence of 20 \selectlanguage{greek}µ\selectlanguage{english}M E2 or 1.0 \selectlanguage{greek}µ\selectlanguage{english}M 4-OHT. Subsequently, PB/UAS/GFP was
transiently introduced into these cells and cloning was performed by
sorting the cell fraction induced for GFP expression under 1.0 \selectlanguage{greek}µ\selectlanguage{english}M 4-OHT
using a cell sorter. For the obtained 26 cell clones, transient gene
introduction of PB/UAS/GFP was performed, followed by culture in the
presence of 1.0 \selectlanguage{greek}µ\selectlanguage{english}M 4-OHT. After 48 h, cell clones with high GFP
expression were selected by observation under a fluorescence microscope
(Supplementary Figure S1A). The same gene introduction of PB/UAS/GFP was
performed for three selected cell clones. After introduction, cells were
evaluated under conditions of 20 \selectlanguage{greek}µ\selectlanguage{english}M E2, 1.0 \selectlanguage{greek}µ\selectlanguage{english}M 4-OHT, and co-addition of
20 \selectlanguage{greek}µ\selectlanguage{english}M E2 and 1.0 \selectlanguage{greek}µ\selectlanguage{english}M 4-OHT (Supplementary Figure S1B). The mean
fluorescence intensity of GFP was measured using a flow cytometer. In
all conditions, the cell clone CHO/GEV\_1 had the highest mean
fluorescence intensity. Therefore, this cell clone was chosen for
subsequent experiments.

\textbf{3.2 Inducible expression of \emph{GFP} reporter gene in
CHO/GEV\_GFP cells}

We next investigated proliferation and gene expression responsiveness
using a \emph{GFP} reporter gene. A transposon vector containing the
GEV-responsive GFP expression unit was introduced into CHO/GEV cells and
integrated into the cell genome through transposition. Upon the addition
of 1.0 \selectlanguage{greek}µ\selectlanguage{english}M 4-OHT to drug-selected cells, GFP expression was induced in
all cells. Therefore, cloning was performed using a cell sorter. All six
tested clones showed equivalent GFP induction and proliferation
responsiveness upon 4-OHT addition. Based on these results, a
representative cell clone (CHO/GEV\_GFP) was used for further analysis.

CHO/GEV\_GFP cells were seeded in a 24-well plate to evaluate cell
proliferation and drug responses (Figure 2). Various concentrations of
E2-containing medium were used, and the medium was changed daily. As a
result, a concentration-dependent decrease in proliferation was observed
(Figure 2A). When 20 \selectlanguage{greek}µ\selectlanguage{english}M E2 was added, cell proliferation was completely
suppressed without decreasing cell viability. In our analysis
of\emph{GFP} expression induction, GFP-positive cells were observed from
the day after the addition of E2, and 80\%--90\% of GFP-positive cells
were observed throughout the culture period at E2 concentrations of 5 \selectlanguage{greek}µ\selectlanguage{english}M
or higher (Figure 2B). However, the mean fluorescence intensity was only
about twice that of the non-additive control, suggesting that the
expression level was insufficient (Figure 2C). Next, we attempted gene
expression induction using 4-OHT, an antagonist of E2. Cell toxicity was
observed at concentrations of 10 \selectlanguage{greek}µ\selectlanguage{english}M or higher (Supplementary Figure S2).
Therefore, we evaluated CHO/GEV\_GFP cells with a low concentration
range of 0.01--1 \selectlanguage{greek}µ\selectlanguage{english}M 4-OHT. As a result, a decreased proliferation rate
was observed and cells were able to proliferate without toxicity (Figure
2D). Analysis of GFP expression inducibility reveals that almost all
cells became GFP-positive from day 1 of culture at concentrations of 0.5
\selectlanguage{greek}µ\selectlanguage{english}M 4-OHT or higher (Figure 2E). The mean fluorescence intensity of GFP
reached its maximum on day 3 of culture, and a 2.5-fold higher
expression was observed with 4-OHT compared with E2 (Figure 2F). This is
attributable to the high affinity of 4-OHT for ERT2 compared with
E2.\textsuperscript{{[}32{]}} Thus, it was found that 4-OHT could induce
higher expression at concentrations more than 10-fold lower than E2.

