Introduction
For decades, therapeutic proteins have been widely used in treatment of
a wide variety of clinical indications, including cancers,
autoimmunity/inflammation, exposure to infectious agents, and genetic
disorders and have formed a huge commercial market. According to the
market financial report disclosed, the market sales of monoclonal
antibodies have increased by 7.2% -18.3% every year since 2013. Based
on the current market value, it is expected to be valued at $137 to
$200 billion by 2022 (Grilo & Mantalaris, 2019). Mammalian cells are
the expression system of choice, based on which 60-70% of biological
drugs are currently produced (Kunert & Reinhart, 2016). However,
mammalian expression systems still harbor limitations in terms of growth
capacity, culture time, and product yield compared to bacterial or
yeast-based production hosts (Tripathi & Shrivastava, 2019). Therefore,
to meet the growing demand for recombinant therapeutic proteins,
improving commercial metrics such as titer, yield, or productivity is at
the core of bioprocess development of mass culture of mammalian cells.
Chinese hamster ovary cells are currently the preferred host cells for
the production of recombinant therapeutic proteins, mainly because CHO
cells have accurate post-transcriptional modifications, e.g., expressing
glycosylated therapeutic proteins that are closest to natural protein
molecules in terms of molecular structure, physicochemical properties,
and biological function (O’Flaherty et al., 2020). Moreover, CHO cells
secrete therapeutical proteins outside the cells, and rarely secrete
their endogenous proteins, which is convenient for the downstream
separation and purification. Over the last 20 years, while it has been
witnessed that with advances in cell line development and bioprocessing,
CHO cells have achieved high yield and quality of monoclonal antibody
(mAb) expression, improving CHO cell productivities remains a major
concern for the production of therapeutic proteins (Mauro Torres &
Dickson, 2022). A variety of strategies based on cell proliferation
control such as low temperature, high osmolarity, or the use of
productivity enhancers such as valeric acid, sodium butyrate, or small
molecule inhibitor, have successfully been implemented to increase cell
specific production rates in CHO cell cultures (Sunley & Butler, 2010).
For example, Alhuthali et al. found that increasing osmolarity by adding
sodium chloride (320 mOsm kg-1 to 470 mOsm
kg-1) can increase cell volume, protein synthesis and
folding capacity following an increase in the volume of organelles such
as the endoplasmic reticulum, thus resulting in a faster rate of protein
secretion (Alhuthali, Kotidis, & Kontoravdi, 2021). In terms of
cytostatic chemicals, Park et al reported that the addition of 1.5 mM
valeric acid to rCHO cell culture can reduce the specific growth rate
(μ) of cells, increase the culture time, and significantly increase the
maximum mAb concentration by 2.9-fold in a dose-dependent manner (0-2.0
mM) (Park, Noh, Woo, Kim, & Lee, 2016). Avello et al. modulated cell
proliferation by adding sodium butyrate (NaBu) to regulate cell cycle
checkpoint (G1/S or G2/M), resulting in a 3-fold increase in the
specific rate of antibody production for recombinant human tissue-type
fibrinogen activator (rh-tPA) (Avello et al., 2017). Although such
cytostatic chemicals can significantly increase antibody yields or
specific cell production rates, these chemicals are somewhat cytotoxic,
promote cell aggregation, and induce apoptosis (Nör et al., 2013).
Moreover, cytostatic chemicals have been reported to adversely affect
the quality of antibodies. For example, the addition of NaBu has been
observed to increase the charge heterogeneity of antibodies and reduce
antibody galactosylation and sialylation (Jong Kwang Hong, Lee, Kim, &
Lee, 2014).
Increasing evidence has shown that temperature downshift of mammalian
cell culture can increase recombinant protein production without
interfering with antibody quality (McHugh, Xu, Aron, Borys, & Li,
2020). For example, in order to maximize productivity for an inducible
CHO cell line expressing rituximab, Mellahi et al. performed the
temperature downshift from 37°C to 30°C during the high cell density
production stage (1 × 107 cells/mL) and achieved the
maximum yield of 1.8 g/L in the 2 L bioreactor (Mellahi et al., 2019).
In perfusion cultivation with recombinant CHO cells for the production
of human factor VIII (FVIII), Juliana et al. achieved a 6-fold increase
in the cellular antibody productivity by combining temperature downshift
from 37°C to 31°C with valeric acid supplementation when the viable cell
density reached 2 × 107 cells/mL (Coronel et al.,
2019). In recent years, there has been increasing research on the
mechanism by which the temperature downshift of CHO cell culture during
the antibody production phase can significantly increase both product
yield and specific cell production rate. For example, Torres et al.
found that the upregulation of two transcriptional regulators, Myc and
XBP1s, is associated with improved cell growth and viability under
low-temperature conditions, and observed synchronous metabolic shift
from production to lactate consumption and from consumption to glutamine
production, which might be a relevant metabolic marker for the increased
antibody production (M. Torres et al., 2018). Wu et al. found that the
increase of cell-specific production rate is consistent with the
increase in pyruvate carboxylase (PC) activity induced by temperature
downshift, and hypothesized that the increase in PC activity is
associated with a more active TCA pathway, which provides more energy
for the cellular production process (Zou, Edros, & Al-Rubeai, 2018).
Recently, Torres et al. explored changes in metabolic profiles including
glucose and amino acids in the presence of low-temperature culture and
found that low temperature exerted an opposite effect on the consumption
of glucose and specific amino acids such as serine, leucine, valine, and
isoleucine. However, no significant effect was observed with respect to
cell specific antibody production rate by CHO cells exposed to the lower
temperature 32°C (Mauro Torres & Dickson, 2022). To the best of our
knowledge, most of the research on the mechanisms associated with
low-temperature strategies is based on changes at the level of some
genes affecting cellular metabolism, or only on the metabolism of major
nutrients such as glucose, glutamine, and lactate, whereas studies on
the effects of low temperature on cellular metabolism are far from
complete. Therefore, it is necessary to fully reveal the metabolic
regulation mechanism by focusing on the changes in the global metabolic
map of CHO cells induced by low temperature perturbation.
Performance monitoring of a mammalian cell-based bioprocess is of
paramount importance to ensure efficient and reliable process control
and thus to improve production performance and product quality. In
recent years, researchers have developed a variety of sensors such as
Raman spectroscopy, near-infrared spectroscopy, live cell sensors, etc.
for online detection of extracellular metabolites, which have improved
the monitoring and analysis of cellular metabolic environmental
parameters during CHO cell culture. For example, online monitoring of
extracellular glucose concentration by Raman spectroscopy and control of
glucose concentration based on a dynamic feed algorithm by adding a feed
medium at a lower glucose set point (~11mM) enables a
dynamic feeding strategy for the CHO cell culture process (Domjan et
al., 2020). Viable cell density can be real-time monitored using
capacitance sensor based on on-line permittivity signals during CHO cell
fed-batch culture (Hofer, Kroll, Barmettler, & Herwig, 2020). Although
these methods are useful for online monitoring of extracellular
environment in CHO cells, there is limited understanding of the
intracellular metabolic activity. To address this, it is vitally
important to develop biosensors that monitor key metabolites and
coenzymes in intracellular metabolic pathways. Such metabolite
biosensors are often defined as genetically encoded proteins or
RNA-based sensors that control protein expression and activity by
regulating transcription rates, translation rates, or post-translational
parameters to produce a detectable phenotype (Liu, Evans, & Zhang,
2015). Among them, protein activity-based biosensors combine metabolite
interactions with enzymes or molecules with fluorescent activity based
on which metabolite level can be quantified (Z. Zhang, Cheng, Zhao, &
Yang, 2020).
