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J. Microbiol. Biotechnol. (2013), 23(11), 1586–1597
http://dx.doi.org/10.4014/jmb.1307.07019
Research Article
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An Investigation Into the Relationship Between Metabolic Responses
and Energy Regulation in Antibody-Producing Cell
Ya-Ting Sun, Liang Zhao, Zhao-Yang Ye, Li Fan, Xu-Ping Liu*, and Wen-Song Tan
The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China
Received: July 10, 2013
Revised: August 12, 2013
Accepted: August 13, 2013
First published online
August 14, 2013
*Corresponding author
Phone: +86-21-64253394;
Fax: +86-21-64252250;
E-mail: [email protected]
pISSN 1017-7825, eISSN 1738-8872
Copyright © 2013 by
The Korean Society for Microbiology
and Biotechnology
Energy-efficient metabolic responses were often noted in high-productive cultures. To better
understand these metabolic responses, an investigation into the relationship between
metabolic responses and energy regulation was conducted via a comparative analysis among
cultures with different energy source supplies. Both glycolysis and glutaminolysis were
studied through the kinetic analyses of major extracellular metabolites concerning the fast and
slow cell growth stages, respectively, as well as the time-course profiles of intracellular
metabolites. In three cultures showing distinct antibody productivities, the amino acid
metabolism and energy state were further examined. Both the transition of lactate from
production to consumption and steady intracellular pools of pyruvate and lactate were
observed to be correlated with efficient energy regulation. In addition, an efficient utilization
of amino acids as the replenishment for the TCA cycle was also found in the cultures with
upregulated energy metabolism. It was further revealed that the inefficient energy regulation
would cause low cell productivity based on the comparative analysis of cell growth and
productivity in cultures having distinct energy regulation.
Keywords: CHO cell, metabolic response, energy regulation, antibody production
Introduction
Therapeutic proteins, as one of the best-selling biologic
drugs, are demanded in considerably large quantities [25].
Their manufacture using mammalian cells has become the
largest ever division in the pharmaceutical industry [1]. In
order to increase both cell mass and productivity, during
the past two decades, great efforts have been paid to
optimize protein-producing cell cultures [27], and many
efficient culture processes have therefore been successfully
developed [6, 8, 9]. Along with the enhanced cell performance
such as increased cell density, cell viability, and production
rate, distinct metabolic responses in these high-yielding
processes have also been noticed. Recently, researches have
further pointed out that cell growth and productivity are
strongly correlated with certain metabolic responses; for
example, the lactate and amino acid metabolisms [7, 16].
However, the recognition of such a correlation calls for a
further detailed investigation into metabolic responses in
both high- and low-productive processes to understand
why cell performance has a close correlation with metabolic
responses.
Some metabolic responses in high-productive culture
processes often displayed a tendency towards more efficient
energy metabolism, whereas in suboptimal processes the
energy metabolism was deregulated [16, 17]. Recently,
based on a proteomic study, the upregulation of proteins
involved in energy metabolism was found to be associated
with the enhancement in cell mass and protein synthesis,
suggesting the critical roles of energy regulation on cell
performance [2, 18]. Moreover, some direct evidences
showed that the high intracellular ATP concentration was
helpful to enhance the synthesis of recombinant protein
[12]. These findings suggest that the energy metabolism
might be a key to understand the correlation between cell
performance and metabolic responses.
It is well known that protein-producing mammalian cells
are usually associated with deregulated central metabolism,
which often leads to disturbed energy regulation. These
cells preferentially undergo aerobic glycolysis, an inefficient
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pathway for energy generation [21]. Compared with the
tricarboxylic acid cycle (TCA cycle), which is the main
mechanism for ATP production, aerobic glycolysis converts
glucose mainly into lactate and produces less amount of
ATP [28]. Besides this, these cells consume an excessive
amount of glutamine, more than their actual needs, with a
large portion metabolized into ammonium or non-essential
amino acids [11]. The inefficient utilization of glutamine
usually impairs its primary function as a backup for the
TCA cycle in energy generation, whereas other anaplerotic
pathways seem trivial owing to the low-level expression of
key enzymes [22]. The deregulated energy metabolism in
protein-producing cells may have a negative influence on
cell performance if the aerobic glycolysis or inefficient
utilization of glutamine predominates the whole cultivation.
Instead, if a metabolic shift towards more efficient energy
metabolism is realized, cell performance should be improved.