\textbf{3.3 Inducible expression of scFv-Fc antibody gene in
CHO/GEV\_scFv-Fc cells}

The functionality of our artificial gene expression system mediated by a
transcription factor containing the estrogen-binding domain of the
estrogen receptor can be confirmed using a reporter gene. Thus, we
attempted to induce the production of a model antibody by adding E2 or
4-OHT (Figure 3). Plasmids containing the UAS/scFv-Fc expression unit
were linearized and introduced into CHO/GEV cells. After selection with
appropriate drugs, we obtained a pool of transformed cells and performed
cloning using the limiting dilution method. Among the 22 clones
obtained, we evaluated six clones that functioned correctly in response
to 0.1 \selectlanguage{greek}µ\selectlanguage{english}M 4-OHT and selected the one with the best cell proliferation
and scFv-Fc production responsiveness. This cell line, designated as
CHO/GEV\_scFv-Fc cells, was further investigated for proliferation and
production induction analysis.

Evaluation of CHO/GEV\_scFv-Fc cell proliferation in the presence of
1.25--5 \selectlanguage{greek}μ\selectlanguage{english}M E2 revealed no significant impact compared with the control
(Figure 3A). Measurement of scFv-Fc production showed a maximum of 1.0
\selectlanguage{greek}μ\selectlanguage{english}g/mL at 5 \selectlanguage{greek}μ\selectlanguage{english}M (Figure 3B), with a specific productivity of 5 pg
cell\textsuperscript{--1} day\textsuperscript{--1} (Figure 3C). Next, we
attempted to induce antibody production using 4-OHT. As 4-OHT
effectively induced reporter gene expression and protein production
levels, we expected a high induction effect for scFv-Fc production as
well. Addition of 4-OHT at concentrations of 0.01--0.1 \selectlanguage{greek}μ\selectlanguage{english}M resulted in
proliferation comparable with the control (Figure 3D). Evaluation of
scFv-Fc production reveals that scFv-Fc gene expression was induced with
0.05 \selectlanguage{greek}μ\selectlanguage{english}M or higher 4-OHT concentrations (Figure 3E), and the
concentration of scFv-Fc increased over the culture period. Following
the addition of 0.5 \selectlanguage{greek}μ\selectlanguage{english}M or 1.0 \selectlanguage{greek}μ\selectlanguage{english}M 4-OHT, the scFv-Fc concentration and
specific productivity reached 4.9 \selectlanguage{greek}μ\selectlanguage{english}g/mL and 24 pg
cell\textsuperscript{--1} day\textsuperscript{--1}, respectively (Figure
3F), which is 5-fold and 4-fold higher compared with E2. We next
examined the combined effect of E2 and 4-OHT addition. With a fixed E2
concentration of 20 \selectlanguage{greek}μ\selectlanguage{english}M, we attempted induction of both the reporter gene
and scFv-Fc gene with various 4-OHT concentrations under conditions of
cell growth inhibition (Supplementary Figure S3A and S3D). Overall
induction levels were lower compared with conditions with 4-OHT alone
(Supplementary Figure S3B-C and S3E-F). Therefore, the addition of 4-OHT
alone to induce production is suitable for antibody production in this
artificial gene expression system.

\textbf{3.4 Suspension culture of CHO/GEV\_scFv-Fc cells in serum-free
medium}

For production of useful substances using CHO cells, suspension culture
with serum-free medium is widely employed. Due to the absence of serum
components, the responsiveness of cell proliferation and exogenous gene
expression to the inducing agent 4-OHT may vary. As the drug response
may become more sensitive compared with conditions using
serum-containing medium, we investigated the impact of 4-OHT
concentration on scFv-Fc production for CHO/GEV\_scFv-Fc cells cultured
in a commonly used serum-free medium (Figure 4). CHO/GEV\_scFv-Fc cells
were seeded at a concentration of 1.0 \selectlanguage{ngerman}× 10\textsuperscript{6} cells/mL
in 2 mL of serum-free medium containing various concentrations of 4-OHT
and cultured in a 24-well cell culture plate. The medium was changed
daily to fresh medium containing 4-OHT, and cells were cultured for 4
days. Cell proliferation and viability are shown in Figure 4A and 4B,
respectively. Only a slight decrease in cell proliferation was observed
at 4-OHT concentrations below 0.1 \selectlanguage{greek}µ\selectlanguage{english}M, and the viability remained close
to 100\% throughout the culture period. In contrast, significant
decreases in cell proliferation were observed, particularly at 0.5 \selectlanguage{greek}µ\selectlanguage{english}M
and 1.0 \selectlanguage{greek}µ\selectlanguage{english}M. Additionally, cell viability was affected with the 1.0 \selectlanguage{greek}µ\selectlanguage{english}M
addition condition, dropping to around 80\% by day 4 of culture. To
evaluate scFv-Fc production over the culture period, we increased the
seeding cell density 10-fold compared with the serum-containing culture.
Under this condition, the scFv-Fc concentration reached 42 \selectlanguage{greek}µ\selectlanguage{english}g/mL (Figure
4C), which is 8.6-fold higher than that of serum-containing culture.
However, specific productivity was decreased in conditions with 0.5 \selectlanguage{greek}µ\selectlanguage{english}M
and 1.0 \selectlanguage{greek}µ\selectlanguage{english}M 4-OHT, which showed good values in serum-containing cultures.
With 0.1 \selectlanguage{greek}µ\selectlanguage{english}M 4-OHT, the maximum specific productivity was 13 pg
cell\textsuperscript{--1} day\textsuperscript{--1} (Figure 4D),
comparable to the production level observed in serum-containing cultures
with the same 4-OHT concentration (15 pg
cell\textsuperscript{--1}day\textsuperscript{--1}). These results
confirm that drug-inducible transgene expression can be achieved even
with serum-free medium, and the optimal concentration of 4-OHT is
approximately 10-fold lower compared with serum-containing culture.