Redox reactions are central to cellular metabolism and play an important
role in key pathways of biological processes, including cellular
maintenance, protein folding, and energy metabolism (Sinharoy,
McFarland, Majewska, Betenbaugh, & Handlogten, 2021). Nonetheless, the
redox state of a cell indicated by the ratio of oxidized/reduced
antioxidants such as NAD+/NADH and GSSG/GSH is highly
dynamic which makes it difficult to be real-time tracked. Reactive
oxygen species (ROS) are the main driver of oxidative stress and are
usually scavenged by the antioxidant glutathione, and two glutathione
molecules are detoxified using NADPH dimerization into GSSG, making the
GSSG/GSH ratio critical in the assessment of redox reactions. As a
hydrogen transmitter for many intracellular reactions, NADPH acts as an
intracellular reducing agent and hydrogen anion donor, and its amount is
also one of the critical parameters characterizing the intracellular
redox state. NAD+ and NADH participate into most of
the redox reactions involved in the pathways of glycolysis, TCA cycle,
and oxidative phosphorylation, and their ratio
NAD+/NADH is an important indicator for evaluating the
intracellular redox state. For example, an increased
NAD+/NADH ratio indicates that the cell is
experiencing oxidative stress, which is a frequent phenomenon occurring
at the onset of antibody production with CHO cell culture (Zhao & Yang,
2016).
Redox imbalance is detrimental to cell growth and product formation. For
instance, Ali et al. found that triggered by intracellular oxidative
stress, detrimental metabolic responses such as endoplasmic reticulum
stress and uncontrolled amino acid-related signaling pathways gave rise
to lactate accumulation and reduced antibody production (Ali et al.,
2019). Handlogten et al. found that as dissolved oxygen tension
increased (20%-75%), intracellular
H2O2 increased and mitochondrial
function was negatively affected due to increased ROS level. As a
consequence, the oxidative stress led to reduced recombinant protein
production (Michael W. Handlogten, Zhu, & Ahuja, 2018). Subsequently,
in an attempt to prevent the productivity loss of monoclonal antibodies
with CHO cell cultures as the oxidative stress arises, Handlogten et al.
monitored the redox potential of the cell culture process using an
online redox electrode and dynamically regulated it via increasing
dissolved oxygen and copper ion concentrations for feedback control (M.
W. Handlogten, Wang, & Ahuja, 2020). However, this approach can only
control changes in redox potential in the culture environment and cannot
precisely quantify intracellular redox state of cells (Grimalt-Alemany,
Etler, Asimakopoulos, Skiadas, & Gavala, 2021). Therefore, it is
essential to develop redox biosensors to monitor intracellular redox
levels in real-time to achieve dynamic feedback regulation. Recently,
Zhu et al. developed a thiol-based fluorescent redox biosensor to
monitor the ratios of reduced and oxidized glutathione (GSH/GSSG) which
is the important antioxidant in CHO cells, and showed the potential of
the biosensor to monitor biological redox processes in real-time.
However, they only potentiated the use of the biosensor for this purpose
and no data regarding the GSH/GSSG ratio were reported in actual CHO
cultivation process (McFarland, Zhu, Sinharoy, Betenbaugh, &
Handlogten, 2022). Furthermore, to the best of our knowledge, few
studies have ever been dealt with the mechanism between in vivo redox
state and antibody productivity during CHO cell cultures.
In this study, we evaluated the differences in cell growth, antibody
titer, antibody quality, and cell-specific antibody productivity between
high-producing (CHO-K1 HP) and low-producing (CHO-K1 LP) cell lines
during fed-batch cultures with the temperature downshift approach. To
gain a comprehensive understanding of temperature-induced differences in
cellular metabolism, extracellular and intracellular metabolomics data
were used to identify key metabolic and related metabolic pathways.
Further, to monitor and validate the observed difference in the
redox-associated metabolic pathway, intracellular
NADH/NAD+ and NADPH real-time fluorescent quantitative
biosensors were developed for this purpose.
Materials and methods
2.1 Cell lines, medium and cell culture
All cell lines used in this study were proprietary CHO suspension cell
lines, involving a low-producing cell line (CHO-K1 LP) and a
high-producing cell line (CHO-K1 HP), derived from the same CHO-K1 host,
stably expressing a mAb. Cells were stably transfected with proprietary
DNA vectors encoding recombinant humanized antibodies using the
glutamine-synthetase (GS) expression system (Lonza, Basel, Switzerland).
The proprietary chemically synthesized basal medium, Dynamis (Thermo
Fisher Science) and the supplemental medium, Efficient Feed™ C+ AGT™
(Thermo Fisher Science) were used in all experiments. CHO cell lines
were revived and expanded in shake flasks using Dynamis medium under
standard conditions of 37°C, 5% CO2, 120 rpm. Cells
were passaged every 3-4 days.
Shake flask and bioreactor Fed-batch culture
Shake flask culture
Fed-batch cultures of HP and LP CHO cells were performed in 250 mL shake
flasks with an initial working volume of 50 mL at 37°C and 5%
CO2 in a shaker with 120 rpm. CHO cells were inoculated
with Dynamis medium at 1 × 106 cells/mL and Efficient
Feed™ C+AGT™ feed medium was added at 10% of the initial culture volume
from day 3 and then every other day, while glucose concentrate was added
to maintain the glucose concentration of 2 ± 0.2 g/L in the culture
medium. The temperature of HP and LP CHO cell cultures was shifted from
37°C down to 33°C at the late stage of exponential cell growth when the
viable cell density reached 1.5 × 107 cells/mL, and
all the other culture conditions remained unchanged.
Bioreactor culture
HP CHO cells were expanded by 500 mL shake flask culture, and the cells
in the exponential growth phase were inoculated into a 1.5 L bioreactor
with an initial cell density of 1 × 106 cells/mL and a
working volume of 800 mL. The culture conditions were set as follows:
pH, 7.05 ± 0.2 (CO2 sparging and 0.2 M
NaHCO3); DO%, 40% ~ 50%; impeller
speed, 50 rpm; airflow rate, 100~200 mL/min. The feeding
strategy and temperature downshift strategy were performed the same as
for the shake flask culture.
Cell cycle analysis
The cell cycle was determined using a flow cytometer (Beckman Coulter).
Daily samples were taken and DNA content was determined using a
propidium iodide (PI) method. Each day 20000 events were recorded inside
the ‘singlet’ population gate defined by a PI-W and a PI-A plot, thus
excluding debris, doublets, and aggregates. Flow cytometry data were
analyzed using FlowJo v10.0 software. Results were presented as the
percentage of cells in G1/G0, S and M phases of the cell cycle.
Metabolite Analysis
Analysis of extracellular metabolites
During the cell culture, samples were taken every 12-hours, and the
concentrations of glucose, lactate and ammonia in the supernatant were
measured using a biochemical analyzer (Roche, Basel, CH). The
concentration of amino acids in the supernatant was measured using an
amino acid analyzer (Sykam, S-433D, Germany).
Intracellular metabolomics analysis
Cells were collected on day 5 and day 8 in both constant and temperature
downshift cultures, with 3 biological replicates per group and
approximately 1×107 cells per sample. The main
processes of the non-targeted metabolomics assay included metabolite
extraction, GC-MS analysis, database analysis, and processing. After
LC-MS detection and database searching, the independence of the samples
was determined using principal component analysis. Orthogonal partial
least squares discriminant analysis (OPLS-DA) was used to screen for
differential metabolites. The screening criteria were a VIP value of
>1 for PC1 in OPLS-DA and a t-test with a p-value
<0.05. Next, differential metabolites were hierarchically
clustered using the R ComplexHeatmap package (2.4.3). The differential
metabolites were analyzed by the KEGG database
(https://www.kegg.jp/kegg/pathway.html). Bubble plots for KEGG analysis
were plotted using the ggplot2 package (3.3.2) (Tables S1 andS2 ).
