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.