To better understand the correlation of cell performance
with metabolic responses, it would be reasonable to
investigate the relationship between metabolic responses
and energy regulation, and further demonstrate the influence
of energy regulation on cell growth and productivity.
However, few studies have tried to elucidate these issues. It
remains elusive which metabolic responses are correlated
with efficient energy regulation and whether efficient energy
regulation has a positive influence on cell growth and
productivity.
To address these issues, the present study was performed
by examining cellular responses to different energy
conditions. Cells were cultured under conditions designed
to have varied energy sources. Three conditions included
(i) deficient energy source (by supplying insufficient glucose);
(ii) abundant energy source (by supplying surplus glucose);
and (iii) limited energy source (by supplying insufficient
glucose and other carbon sources). Cell growth and
productivity were monitored and the metabolic responses
of glycolysis and glutaminolysis were examined regarding
both fast and slow growth stages, respectively. Then amino
acid metabolism as well as intracellular energy status was
further investigated. Through a comparative analysis, the
relationship between metabolic responses and energy
regulation as well as the influence of energy regulation on
cell growth and productivity was elucidated.
Materials and Methods
Cell Culture
A DHFR-CHO cell line expressing a chimeric anti-human CD20
monoclonal antibody was used in this study [4]. Cells were
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routinely maintained in a protein-free cell culture medium, which
was composed of a mixture of DMEM/Ham’s F12 (1:1, (v/v);
Gibco) supplemented with vitamins, nucleic acids, ethanolamine,
sodium selenite, ferric citrate, and Pluronic F-68. This basal medium
containing 7.8 mM glucose and 1 mM pyruvate was further
supplemented with glutamine at the final concentration of 6 mM.
This basal medium was then added with various energy sources
for different conditions.
Four batch cultures were performed under different conditions:
(i) deficient energy source with an insufficient low concentration
of glucose (supplemented with 2.2 mM glucose) (designated as
LG with final 10 mM glucose); (ii) abundant energy source with a
sufficient high concentration of glucose (supplemented with
32.2 mM glucose) (HG with final 40 mM glucose); (iii) limited
energy source with a low concentration of glucose (supplemented
with 2.2 mM glucose) but further supplemented with either an
monocarboxylate (pyruvate, 9 mM) (LG+Pyr with final 10 mM
glucose and 10 mM pyruvate) or hexose (galactose, 25 mM) (LG+Gal
with final 10 mM glucose and 25 mM galactose). In addition, a
fed-batch culture was carried out on the basis of LG+Gal culture
(designated as FB-Gal), wherein the only difference from the LG+Gal
culture was that a feeding medium composed of concentrated
amino acids and vitamins was supplemented once at the time
point of 72 h. All reagents were purchased from Sigma-Aldrich.
Exponentially growing cells were inoculated at a density of 45 × 105 cells/ml. All cultures were carried out in 50 ml cell culture
tubes (TPP, Switzerland) with a working volume of 20 ml and
agitated at 220 rpm on a rotary shaker (Adolf Kühner, Switzerland).
Cells were incubated at 37oC with 5% CO2 and 85% relative
humidity. Daily sampling was performed for cell counting and
metabolite measurements. Cell density and viability were determined
with a hemacytometer using trypan blue staining. Cell size was
examined through visually comparing cells against the scale on
the hemacytometer and found to be approximate 14 µm in diameter,
thus giving a single cell volume of 1.44 × 10-9 ml [24].
Metabolite Measurements and Quantification of Antibody
Concentration
Extracellular metabolites. Concentrations of glucose, glutamine,
glutamate, lactate, and ammonium in culture medium were measured
on a BioProfile 400 Analyzer (Nova Biomedical). The concentration
of pyruvate in the culture medium was determined using commercial
assay kits following the manufacturer’s instructions (Nanjing
Jiancheng Bioengineering Institute, China). Concentrations of
amino acids were quantified using HPLC (1525 Binary HPLC
Pump; 717 plus Autosampler; 2475 Multi λ Fluorescence Detector;
Waters) equipped with a reversed-phase column (Nova-Pak C18
4 µm, 3.9×150 mm; Waters), following the pre-column derivation
using the AccQ·Fluor reagent kit (Waters).
Intracellular metabolites. Intracellular ATP was directly quantified
with a bioluminescent method using an ATP assay kit (Beyotime
Institute of Biotechnology, China). Intracellular NADH and NAD+
were measured using an NAD+/NADH quantification kit (BioVision,
Metabolic Response and Energy Regulation
USA). These measurements were all carried out according to the
manufacturers’ protocols.