Analysis of antibody concentration (Titer)
The antibody concentration was determined by high-performance liquid
chromatography (Shimadzu) with a ProteinA-5PW 4.6×3.5, 20 μm (Tosoh,
Japan) column using the following detection method: mobile phase A: 50
mM sodium phosphate/150 mM sodium chloride, pH 7.0; mobile phase B: 100
mM glycine/150 mM sodium chloride, pH 2.5. Gradient elution
(Table S3 ), injection volume 20 μL, column temperature 30°C, UV
detection at 280 nm. Sample concentrations were determined using
standard curves generated by linear regression analysis.
Physicochemical analysis
Cell supernatants were collected on the last day of culture and
antibodies were purified using HiTrap MabSelect PrismA (Cytiva)
(Table S4 ). For molecular size heterogeneity, samples were
detected using TSK G3000WXL 7.8 × 300 nm, 5 μm (Tosoh, Japan). In
addition, purity was analyzed by CE-SDS under non-reducing conditions
using a capillary electrophoresis instrument. For charge heterogeneity,
samples were analyzed using a full column imaging capillary isoelectric
focusing electrophoresis instrument (RJBIO iCE280 Advance)
(Tables S5 and S6 ).
Analysis of intracellular ROS
According to the protocol provided by the manufacturer, the amount of
ROS produced by cells under the experimental conditions was determined
using the Reactive Oxygen Species Assay Kit (E004-1-1, Nanjing
Jiancheng, Nanjing, CN). Briefly, the fluorescent dye DCFH-DA
(2,7-Dichlorofuorescin Diacetate) itself was non-fluorescent and can
freely cross the cell membrane. When it entered the cell, it is
hydrolyzed by the relevant intracellular esterases to DCFH
(Dichlorofluorescin), which could not permeate the cell membrane and
accumulates in the cell. Intracellular reactive oxygen species oxidize
the non-fluorescent DCFH to form green fluorescent DCF, which was
detected by flow cytometry (Beckman Coulter) using 2000 events per
sample, with the excitation wavelength of 485 nm and the emission
wavelength of 525 nm.
Analysis of real-time fluorescence quantitative biosensor
SoNar, iNapc, and iNap1 sensors were infected into CHO cells by
recombinant lentivirus and adenovirus, respectively, and positive cells
capable of stable expression were screened using puromycin
(Figures S1 and S2 ). SoNar and iNap1 sensors were
constructed for quantifying intracellular NAD+/NADH
ratio and intracellular NADPH amount, respectively, while iNapc sensor
was used for correcting pH perturbations in culture. All intracellular
biosensors were correctly characterized (Figures S3-S5 ). All
samples were taken every 24 hours and biosensor expressions were
detected by flow cytometry (Beckman Coulter), with 2000 events collected
per sample and the detection channels were FITC and V525-KrO-A. However,
the fluorescence of these biosensors is affected by pH fluctuations when
excited at 488 nm, and is more pH resistant at 405 nm, so it needs to be
pH corrected. The formulae were as follows.
\begin{equation}
\frac{F_{405nm}}{F_{488nm}}\ pH\ corrected=\frac{\text{SoNar\ }F_{405nm}-Blank\ F_{405nm}}{\begin{matrix}\text{SoNar\ }F_{488nm}-Blank\text{\ F}_{488nm}\\
\end{matrix}}/\frac{\text{iNapc\ }F_{405nm}-Blank\ F_{405nm}}{\begin{matrix}\text{iNapc\ }F_{488nm}-Blank\ F_{488nm}\\
\end{matrix}}\nonumber \\
\end{equation}\begin{equation}
\frac{F_{405nm}}{F_{488nm}}\ pH\ corrected=\frac{iN\text{ap}1\ F_{405nm}-Blank\ F_{405nm}}{\begin{matrix}iN\text{ap}1\ F_{488nm}-Blank\text{\ F}_{488nm}\\
\end{matrix}}/\frac{\text{iNapc\ }F_{405nm}-Blank\ F_{405nm}}{\begin{matrix}\text{iNapc\ }F_{488nm}-Blank\ F_{488nm}\\
\end{matrix}}\nonumber \\
\end{equation}Statistical analysis
All data in this study were expressed as mean ± standard deviation (mean
± SD). Significance analysis was performed using GraphPad Prism 8.3.0,
(*) p<0.05, (**) p<0.01, (***) p<0.001 and
(****) p<0.0001.
- Results
- Effect of temperature downshift on cell growth, antibody titer, and
antibody quality of CHO cells in shake flasks
- Effect of temperature downshift on cell growth
To experimentally investigate the effect of temperature downshift on
cell metabolism and antibody production performance, we performed
fed-batch cultures of two CHO cell lines (HP and LP), where cells were
grown at 37°C and then subjected to temperature downshift to 33°C when
the live cell density reached 1.5 × 107 cells/mL. We
found that temperature downshift imposed a significant impact on CHO
cell growth, antibody titer, and cell metabolism.
As shown in Figure 1 , the culture constantly cultivated at 37°C
exhibited the highest viable cell density (VCDmax) and
the specific cell growth rate (μ). After the two cell lines were
inoculated into the fresh medium, there was no significant stagnation
period in cell growth, but rather a direct entry into the exponential
growth phase, with viable cell density rapidly reaching the maximum on
day 6 (HP:1.8 × 107 cells/mL; LP:2 ×
107 cells/mL) and cell viability gradually decreasing
below 90% as the culture time progressed. In contrast, after performing
the temperature downshift, the maximum viable cell density and
cell-specific growth rate decreased significantly (around 1.5 ×
107 cells/mL for both HP and LP CHO cells), while at
the same time showing an increase in cell viability and longer culture
time, with cell viability above 90% throughout the cultivation period
(Figure 1a , b ). This is consistent with the previously
observed common features such as slow cell growth, increased cell
viability, and long cell survival time of CHO cell culture exposed to
low temperatures (Mauro Torres & Dickson, 2022). Meanwhile, the
characteristics of cell growth in low-temperature cultures were
reflected in the cell cycle. As the temperature shifted down, the
percentage of cells in the G0/G1 phase gradually increased until the
maximum value reached on the last day of culture (HP:79.6%; LP:81.3%);
cells in S phase gradually decreased as the culture progressed, with HP
cells reaching the lowest percentage of 9.2% on day 10 and LP cells
reaching the lowest percentage of 8.4% on day 9; the percentage of
cells in the G2 phase remained relatively constant, with a slight
decrease in the later stages. Throughout the culture, the vast majority
of cells were in G0/G1 phase, especially in the late and stable phases
of exponential growth, thus establishing a trend of G0/G1
synchronization (Figure 1c , d ). Hence, temperature
downshift can cause cells to arrest at G0/G1 phase, thus providing a
possibility for the transition from cell proliferation to antibody
production (Tossolini, Lopez-Diaz, Kratje, & Prieto, 2018).
Effect of temperature downshift on antibody titer
The effect of temperature downshift on antibody titer was significant
(p<0.001), with the low-temperature culture showing higher
antibody yield than the constant temperature culture. As shown inFigure 1f , after downshifting the temperature, the final
antibody titers of HP and LP was 2.40 g/L and 1.76 g/L, which was about
1.5 and 1.3 times higher than that of the constant temperature culture,
respectively. The temperature was decreased from 37°C to 33°C on day 5,
and the difference in antibody titer between temperature downshift and
constant-temperature culture was not significant during the early stage
of low-temperature culture (day 5-day 8) but increased significantly
from day 8 on (Figure 1e ).