\textbf{3.5 scFv-Fc production by CHO/GEV\_scFv-Fc cells in
semi-continuous culture at high density}

Next, we attempted continuous production under high-cell-density culture
conditions with 4-OHT induction using a bioreactor tube (Figure 5). As
described above, our results reveal that antibody production could be
induced without significantly impacting cell proliferation under the 0.1
\selectlanguage{greek}µ\selectlanguage{english}M 4-OHT condition. We also found that the maximum concentration and
specific productivity of scFv-Fc could be achieved within 3 to 4 days
after adding 4-OHT. Following pre-culture for cell proliferation without
4-OHT, we initiated suspension cultures with high cell density by
switching to 4-OHT-containing medium. Semi-continuous culture was
carried out for more than 2 weeks to evaluate the stability of induced
scFv-Fc production under the 0.1 \selectlanguage{greek}µ\selectlanguage{english}M 4-OHT condition. When seeded at a
density of 1.0 \selectlanguage{ngerman}× 10\textsuperscript{7} cells/mL, cell proliferation was
observed (Figure 5A) but a decreased level of cell viability was
maintained around 80\% (Figure 5B). scFv-Fc concentrations demonstrate
stable production in the medium at an average of 0.44 ± 0.05 g/L over
the duration of culture (Figure 5C), with a specific productivity of 37
± 4 pg cell\textsuperscript{--1} day\textsuperscript{--1} (Figure 5D).
In contrast, with a seeding density of 0.5 ×
10\textsuperscript{7}cells/mL, nearly 100\% viability was maintained
throughout the culture period (Figure 5B) and scFv-Fc production at a
concentration of 0.31 ± 0.03 g/L (Figure 5C) and specific productivity
of 57 ± 5 pg cell\textsuperscript{--1} day\textsuperscript{--1} (Figure
5D) was stably achieved. Analysis of metabolites in the spent medium
indicate glucose concentrations of 4.3 ± 0.9 mM and 2.0 ± 0.7 mM for
seeding densities of 0.5 × 10\textsuperscript{7} cells/mL and 1.0 ×
10\textsuperscript{7} cells/mL, respectively (Figure 5E). Lactate
concentrations were measured to be 34 ± 6 mM (0.5 ×
10\textsuperscript{7} cells/mL) and 66 ± 7 mM (1.0 ×
10\textsuperscript{7} cells/mL) (Figure 5F). Conversion rates from
glucose consumption to lactate production were estimated to be 52\% (0.5
× 10\textsuperscript{7} cells/mL) and 100\% (1.0 ×
10\textsuperscript{7}cells/mL).

scFv-Fc protein produced in the medium of semi-continuous cultures was
analyzed using SDS-PAGE. The results show that bands of the expected
molecular weight were detected under both reducing (Figure 6A and 6C)
and non-reducing (Figure 6B and 6D) conditions, indicating that the
protein was produced in an intact form (Figure 6).

Taken together, these results demonstrate that our estrogen
receptor-based inducible expression system can be applied for
high-cell-density semi-continuous culture to stably produce recombinant
proteins for over 2 weeks.

\textbf{4 DISCUSSION}

Recombinant protein production in \emph{Escherichia coli} cells has been
carried out using \emph{lac} -based inducible
promoters.\textsuperscript{{[}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,\textsuperscript{{[}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 \selectlanguage{greek}µ\selectlanguage{english}M
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\textsuperscript{--1}day\textsuperscript{--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\textsuperscript{--1}
day\textsuperscript{--1}).