Intracellular pyruvate and lactate were extracted as previously
described [13]. Briefly, sampled cell preparations were immediately
centrifuged and the supernatant was discarded. To lyse cells, cold
perchloric acid solution was added to the cell pellets, followed by
neutralization with KOH and K2HPO4 solutions. The whole
process was carried out at 0oC. Intracellular concentrations of
pyruvate and lactate were determined after extraction using
commercial assay kits following the manufacturer’s instructions
(Nanjing Jiancheng Bioengineering Institute, China).
Antibody concentration. The concentration of the chimeric
anti-human CD20 monoclonal antibody secreted by cells in the
culture medium was determined using an ELISA method.
Calculation of Specific Consumption/Production Rates of Metabolites
or the Antibody
Specific rates of consumption and/or production of metabolites
or the anti-human CD20 antibody were calculated by plotting
measured concentrations against the respective integral of viable
cell concentration (IVCC). The respective average specific rates
were equal to the slopes of the linear fitted curves.
Calculation of Relative Standard Deviation of Each Amino Acid
and Intracellular Metabolite
The relative standard deviation (RSD) of each amino acid and
intracellular metabolite was evaluated during the establishment
of the analytical method, which was assumed as a constant
characteristic parameter of statistical distribution and calculated
as follow:
n
(Xi – X)
∑
i=1
2
-------------------------n–1 RSD ≈ ----------------------------X
X : measurement value for one sample
X : average of repeated measurements
n : repeated n times of measurement, n ≥ 20
Results and Discussion
Cell Growth and Antibody Production
Cell growth and production profiles for all cultures
including the fed-batch culture (FB-Gal) were obtained and
are summarized in Fig. 1. Within the initial 48 h in culture,
cell growth appeared similar under all conditions (Fig. 1A).
After 48 h, the growth of cells halted both with deficient
energy source (LG) and limited energy source supplemented
with pyruvate (LG+Pyr), accompanied by a drastic decrease
in cell viability after 72 h (Fig. 1B). In contrast, cells kept
growing under the conditions of abundant energy source
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(HG) and limited energy source supplemented with galactose
(including LG+Gal and FB-Gal), and cell viability was
maintained above 98% during 48-120 h in these cultures
(Figs. 1A and 1B). When plotting the logarithm of viable
cell density against culture time, two distinct growth stages
with apparently different growth behaviors could be
discerned (as shown in Fig. 1C and denoted as I & II,
respectively). The respective specific growth rates of these
two stages for all cultures are also summarized in Table 1.
Based on the specific growth rates, a fast growth stage for
all cultures (0-48 h) and a slow growth stage (48-120 h) for
HG, LG+Gal, and FB-Gal cultures were established. However,
in LG and LG+Pyr cultures, the slow growth stage was not
present since cells could not thrive at all after 48 h.
The production profile of the monoclonal antibody in all
cultures is shown in Fig. 1D and the corresponding
production rates are listed in Table 1. Within the initial
72 h, the production rates were lower in LG and LG+Pyr
than those in HG, LG+Gal, and FB-Gal, which was probably
due to the early cell death in the latter two cultures.
However, after 72 h, the antibody production rate declined
significantly by 43.6% and 47.8% in HG and LG+Gal
cultures, respectively, while a constant rate was sustained
in FB-Gal culture in the whole cultivation process (Table 1).
Metabolic Responses of Glycolysis and Glutaminolysis
As shown in Fig. 2, the concentrations of key metabolites
involved in both glycolysis and glutaminolysis were
plotted against IVCC and the corresponding specific rates
are summarized in Table 2. The metabolic responses under
different culture conditions were analyzed with respect to
distinct growth stages.
In the fast growth stage, the metabolites in glycolysis,
including glucose, pyruvate, and lactate, were measured in
all cultures (Figs. 2A-2C and Table 2). It was found that
when cells were exposed to deficient (LG) or abundant energy
source (HG), the consumption of glucose was similar,
showing almost identical specific rates. However, the
downstream metabolites in glycolysis, including pyruvate
and lactate, demonstrated differential responses under these
two conditions (Figs. 2B and 2C). Whereas an overflow of
extracellular pyruvate at a rate of 0.13 mmol 109 cells-1·day-1
took place in HG culture, which was accompanied by a
significant increase in its intracellular concentration, LG
culture cells consumed pyruvate at a rate of 0.11 mmol
109 cells-1·day-1 and the intracellular pyruvate concentration
remained relatively constant at a lower level (Figs. 3A and
3B and Table 2). In the meantime, both extra- and
intracellular lactate were at higher levels in HG culture
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Fig. 1. Time profiles of cell growth and productivity.