With respect to the cell specific antibody production rate
(qp), it could be divided into two stages, cell
proliferation stage and antibody production stage. In the early stage of
culture (day 0-day 5), cells proliferated rapidly, and few antibodies
were synthesized at this stage, while integrated viable cell density
(IVCD) rose rapidly, resulting in lower qp, and there
was no significant difference in qp between the two
culture environments. IVCD refers to the integral of viable cell density
over time, which indicates the cell growth status and proliferation. In
the antibody generation stage (day 5-day 10), the viable cell density
reached the peak and thereafter maintained at a lower value relative to
the maximum VCD, and qp was rapidly increased. Compared
to constant temperature culture, the qp of HP and LP CHO
cell cultures undergoing temperature downshift was increased by 81.2%
and 35.5% to 30.8 and 18.7 pg/cell/day, respectively (Figure
1g , h ). Taken above, temperature downshift imposed a
significant effect on antibody titer, and the increase in antibody titer
was mainly ascribed to an increase in the specific antibody production
rate.
Analysis of extracellular metabolites
The above significant differences in cell growth and antibody titer upon
temperature downshift can in principle give rise to the changes in the
concentrations of extracellular glucose, lactate, ammonia, and amino
acids. Figure 2 shows the changes in the concentrations of
glucose, lactate, and ammonia under both constant and temperature
downshift cultures. The results showed that the changes in lactate and
ammonia concentrations were significantly different under different
culture modes. To maintain sufficient carbon source for cell growth and
product formation during the culture, the glucose concentration was
maintained above 2 g/L by feeding of glucose concentrate (Figure
2e ). From day 5 on, the glucose consumption rate (qs)
was slightly higher in constant temperature than in the temperature
downshift culture, mainly because low temperature inhibited cell
metabolism and thus metabolite consumption rate was also significantly
lower, which was consistent with the phenomenon of lower
qs observed in the low-temperature production of human
recombinant clotting factors (Coronel et al., 2019).
In contrast, the differences in lactate metabolism were more pronounced,
as lactate in the constant temperature culture tended to gradually
increase, reaching a maximum of 1.83 g/L on day 3, after which the
lactate concentration was maintained at around 1.8 g/L. However, the
lactate metabolism showed a trend of increasing and then decreasing in
the temperature downshift culture, indicating that the cells reabsorbed
lactate as the carbon source after lowering the temperature, and the
cell metabolism switched from lactate production to lactate consumption
type, reaching a minimum value of 0.3 g/L on day 10 (Figure
2a ). During mammalian cell culture, higher concentrations of lactate
can inhibit cell growth and negatively affect antibody production and
quality (Wilkens, Altamirano, & Gerdtzen, 2011), whereas
low-temperature culture reduces the concentration of lactate in the
culture by shifting the cells from lactate production to lactate
consumption, which is one of the advantages of downshifting culture
temperature (McHugh et al., 2020). Changes in the type of lactate
metabolism are dependent on the intracellular redox state, which is an
important factor in determining the direction of the reaction between
pyruvate and lactate, implying that changes in redox potential might
drive lactate consumption.
The ammonia concentration increased gradually throughout the culture
process and could reach 4 mmol/L in the constant temperature culture at
the end of the culture, while the ammonia concentration increased slowly
in the temperature downshift culture, reaching about 3 mmol/L on day 12,
which was significantly lower than that in the constant temperature
culture (Figure 2c ). Different cell lines might have different
degrees of tolerance to ammonia, and Chitwood et al. showed that ammonia
concentrations of 4-8 mmol/L may severely inhibit normal cell growth
(Chitwood et al., 2021). However, this threshold was not reached in both
culture modes, thus implying there was no adverse effect on cell growth.
Figure 3 shows the concentration of extracellular amino acids
in both constant and temperature downshift culture. Compared with
constant temperature culture, the consumption of extracellular amino
acids such as valine, cystine, leucine, isoleucine, glutamic acid,
aspartic acid, asparagine, and serine decreased after downshifting
temperature; In glutamate-based media, asparagine and serine play a
central role in the provision of nitrogen sources (Duarte et al., 2014).
Consistent with this, as shown in Figure 3 , asparagine was the
most consumed amino acid, and aspartic acid was generated through
asparagine deamidation, however, which was still continuously consumed
and did not accumulate, indicating that it was mostly used for cell
growth and product synthesis. This is consistent with previous findings
that aspartate consumption has been observed to be positively associated
with antibody productivity (Yao et al., 2021). The consumption of
cystine in temperature downshift cultures was significantly lower than
in the control, which indicated there was more pronounced metabolic
stress in the presence of high temperature. Furthermore, sufficient
extracellular cysteine concentration under temperature downshift culture
might allow to cope with high productivity associated stress. For
example, Ali et al. showed that sufficient cysteine in the medium can
support high productivity (Ali et al., 2019), mainly due to the
deleterious effects of cysteine depletion on antioxidants (GSH, taurine)
and increased accumulation of ROS in the endoplasmic reticulum (ER),
which further leads ER stress as well as oxidative stress. Consistent
with higher antibody productivity under temperature downshift cultures,
increased consumption of proline, lysine, arginine was observed in spite
of CHO cell line used in this study. On the contrary, alanine and
glycine were observed to be gradually increased throughout the
cultivation, and their secretions were positively correlated with
culture temperature. This accumulation had previously been found in
association with the higher consumption of asparagine (Selvarasu et al.,
2012).
Scale comparability of temperature downshift strategy with
high-producing CHO cell line in the 1.5 L bioreactor
Bioreactor scale-up with temperature downshift strategy
Cell culture in shake flasks is usually achieved by surface aeration for
oxygen supply and pH control, and oxygen tends to be a limiting factor
when viable cell densities are high (1 × 107cells/mL). Moreover, pH cannot be controlled within a stable range due
to active cell metabolism and high lactate production as a result of
high viable cell densities. To address these limitations, we scaled up
the temperature downshift strategy with the HP CHO cell line to a 1.5 L
bioreactor and compared the differences in cell growth, antibody titer,
antibody quality and metabolic regulation across the scales.
As shown in Figure 4 , the measured viable cell density,
cellular vitality, and antibody titer are given. In the constant
temperature culture, the viable cell density in the reactor reached a
maximum of 1.6 × 107 cells/mL on day 8, which was 11%
lower than that in the shake flask (1.8 × 107cells/mL) (Figure 4a ), and the cell viability decreased rapidly
from day 8 until the end of the culture on day 12 when it dropped below
80% (Figure 4b ). However, under temperature downshift
cultures, the viable cell density reached a maximum of 2.3 ×
107 cells/mL on day 6, which was 1.4 times higher than
that in the shake flask, and the cell viability remained above 95%
throughout the culture period until the end of the culture, which was
slightly higher than that in the shake flask. This showed that the
difference in cell growth during bioreactor fed-batch culture was not
only dependent on different temperature control strategies but also on
different CHO cell lines with differential metabolic capacities such as
antibody productivity across the scales. In addition, high cell-density
CHO cells in general have extremely high oxygen demand, whereas
dissolved oxygen is very likely to be limited in shake flask, which
aggravates lactate accumulation and reactive oxygen species (ROS)
generation (Michael W. Handlogten et al., 2018), which may be one of the
reasons for limiting cell growth. In contrast with this, dissolved
oxygen in the bioreactor was controlled between 40% and 50% to provide
sufficient oxygen for cell metabolism, thus increasing the density of
living cells. However, the highest viable cell density and cell
viability in the bioreactor were always lower than those in the shake
flask under the constant temperature culture, which may be caused by the
accumulation of by-products that inhibited cell growth.
Figure 4c shows the antibody titer of the cells in the shake
flask system and the reactor system. The antibody titer in the
bioreactor was significantly higher than in the shake flask, regardless
of cultivation mode (Figure 4d ). In the bioreactor system, the
final titer of the antibody in the constant temperature culture was 2.5
g/L, 1.36 times that of the shake flask culture; while in the
temperature downshift culture, the final titer of the antibody was 3.3
g/L, 1.4 times that of the shake flask culture, and also 1.32 times that
of the constant temperature culture. The highest qpunder temperature downshift culture was 38 pg/cell/day, which was 1.5
times higher than the constant temperature culture (Figure 4d,
e ).