In this gene induction system, GFP-positive cells with over 95\%
efficiency were observed from day 1 after 0.5 \selectlanguage{greek}µ\selectlanguage{english}M 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\textsuperscript{{[}32{]}}derived from the ligand-binding domain of
human ERalpha (amino acids 282--595; Accession No.
NP\_000116)\textsuperscript{{[}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.\textsuperscript{{[}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)\textsuperscript{{[}35{]}} of human ER[?] in
ERT2. Nuclear translocation of proteins can be controlled by the type of
nuclear localization signal, mutations, or their
placement.\textsuperscript{{[}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 \selectlanguage{ngerman}× 10\textsuperscript{7}cells/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 × 10\textsuperscript{7} 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\textsuperscript{--1}day\textsuperscript{--1}. With a seeding cell
density of 1.0 × 10\textsuperscript{7} 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 × 10\textsuperscript{7} 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\textsuperscript{--1}day\textsuperscript{--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.\textsuperscript{{[}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,\textsuperscript{{[}32{]}}transposase,\textsuperscript{{[}40{]}}
transcription factors,\textsuperscript{{[}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\textsuperscript{{[}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.

\textbf{ACKNOWLEDGMENTS}

We thank Edanz (https://jp.edanz.com/ac) for editing a draft of this
manuscript.

\textbf{DATA AVAILABILITY STATEMENT}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

\textbf{CONFLICT OF INTEREST STATEMENT}

The authors declare no commercial or financial conflict of interest.

\textbf{AUTHOR CONTRIBUTIONS}

Conceptualization, M.K.; methodology, M.R.R., Y.K., and M.K.;
validation, Y.K. and M.K.; formal analysis, M.R.R., Y.K., K.S., and
M.K.; investigation, M.R.R., K.S., S.C. and Y.A.; resources, Y.K. and
M.K.; data curation, Y.K. and M.K.; writing---original draft
preparation, M.R.R., Y.K., and M.K.; writing---review and editing,
M.R.R., Y.K., and M.K.; visualization, M.R.R., Y.K., and M.K.;
supervision, M.K.; project administration, M.K.; funding acquisition,
M.K. All authors have read and agreed to the published version of the
manuscript.

\textbf{FUNDING}

This work was supported by AMED under Grant Number JP20ae0101054h003 and
JSPS KAKENHI Grant Number JP20H00322.