(A) Viable cell density; (B) logarithmic value of viable cell density (I, II, III represents three culture span); (C) cell viability; (D) antibody
concentration in culture LG ( ■ ), HG ( ● ), LG+Pyr ( □ ), LG+Gal ( ○ ), and FB-Gal (×). The error bar represents standard deviation of three
independent experiments (same for Figs. 2 and 3).
than those in LG culture, which was consistent with the
fact that the production rate of extracellular lactate in HG
culture was 50.2% greater (Figs. 3C and 3D and Table 2). In
addition, when cells were subjected to limited energy
supply with different carbon sources (i.e., LG+Gal, FB-Gal,
and LG+Pyr), the metabolic responses of glycolysis also
appeared different. When galactose was used (LG+Gal and
FB-Gal), the glycolysis was similar to that in LG culture.
However, when cells were given pyruvate (LG+Pyr), the
glucose consumption rate was 44.5% lower than that in
Table 1. Specific growth rates and monoclonal antibody production rates in different cultures.
Composition of energy sourcesa
Culture
Specific growth rate
h-1
0-48 h
LG
10 mM glucose
0.023
HG
40 mM glucose
0.024
48-120 h
Monoclonal antibody production rate
mg/(109 cells-day)
0-72 h
8.13 (0-96 h)
0.0094
11.33
10 mM glucose & 10 mM pyruvate
0.018
10 mM glucose & 25 mM galactose
0.023
0.011
11.59
FB-Gal
10 mM glucose & 25 mM galactose
feeding of amino acids and vitamins at 72 h
0.023
0.01
10.23 (0-168 h)
The concentration of energy source for specific culture refers to the final concentration in the medium.
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6.39
8.25 (0-120 h)
LG+Pyr
LG+Gal
a
72-168 h
6.05
Metabolic Response and Energy Regulation
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Fig. 2. Concentrations of extracellular metabolites versus integral of viable cell concentration (IVCC).
(A) Glucose; (B) pyruvate; (C) lactate; (D) glutamine; (E) glutamate; and (F) ammonium in culture LG ( ■ ), HG ( ● ), LG+Pyr ( □ ), LG+Gal ( ○ ), and
FB-Gal (×). The solid and dotted lines represent the relevant slopes before and after 48 h, respectively.
Gal). In HG culture, the uptake of glucose became slower
compared with that in the fast growth stage (Figs. 2A and
2D). At this stage, even though the glucose concentration
was still above 10 mM, the glucose consumption rate
declined by 80% in HG culture (Fig. 2A and Table 2). In
LG+Gal and FB-Gal cultures, since glucose was exhausted
within the first 48 h (Fig. 2A), cells should have turned to
galactose for glycolysis. However, the consumption profile
of galactose was not examined in the present study.
However, according to a previous report, cells generally
consume a lower quantity of galactose than glucose [3]. In
consistent with the decelerated consumption of carbon
sources, the consumption rate of glutamine at the slow
growth stage also decreased by 55.8% in HG culture and
18.4% in both LG+Gal and FB-Gal (Table 2) compared with
LG+Gal (FB-Gal) culture and the consumption rate of
extracellular pyruvate was increased significantly (by 8.8folds compared with LG culture), whereas no obvious
uptake was noted in LG+Gal (FB-Gal) culture (Table 2). On
the other hand, the metabolites in glutaminolysis, including
glutamine, glutamate, and ammonium, were also monitored
(Figs. 2E-2G and Table 2). Compared with the glycolysis,
glutaminolysis did not show much difference among different
culture conditions (Table 2). However, both the uptake and
accumulation rates of glutamine and ammonium in
LG+Pyr culture were found to be the lowest among all the
cultures, which implicated the slowest glutaminolysis.
As cells entered the slow growth stage, significant
metabolic shifts were observed under the conditions of
abundant (HG) and limited energy source (LG+Gal and FB-
Table 2. Specific rates of metabolites consumption/production in 0-48 h/48-120 h (negative value represents consumption).