Figure 5 shows the extracellular concentrations of glucose,
lactate and ammonium. It is apparent that glucose concentration in shake
flask and bioreactor showed no significant difference while significant
differences were observed in lactate and ammonium. In the constant
temperature culture, lactate was continuously produced as the
cultivation progressed, and the concentration of lactate rapidly
increased from day 5, reaching more than 3 g/L, until the end of the
culture when the lactate concentration reached 3.5 g/L (Figure
5b ), which was about twice as high as that of the shake flask culture.
Although the same trends of ammonia were observed in shake flask and
bioreactor, ammonia was less excreted upon temperature downshift and
accumulated 1.67 times higher at the end of the culture in shake flask
than in the bioreactor (Figure 5c , Figure 2c ). Due to
sufficient oxygen supply in the bioreactor, the active glycolytic
pathway consumed large amounts of glucose and produced more lactate,
which negatively affected cell growth and cell viability. This may
partly explain the lower viable cell density and cellular vitality
observed in the bioreactor than in shake flask under constant
temperature conditions. However, under temperature downshift culture,
the lactate concentration decreased gradually from 1.6 g/L to 0.7 g/L at
the beginning of day 5 upon temperature downshift, and this phenomenon
was also observed in the shake flask culture but became more pronounced
in the bioreactor. Taken above, temperature downshift promoted the CHO
cell growth and antibody production with less accumulation of metabolic
byproducts such as lactate and ammonia.
3.2.2 Effect of temperature downshift on antibody quality
Charge heterogeneity and
molecular size heterogeneity are two key attributes for evaluating the
quality of antibodies. In particular, acidic charge variants may affect
the therapeutic efficacy, studies have shown that increases in acidic
charge variants typically result in decreased tissue retention and
increased whole body clearance (Boswell et al., 2010). As shown inFigure 6 , temperature downshift significantly affected the
proportion of acidic and basic charge variants of the antibodies
compared to the constant temperature culture. The results showed that
under the constant temperature culture, the proportion of the acidic
charge variant and the basic charge variant of HP was 50.66% and
17.91%, respectively, and the main peak was accounted for 31.43%. It
can be seen that the charge variants are nearly 1.6 times higher than
that of the main peak. However, the proportion of antibody charge
variants was changed significantly under temperature downshift culture,
with the acidic charge variant decreasing to 43.4% while the basic
charge variant increasing to 21.37%. Apparently, temperature downshift
significantly reduced the acidic charge variant content and increased
the basic charge variant content. This finding was consistent with the
previous study by Zhang et al. who showed that lowering temperature
reduced the content of acidic charge variants while increased the
content of basic charge variants (X. Zhang et al., 2015). Possible
explanation regarding this change is that the decrease in temperature
reduces the expression of alkaline carboxypeptidase, and the C-terminal
lysine could not be completely excised, thus increasing lysine variants,
which lead to an increased in basic charge variants. On the other hand,
elevated acidic variants may be associated with oxidative stress; Chung
et al. found that increased antibody acidic peaks were associated with
increased intracellular ROS activity and increased supernatant hydrogen
peroxide concentration, both of which are characteristic of oxidative
stress, and that reducing cellular oxidative stress would reduce the
formation of acidic peaks (Chung et al., 2019). Hence, it can be
inferred that temperature downshift in this study might influence
intracellular reactive oxygen species levels and cellular oxidative
stress which then affect antibody quality.
Molecular size heterogeneity is also one of the key qualitative
attributes of antibodies, with the formation of aggregates, as well as
fragmentation, being an important factor contributing to antibody
molecular size heterogeneity. As shown in Table 1 , the high
molecular-weight species (HMW) in HP CHO cells was 24.4% and the main
peak (MP) was 75.6% under constant temperature culture; a slight
increase in the HMW% in HP CHO cells could be observed at 27.5% under
temperature downshift culture, and its MP still exceeded more than 70%.
This was consistent with the findings of Gomez et al. that mRNA levels
of antibody heavy chain (HC) and light chain (LC) increased 24-fold at
lower temperatures, while endoplasmic reticulum (ER) folding capacity is
reduced, so it can be assumed that low-temperature culture increases
antibody aggregate formation in CHO cells through increased LC and HC
transcripts and a limited ER mechanism (Gomez et al., 2012; Gomez et
al., 2018). The formation of aggregates accounted for the majority of
the formation relative to fragments, while there is almost absence of
fragmentation. Antibody fragmentation was further explored using
capillary electrophoresis, and it was found that MP exceeded more than
96% and the heavy-heavy-light chain (HHL) was lower than 3% regardless
of the temperature control mode. However, it should be noted that the MP
and HHL were accounted for 97.2 and 2.1%, respectively, under
temperature downshift conditions. Taken together, the results
demonstrated good quality properties of the antibodies produced by CHO
cells in this study, with only a few aggregates and fragments formed,
which hardly affect the efficacy and safety of the antibodies. In
addition, temperature downshift somewhat benefits the formation of
antibody with homogeneous molecular size.
Intracellular metabolomics analysis upon temperature downshift
To investigate the global response of cell metabolism to temperature
downshift, we measured the metabolome by untargeted metabolomics
techniques on day 5 and day 8 with two different temperature settings.Table 2 shows 95 and 107 differential metabolites on day 5 and
day 8 for temperature constant and downshift conditions, respectively.
The results of the principal component analysis showed good
reproducibility within sample groups and significant variability between
sample groups, thus supporting subsequent data analysis (Figure
S6a ). All the differential metabolites were firstly analyzed using KEGG
pathway enrichment, and about 50 key metabolites in the pathway with a
high impact on cell growth and antibody production were selected for
hierarchical clustering according to the order of significance of the
KEGG pathway enrichment. As shown in Figure 7 , there were
significant differences in the metabolites involved in the central
carbon metabolism, glutathione metabolism, lipid metabolism, and purine
pyrimidine metabolism of CHO cells before and after downshifting the
culture temperature (Figure S6 and S7 ).
The TCA cycle and pentose phosphate pathway are major pathway associated
with energy metabolism and NADPH generation, which are highly active
during peak antibody production. Intermediates of the TCA cycle are also
used as precursors for many biomolecules, and CHO cells require the
active TCA cycle to cope with the high energy demands of recombinant
protein production and maintain cell growth (Dhami, Trivedi, Goodacre,
Mainwaring, & Humphreys, 2018). As shown in Figure 7 ,
intermediates of the TCA cycle including citric acid, isocitric acid,
and α-ketoglutarate were significantly upregulated after temperature
downshift, followed by citric acid and isocitric acid entering the
cytoplasm via the shuttle system and subsequently entering the lipid
metabolism pathway. Meanwhile, the conversion of α-ketoglutarate to
glutamate was upregulated, but its products proline and arginine were
significantly downregulated, probably because most glutamates entered
the GSH pathway and were used for the synthesis of glutamyl-cysteine,
moreover, the glycolytic activity decreased after temperature downshift,
leading to a decrease in the content of 3-phosphoglycerate, the
precursor of glycine serine. Besides, in the purine nucleotide synthesis
pathway, xanthine nucleotide (XMP), hypoxanthine nucleotide (IMP), and
their precursors hypoxanthine, xanthine, and adenosine were all
upregulated, probably due to the active metabolism of the pentose
phosphate pathway, which produced large amounts of ribonuclease
5-phosphate and NADPH and contributed to the scavenging of ROS generated
in late cell culture. Likewise, downregulation of the metabolites
adenosine monophosphate (AMP), adenosine diphosphate (ADP), and
guanosine monophosphate (GMP) was observed, mainly for the generation of
adenosine triphosphate (ATP) and guanosine triphosphate (GTP) for energy
supply, supporting cell growth, protein synthesis or to participate in
some signaling pathways and replenish the TCA cycle. Thus, after
downshifting the temperature, cells can provide the precursors of many
biomolecules by enhancing the TCA cycle, the pentose phosphate pathway
and purine nucleotide pathway to support cell growth and antibody
production. To agree with this, one previous study has shown that
enhancing the TCA cycle can increase the production of recombinant
proteins and meanwhile reduce the accumulation of lactate (Templeton,
Dean, Reddy, & Young, 2013). In addition, the significant
down-regulation of amino acids such as arginine, proline, valine, and
isoleucine after lowering temperature indicates an increased
intracellular consumption of these amino acids, which greatly increases
the concentration of TCA cycle intermediates (Huang et al., 2020).