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\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1} Estrogen receptor-based artificial gene expression
system.\textbf{(A)} Vector constructs of the Gal4-ERT2-VP16 (GEV)
expression vector (PB/chEF1\selectlanguage{greek}α/\selectlanguage{english}GEV\_Hyg) and gene-of-interest (GOI)
expression vectors (PB/UAS/GFP\_Zeo and PB/UAS/scFv-Fc\_Zeo).
P\selectlanguage{greek}\textsubscript{chEF1α}, \selectlanguage{english}Chinese hamster-derived elongation factor 1\selectlanguage{greek}α
\selectlanguage{english}promoter; Gal4-ERT2-VP16 (GEV), estrogen receptor-based artificial
transcription factor; Gal4, DNA binding domain of yeast transcription
factor; ERT2, ligand binding region of estrogen receptor mutant;
P\textsubscript{TK}, thymidine kinase promoter; Hyg, hygromycin
resistance gene; UAS, Gal4-linked upstream activation sequence;
P\textsubscript{min}, Ad major late promoter minimal region; GOI, target
gene; GFP, green fluorescent protein; anti-prion single-chain antibody
fused with the Fc-region of human IgG1 (scFv-Fc); P\textsubscript{SV40},
SV40 promoter; Zeo, zeocin resistance gene. \textbf{(B)} GEV expressed
under a constitutive promoter accumulated in the cytoplasm. In the
presence of \selectlanguage{greek}β-\selectlanguage{english}estradiol (E2) or 4-hydroxytamoxifen (4-OHT), GEV
translocates into the nucleus and binds to UAS-responsive elements. The
activated GEV induces expression of the target gene (scFv-Fc) under
control of the UAS/P\textsubscript{min} artificial promoter. Translated
scFv-Fc protein is extracellularly secreted.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2} Inducible expression of reporter gene using E2 or
4-OHT. Growth responses of CHO/GEV\_GFP cells upon the addition of E2
\textbf{(A)} or 4-OHT \textbf{(D)} . Symbols are indicated as follows.
\textbf{(A)} Gray squares, no addition; closed squares, 1.25 \selectlanguage{greek}µ\selectlanguage{english}M; closed
triangles, 2.5 \selectlanguage{greek}µ\selectlanguage{english}M; closed circles, 5 \selectlanguage{greek}µ\selectlanguage{english}M; closed diamonds, 10 \selectlanguage{greek}µ\selectlanguage{english}M; open
circles, 20 \selectlanguage{greek}µ\selectlanguage{english}M.\textbf{(D)} Gray squares, no addition; closed squares,
0.01 \selectlanguage{greek}µ\selectlanguage{english}M; closed triangles, 0.05 \selectlanguage{greek}µ\selectlanguage{english}M; closed circles, 0.1 \selectlanguage{greek}µ\selectlanguage{english}M; closed
diamonds, 0.5 \selectlanguage{greek}µ\selectlanguage{english}M; open circles, 1 \selectlanguage{greek}µ\selectlanguage{english}M. Percentage of GFP-positive cells
at each concentration of E2 \textbf{(B)} or 4-OHT \textbf{(E)} . Open
columns, day 1; light gray columns, day 2; dark gray columns, day 3;
closed columns, day 4. Mean green fluorescence intensity at each
concentration of E2 \textbf{(C)} or 4-OHT \textbf{(F)} . The distinction
of columns is the same as \textbf{(B)} and \textbf{(E)} .\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3} Inducible expression of scFv-Fc gene using E2 or
4-OHT. Growth response of CHO/GEV\_scFv-Fc cells upon the addition of E2
\textbf{(A)}or 4-OHT \textbf{(D)} . scFv-Fc concentrations in media of
cells cultured with each concentration of E2 \textbf{(B)} or 4-OHT
\textbf{(E)} . Specific production rate at each concentration of E2
\textbf{(C)} or 4-OHT \textbf{(F)} . The distinction between symbols
\textbf{(A and D)}and columns \textbf{(B, C, E and F)} is the same as
described for Figure 2.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4} Serum-free culture of CHO/GEV\_scFv-Fc cells with the
addition of 4-OHT. Growth response \textbf{(A)} and
viability\textbf{(B)} during culture. Symbols: gray squares, no
addition; closed squares, 0.01 \selectlanguage{greek}µ\selectlanguage{english}M; closed triangles, 0.05 \selectlanguage{greek}µ\selectlanguage{english}M; open
circles, 0.1 \selectlanguage{greek}µ\selectlanguage{english}M; closed diamonds, 0.5 \selectlanguage{greek}µ\selectlanguage{english}M; closed circles, 1 \selectlanguage{greek}µ\selectlanguage{english}M. scFv-Fc
concentration\textbf{(C)} and specific production rate \textbf{(D)}
during culture. The distinction of columns is the same as described for
Figure 2.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5} Continuous production of scFv-Fc in semi-continuous
cultures with high cell density. CHO/GEV\_scFv-Fc cells (10 mL) induced
with 0.1 \selectlanguage{greek}µ\selectlanguage{english}M 4-OHT were seeded in 50-mL bioreactor tubes at cell
densities of 0.5 \selectlanguage{ngerman}× 10\textsuperscript{7} cells/mL and 1.0 ×
10\textsuperscript{7} cells/mL. Cells were counted the day after
seeding. The same seeding density was prepared in fresh medium
containing 0.1 \selectlanguage{greek}µ\selectlanguage{english}M 4-OHT and then reseeded. \textbf{(A)} Viable cell
density. \textbf{(B)} Viability. \textbf{(C)} scFv-Fc production
concentration. \textbf{(D)} Specific production rate.
\textbf{(E)}Glucose concentration. \textbf{(F)} Lactate concentration.
Symbols: open circles, 0.5 \selectlanguage{ngerman}× 10\textsuperscript{7} cells/mL; closed
circles, 1.0 × 10\textsuperscript{7} cells/mL.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6} SDS-PAGE analysis of medium samples harvested from
semi-continuous cultures. Medium samples (10 \selectlanguage{greek}µ\selectlanguage{english}L) from cultures seeded at
cell densities of 0.5 \selectlanguage{ngerman}× 10\textsuperscript{7} cells/mL \textbf{(A,
B)}and 1.0 × 10\textsuperscript{7} cells/mL \textbf{(C, D)} were applied
to SDS-PAGE analysis under reducing \textbf{(A, C)} and
non-reducing\textbf{(B, D)} conditions. Lane MW, molecular weight
markers; lanes 1--7, medium samples harvested at day 2 (lane 1), day4
(lane 2), day 6 (lane 3), day 8 (lane 4), day 10 (lane 5), day 12 (lane
6), and day 14 (lane 7); lane N, fresh culture medium; lane P, purified
scFv-Fc (2 \selectlanguage{greek}µ\selectlanguage{english}g).

\selectlanguage{english}
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\end{document}