The glutathione metabolic pathway plays a central role in the
maintenance of redox balance in mammalian cells, and there is a
correlation between GSH metabolism and cellular productivity. Previous
studies have shown that high-yielding cell lines have more intracellular
GSH levels than low-yielding cell lines and upregulate GSH metabolism
(Chong et al., 2012; Orellana et al., 2015), while reduced intracellular
GSH levels result in reduced protein productivity (Chevallier, Schoof,
Malphettes, Andersen, & Workman, 2020). Consistent with these findings,
as shown in Figure 7 , the intermediate metabolites of the
glutathione metabolic pathway, including glutathione and its precursor
glutamate and glutamate-cysteine, were generally upregulated on day 8,
in contrast with the downregulation of the glutathione product
L-glutamyl-L-alanine. Intracellular GSH levels were significantly
upregulated, however, no significant changes in the glutathione pathway
were observed on day 5, probably due to downregulation of the product
3-phosphate hydroxypyruvate of S-glutathione-L-cysteine in the cysteine
and methionine pathways, resulting in the upregulation of its other
product glutathione. The glutathione pathway is an important pathway in
the mediation of oxidative stress, and in particular, glutathione plays
an important role in the detoxification of ROS generated by
mitochondria. It is therefore hypothesized that up-regulation of the GSH
metabolic pathway upon temperature downshift in late CHO cell culture
may be involved in counterbalancing oxidative stress and maintaining
redox balance. Lipid metabolism has an important impact on cell growth
and is a key pathway for energy storage, signal communication, protein
production and secretion. After temperature downshift, as shown inFigure 7 , the metabolites involved in the lipid metabolism
pathway, including choline metabolism and glycerophospholipid
metabolism, changed significantly on day 5 and day 8. In the
cytidylphosphorylcholine (CDP-choline) pathway, an intermediate
metabolic pathway of glycerophospholipid metabolism, the levels of
choline and its product phosphorylcholine were down-regulated, whereas
the levels of the intermediate product CDP-choline were up-regulated,
probably due to feedback regulation of the precursor of CDP-choline by
phosphatidylcholine. The levels of phosphorylcholine, a precursor of
diacylglycerol (DG), were down-regulated, leading to the down-regulation
of DG, which involves the synthesis of important membrane phospholipids,
and the down-regulation of the product phosphatidylcholine (PC).
Meanwhile, we found that there was up-regulation of phosphatidic acid
(PA), a precursor of DG in another synthetic pathway, probably due to
the entry of DG into other lipids such as triglycerides (TG). The main
reason for the down-regulation of cellular lipid metabolism after
temperature downshift might be low temperature adaptation. The fluidity
of lipid molecules in cell membranes decreased at low temperature, and
cells respond to low temperature by increasing the content of
unsaturated fatty acids in membrane phospholipids, thereby maintaining
fluidity under low temperature conditions. In addition, lipid
composition appeared to be altered, which may account for the
reprogramming of lipid metabolism (Fan & Evans, 2015).
Intracellular reactive oxygen species (ROS) levels
Combined with metabolomic analysis, it was evident that the glutathione
pathway and its intermediate metabolites were significantly upregulated
in the late stages of temperature downshift culture, presumably with
changes in redox metabolism. Next, the intracellular redox state after
temperature downshift was investigated by monitoring the relative
content of intracellular reactive oxygen species (ROS). As shown inFigure 8 , the shake flask results showed that intracellular ROS
level depended on different cell lines and different temperature
settings while temperature downshift significantly reduced the
intracellular ROS level. Furthermore, it can be seen that intracellular
ROS level in both HP and LP CHO cells increased with cultivation age but
there was no significant difference between two cell lines with constant
temperature settings. Recent studies have shown that the accumulation of
ROS can cause oxidative stress in cells, affecting cell growth and
antibody expression (Michael W. Handlogten et al., 2018), which may be
ascribed to the increased energy demand of cellular repair pathways, and
at the same time, high ROS levels induce more production of lactate,
which further induces cell death (Chevallier, Andersen, & Malphettes,
2020). However, the intracellular ROS level upon temperature downshift
was significantly lower than in the constant-temperature culture, which
implied a lesser oxidative stress and a more balanced intracellular
redox state.
In vivo monitoring of redox state with real-time quantitative
fluorescent biosensors
The previous results indicate that temperature downshift is instrumental
in improving antibody productivity, which is likely to be associated
with the capability of scavenging cellular oxidative stress and
maintaining balanced redox state. As is known, NAD+,
NADH, and NADP+, NADPH have important roles in
intracellular catabolism and anabolism, and their alterations can often
lead to abnormal energy metabolism and are important for maintaining the
intracellular redox state. To experimentally verify this, in this work,
we developed real-time quantitative fluorescent biosensors to analyze
the intracellular NAD+ /NADH ratio and NADPH level and
thus to understand the effect of temperature downshift on the CHO cell
intracellular redox-associated response.
3.5.1 Impact of biosensor expression on biological processes
To assess whether the expression of SoNar, iNapc, and iNap1 affects
biological processes, a 12-day fed-batch culture was performed, using
both transfected and untransfected CHO cell lines. As shown inFigure 9 , sensor expression in general did not place
significant effect on viable cell density, viability, antibody titer and
quality for both HP and LP CHO cell lines in shake flasks. Viable cell
density did not differ significantly from the control throughout the
culture, and cell viability was maintained above 95% in all cases.
Antibody titers were virtually unaffected by sensor expression, and the
final titer at the end of the culture was close to that of the control.
In addition, the quality of the antibodies including charge
heterogeneity and molecular size heterogeneity was also
indistinguishable from the control. Taken together, all these results
indicate that intracellular biosensor expression does not affect overall
process performance.
3.5.2 Changes in intracellular NAD+/NADH ratio upon
temperature downshift
To explore the changes in intracellular redox levels after downshifting
the culture temperature, transfected cells expressing
NAD+/NADH and NADPH probes were used for a 11-day
fed-batch culture for antibody production.
Figure 10 shows the changes in NAD+/NADH
ratio, where the ratio of F405nm/F488nm is negatively correlated with
NAD+/NADH ratio (Figure S8a ). The
intracellular NAD+/NADH ratio gradually decreased
during the fed-batch culture; after downshifting the culture temperature
on day 5, the results showed that the NAD+/NADH were
significantly lower than at constant temperature, while the increase in
the NAD+/NADH ratio implied that oxidative stress was
generated (Zhao et al., 2015). The gradual decrease in intracellular
NAD+/NADH upon temperature downshift may be caused by
the reduced glucose uptake and reduced glycolytic activity, while the
active mitochondrial metabolism consumes large amounts of NADH for
maintaining cell growth and product expression, resulting in lower NADH
levels and the inability of glycolysis to meet the needs of mitochondria
leading to an intracellular redox imbalance. Thus, prompting the cell to
produce large amounts of NADH by consuming lactate maintains the
cellular redox balance, and this phenomenon is consistent with the
findings by Hartley et al. that intracellular lactate metabolism is
driven by redox reactions (Hartley, Walker, Chung, & Morten, 2018).
This is indeed observed in CHO cell cultures exposed to temperature
downshift, and cellular metabolism shifted from a lactate-producing to a
lactate-consuming pattern (Figure 5 ) and less ROS was produced
to mitigate severe oxidative stress (Figure 8 ). Furthermore,
the NAD+/NADH ratio was significantly lower upon
temperature downshift, which means that NADH was produced rather than
consumed in cytosol, resulting in higher levels of oxidative
phosphorylation, when the energy metabolism in the cells was in an
efficient state (Templeton et al., 2013). However, on the one hand, the
higher NAD+/NADH ratio along with the higher rate of
lactate production under constant temperature culture indicated that a
lower level of oxidative phosphorylation occurred at this time, and on
the other hand, it indicated that the redox balance was difficult to be
maintained in the cells and oxidative stress was generated.
3.5.3 Changes in intracellular NADPH level upon temperature downshift
The change in intracellular NADPH level under two different temperature
settings is shown in Figure 11 , where the ratio of
F405nm/F488nm has a positive correlation with intracellular NADPH level
(Figure S8b ). As the culture progressed, the intracellular
NADPH level of LP CHO cell showed a decreasing trend and reached the
lowest value at the end of the culture. In contrast with this, the
intracellular NADPH level of LP CHO cell undergoing temperature
downshift was significantly higher than that in the constant temperature
culture, especially in the middle of the culture. Furthermore,
intracellular NADPH level of LP CHO cell tended to equilibrate upon
temperature downshift, demonstrating the ability to maintain redox
homeostasis in the cells; whereas a decreasing trend of intracellular
NADPH level was observed in the constant temperature culture. The
conversion between GSH and GSSG is controlled by GSH reductase using
NADPH, so a decrease in intracellular NADPH levels most likely leads to
a decrease in intracellular GSH level (Darja et al., 2016). As mentioned
in the results above, a large amount of ROS was produced in the cells
during the late stage of constant temperature culture, which inhibited
cell growth and antibody synthesis. Toxic substances such as ROS and
hydrogen peroxide are normally scavenged by antioxidants such as GSH,
the decrease in GSH levels most probably led to a high accumulation of
ROS, which resulted in oxidative stress. Taken above, the decrease in
intracellular NADPH levels may explain this phenomenon.
As shown in Figure 11 , we observed in HP CHO cells that there
was a slowly decreasing trend of intracellular NADPH levels despite
downshifting the culture temperature, which may be ascribed to
individual differences between cell lines with different metabolic
capacities. Even so, the intracellular NADPH level upon temperature
downshift was slowly decreased and significantly higher than that of
constant temperature culture, which proved that more reducing power in
the form of NADPH was provided intracellularly to promote the conversion
of GSSG into GSH and reduce the accumulation of GSSG, and more
efficiently remove intracellular ROS. Likewise, glutathione metabolism
is related to lipid metabolism, especially de novo cholesterol synthesis
pathway, which also plays an important role in protein secretion. An
increase in cholesterol synthesis capacity can increase the secretion
capacity of CHO cells to improve their productivity, and this process
relies on NADPH supply (Loh, Yang, & Lam, 2017). As a result,
temperature downshift also benefits CHO cell growth and antibody
production by means of providing more NADPH involved in ROS scavenging
and protein secretion system.
Discussion
During CHO cell culture, the temperature is one of the key environmental
variables affecting cell growth and antibody production. In this study,
to explore the effect of temperature downshift on the metabolic
phenotype of CHO cells, we first compared the effects of downshifting
the culture temperature on cell culture processes from the perspective
of cell growth, antibody expression, cell cycle distribution,
intracellular metabolite level and intracellular redox associated
system. As expected, we found that temperature downshift inhibited cell
growth, arrested the cell cycle, and significantly increased
intracellular antibody titers and specific production rates. At the same
time, based on metabolomics data analysis, we found significant
differences in energy metabolic pathways, glutathione pathways, and
lipid metabolic pathways in the later stages of low-temperature culture.
Then, we developed real-time quantitative fluorescent biosensors
including NAD+/NADH and NADPH probes to quantitatively
verify and further analyze the metabolic regulation mechanism upon
temperature downshift. The results showed that the downshifting the
culture temperature increased antibody titer and specific production
rate, which was closely related to the ability of cells to maintain
redox balance.
Effect of cell cycle on cell growth and antibody titer
First, we evaluated the effect of temperature downshift on cell growth
and antibody production at both shake flask and bioreactor level. Cell
culture performance was improved by downshifting the culture temperature
from 37°C to 33°C when cell densities achieved 1.5 ×
107 cells/mL. The results showed that temperature
downshift benefited antibody production (Figures 1e and4e ) and also had a favorable effect on antibody quality
(Figure 6 ), which was not only a consequence of the longer
culture time but also correlated with a higher rate of cell specific
production (Figures 1f and 4f ). Meanwhile, we also
observed slow cell growth (Figures 1a and 4a ),
suggesting a correlation between increasing the specific production rate
of antibody and decreasing cell growth rate. Previous studies have shown
that some cold shock proteins such as CIRP (cold-inducible binding
protein) and RBM3 (RNA-binding motif protein 3) are significantly
upregulated under low-temperature induction, both of which are involved
in transcriptional and translational regulation, and that overexpression
of both proteins significantly increases recombinant protein production
while blocking cell growth (Tossolini et al., 2018). Our study revealed
that cell growth arrest may be associated with a cell cycle arrest in
the G0/G1 phase, which is considered to be the optimal phase for
increasing recombinant protein production. Several ribosomal
biosynthesis genes and protein translation genes were found to be highly
expressed in the G1 phase (Kumar, Gammell, & Clynes, 2007). In
addition, low temperature may act through upregulation of the P53-P21
pathway, whose downstream gene targets may be responsible for blocking
the cell cycle as well as controlling cell proliferation to protect
damaged or stressed cells from apoptosis, thus providing a time window
to repair the damage and eliminate the stress (Bedoya-Lopez et al.,
2016; Roobol et al., 2011). On the other hand, our results suggest that
temperature downshift plays an important role in regulating carbon
metabolism, lipid metabolism, glutathione metabolism, and energy
metabolism in the cell.
Interactions between pyruvate, lactate metabolism, and
NAD+/NADH ratio upon temperature downshift
We observed that temperature-induced effects on cell growth and antibody
expression are also reflected in cellular metabolic rearrangement,
especially lactate metabolism. Low temperature induces a shift from a
lactate-producing to a lactate-depleting phenotype thereby reducing the
accumulation of lactate in the culture environment (Figures 2aand 5b ). Lactate has been widely reported as metabolic
by-product that inhibits cell growth during cell culture, while it plays
a central role in regulating cellular redox state (Martínez-Monge et
al., 2019). Consistent with previous study, our results showed that when
lactate was consumed, cell growth decreased and specific consumption
rates of glucose and amino acids were significantly reduced, while
product titers were increased (M. Torres et al., 2018). To elucidate
what triggers this phenomenon, it is crucial to understand the
relationship between redox metabolism and lactate metabolic switch.
It has been shown that the oxidation of lactate to pyruvate balances the
intracellular redox state to maintain the rate of oxidative
phosphorylation, so changes in the intracellular redox state are
directly influenced by the competition between glycolysis and oxidative
phosphorylation (Hartley et al., 2018). The NAD+/NADH
ratio, and intracellular pyruvate level are the driving forces for the
switch from aerobic glycolytic metabolism to hyperoxidative metabolism
in CHO cells, and changes in lactate levels vary with these parameters
(Locasale & Cantley, 2011; Vappiani et al., 2021). Usually, in the
rapid glycolysis stage, the reducing power NADH is continuously
generated, and the reduction of pyruvate to lactate can quickly balance
the intracellular redox state to maintain the rate of glycolysis; when
the rate of oxidative phosphorylation is rapid, intracellular NADH
continuously enters the mitochondria to provide reducing power, and
redox balance is then maintained by the consumption of lactate to
produce NADH. Therefore, it can be considered that the redox balance
drives the consumption of lactate, which is consistent with our
findings. We observed a decrease in the intracellular
NAD+/NADH ratio after lowering the culture temperature
(Figure 10 ), probably due to the reduced rate of intracellular
glucose consumption after temperature downshift (Figures 2e and5a ), which limits the glycolytic pathway and reduces NADH
production. When the cell supplies the oxidative phosphorylation pathway
by promoting lactate metabolism to generate NADH, the free intracellular
NADH level will be increased. Our experimental results support the
speculation of Torres et al. that an imbalance in the
NAD+/NADH ratio could explain the metabolic transition
from a lactate-producing state to a lactate-consuming state (M. Torres
et al., 2018). Thus, it can be concluded that promoting lactate
consumption is an important reason for the ability of temperature
downshift to maintain redox balance, and this metabolic shift is likely
to be a key metabolic indicator for CHO cells to improve antibody
production.
Interaction between the glutathione pathway and intracellular NADPH
level upon temperature downshift
Previous studies suggest that the intracellular redox state may be a key
factor in prolonging cell viability and maintaining specific production
rates (Hecklau et al., 2016). As cell culture progressed, the
intracellular ROS level in the constant temperature culture also
increases, which is significantly higher than that in the temperature
downshift culture (Figure 8 ). The accumulation of ROS shows the
sign of oxidative stress in cells (Chevallier, Andersen, et al., 2020).
A recent study showed that insufficient cysteine supply negatively
affects antioxidants such as glutathione and taurine, and then oxidative
stress takes place, resulting in apoptosis, decreased antibody titers,
and differential expression of proteins (Ali et al., 2019). Our results
showed that cysteine concentrations were slightly higher in the
temperature downshift culture than in the constant temperature culture,
but the extracellular cysteine was not depleted in both conditions
(Figure 3 ), which suggested that reduction in cysteine may not
be the reason responsible for inducing oxidative stress in the cells.
The redox balance mechanism consists of NADH, NADPH, and glutathione
(GSH) acting in concert to adapt to oxidative stress and restore
reducing equivalents (J. K. Hong et al., 2020). Glutathione is the main
cellular antioxidant and can detoxify cells by directly scavenging free
radicals or other ROS. Hence, the accumulation of glutathione oxide
(GSSG) is another important marker of oxidative stress, which has been
observed to be associated with growth arrest and apoptosis (Selvarasu et
al., 2012). In agreement with this, our metabolomic analysis showed that
the intracellular glutathione pathway was significantly up-regulated
upon temperature downshift compared to the constant temperature culture
(Figure 7 ), indicating that the cells were relieved of
oxidative stress by enhancing the antioxidant metabolism pathway and
that intracellular ROS was maintained at a lower level in the late
low-temperature culture (Figure 8 ).
In addition, upregulation of the glutathione pathway may be associated
with increased cellular-specific production rates. Omics studies have
confirmed that the GSH pathway is up-regulated in high-yielding CHO
cells. For example, significant up-regulation of proteins involved in
the GSH pathway was observed in high-yielding cell lines using a
proteomic approach (Darja et al., 2016; Orellana et al., 2015). The
reasons for this are manifold; the productivity of CHO cells may be
limited by folding in the endoplasmic reticulum (ER) and an oxidative
environment to introduce disulfide bonds into secreted proteins is
necessary for protein assembly (M. Torres, Akhtar, McKenzie, &
Dicksons, 2021). Therefore, we believe that the higher glutathione
pathway in a low-temperature environment not only maintains redox
balance against oxidative stress but also is associated with endoplasmic
reticulum folding, resulting in higher secretion rates and higher
antibody titers.
Real-time monitoring of intracellular NADPH level showed that as the
culture progressed, intracellular NADPH amount increased upon
temperature downshift (Figure 13 ). NADPH is an important
coenzyme of the intracellular glutathione system, reactivating
peroxidase and thioredoxin reductase-mediated production of thioredoxin
by promoting the glutathione reductase (GR)-mediated regeneration
process of GSH. Our results showed a continuous decrease in
intracellular NADPH level under constant temperature culture conditions,
which may also account for the difficulty in maintaining redox balance
in the late cultivation stage.
Temperature-induced effects on lipid metabolic pathways
Lipids are major components of cell membranes, making them a key part of
cellular metabolic activity, involved in energy metabolism, cell growth
and maintenance, and protein secretion through vesicle formation and
transport (Budge et al., 2020). Previous study has showed that
temperature, redox state, and cellular steroid levels can be manipulated
to regulate intracellular lipid balance, thereby promoting cell growth
and antibody production (Han & Kaufman, 2016). For example, the
unfolded protein response (UPR) can regulate the amount of intracellular
ER through protein and lipid synthesis, and overexpression of its key
regulator XBP1 has been shown to expand the endoplasmic reticulum of CHO
cells and has been successfully used to increase recombinant protein
production (Tigges & Fussenegger, 2006). The overexpression of XBP1 was
also found in CHO cells during low-temperature culture (M. Torres et
al., 2021), It can therefore be suggested that low-temperature induction
of XBP1 overexpression increases the area of endoplasmic reticulum,
thereby increasing antibody production.
The lipid metabolic pathway was significantly down-regulated after
lowering the temperature, both on day 5 and day 8 (Figure 7 ).
Both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are
major components of cell membranes and intracellular transport vesicles,
and their synthesis may be important for cell growth and protein
production. Down-regulation of phosphatidylcholine after temperature
downshift activated P53 and subsequently P21 expression, a signaling
pathway associated with a cell cycle arrest in response to impaired DNA
replication. Therefore, a decrease in cell membrane composition may be
partly responsible for cell arrest in the G1 phase. In addition, Kentaro
et al. reported that the addition of phospholipid synthesis precursor
phosphatidic acid to the culture medium would contribute to cell
proliferation (Sakai, Matsunaga, Hayashi, Yamaji, & Fukuda, 2002).
Currently, there are few studies on lipid metabolism in fed-batch
cultures of CHO cells, which affects cell growth and antibody
expression. In the future, systematic studies could be carried out on
lipid metabolism upon temperature downshift.
Conclusions
In this study, we optimized the CHO cell fed-batch culture process based
on a temperature downshift approach, which significantly increased
antibody titer and cell-specific production rate, and maintained good
quality attributes. The results suggested that the cellular ability to
maintain balanced redox state likely being the main reason account for
this improvement. Based on metabolomics data, we explored the mechanisms
of temperature-induced differences in cell metabolism and searched for
key regulatory metabolites and associated metabolic pathways. We found
that the up-regulation of central carbon and energy metabolism pathways
after temperature downshift may provide the large amount of energy
required for increased recombinant protein production; down-regulation
of lipid metabolic pathways may be associated with cell growth
retardation. In addition, up-regulation of the glutathione pathway may
be responsible for maintaining cellular redox balance.
We further developed fluorescent quantitative biosensors to explore the
intracellular redox level upon temperature downshift and analyzed the
metabolic mechanism related to the redox reaction in cells induced by
temperature based on the intracellular NAD+/NADH ratio
and intracellular NADPH level. To our knowledge, this is the first time
that a fluorescent biosensor has been used for real-time monitoring of
CHO cell fed-batch culture processes. We found that the elevation of
NAD+/NADH ratio after lowering temperature promotes
the switch of cellular metabolism from lactate-producing to
lactate-consuming types, reducing extracellular lactate concentration
and maintaining intracellular redox balance. Furthermore, the increased
intracellular NADPH level may support the upregulation of the
glutathione pathway, scavenging the intracellular accumulated ROS.
Overall, the application of fluorescent biosensors enables us to gain an
immediate and accurate insight into intracellular redox-related
metabolite changes. In the near future, it is necessary to combine
online detection instruments for continuous sampling to achieve
real-time observation during the entire culture process.