Specific consumption/production rate
mmol/(109 cells-day)
Glucose
LG
-5.57
HG
-6.33
LG+Pyr
Pyruvate
-0.11 (0-96 h)
-1.44
-3.21 (0-72 h)
0.13
-0.14a
-1.28 (0-120 h)
Lactate
Glutamine
9.94
14.93
-1.54
--b
-1.45
9.77
Glutamatec
--
-0.64
0.14
-1.37
Ammonium
1.59
-0.17
0.19 (0-96 h)
1.52
--
1.05
LG+Gal
-5.84
0
--
-0.042
10.39
-1.02
-1.58
-1.32
0.13
-0.17
1.57
--
FB-Gal
-5.73
0
--
-0.052
10.73
-1.6
-1.62
-1.29
0.15
--
1.58
--
a
The specific consumption rate of pyruvate in HG culture was fitted after 72 h.
b
c
“--” stands for rates with no distinct consumption or production trend.
The specific rates of glutamate in LG+Gal and FB-Gal cultures were fitted within 0-72 h/72-120 h.
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Fig. 3. Time profiles of extracellular and intracellular metabolites concentrations.
(A) and (B) Extra-/intracellular pyruvate concentrations; (C) and (D) extra-/intracellular lactate concentrations in culture LG ( ■ ), HG ( ● ),
LG+Pyr ( □ ), LG+Gal ( ○ ), and FB-Gal (×). The arrow indicates that the value of the open square is according to the Y-axis on the right side.
those in the fast growth stage, respectively. Despite the
consistent reduced uptake of nutrients in all these three
cultures, the metabolic responses of pyruvate, lactate, and
glutamate varied at the slow growth stage. Under the
condition of abundant energy source (HG), cells started to
take up pyruvate in HG culture after 72 h (Fig. 3A) and at
the same time extracellular lactate ceased to accumulate
(Fig. 3C). Meanwhile, the intracellular concentrations of
pyruvate and lactate dropped dramatically after 96 h in HG
culture (Figs. 3B and 3D). Under the condition of limited
energy source (LG+Gal and FB-Gal), pyruvate was also
consumed but at a much lower rate, which was nearly one
third of that in HG culture (Figs. 2B, 3A, and Table 2).
However, different from that in HG culture, a steady
uptake of lactate was observed after 48 h in both LG+Gal
and FB-Gal cultures (Fig. 3C). In addition, in comparison
with that in LG+Gal culture, the consumption rate of
lactate was increased by 64.5% in FB-Gal culture (Table 2),
suggesting that the feeding of amino acids and vitamins
promoted the uptake of lactate. This faster uptake caused
the exhaustion of lactate in the end of FB-Gal culture
(Fig. 2C). Moreover, the intracellular concentrations of
pyruvate and lactate remained relatively steady in the slow
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growth stage in both LG+Gal and FB-Gal cultures when
compared with those in HG culture (Figs. 3B and 3D). On
the other hand, consistent with the decreased consumption
of glutamine, the accumulation of ammonium halted in all
three cultures at the slow growth stage (Figs. 2A and 2F). In
contrast, whereas the glutamate remained unchanged in
FB-Gal culture, in both HG and LG+Gal cultures, glutamate
was consumed from the previous accumulation in the fast
growth stage (Fig. 2E).
Amino Acid Metabolism and Intracellular Energy States
The time-course profiles of amino acids in HG, LG+Gal,
and FB-Gal cultures are shown in Fig. 4. Among the major
amino acids, in HG and LG+Gal cultures, similar
concentrations of both arginine and threonine were found
with no obvious consumption or production, and these two
amino acids also maintained steady concentrations in FBGal culture after the feeding point (data not shown). In
contrast, during the whole cultivation process, other amino
acids, except proline, glycine, and alanine, were consumed
gradually to a similar extent in the two batch cultures (HG
and LG+Gal). Meanwhile, cysteine, leucine, isoleucine,
valine, and histidine were found at lower levels in LG+Gal
Metabolic Response and Energy Regulation
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Fig. 4. Relative amino acids concentrations (to 0 h) in the early, middle, and end of cultivation.
LG (square blocks); LG+Gal (shaded blocks); FB-Gal (black blocks). The arrow indicates that the value of the black blocks is according to the Y-axis
on the right side.
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culture, suggesting more utilization of these amino acids
than in HG culture. Compared with the LG+Gal culture,
the one-shot feeding at 72 h in FB-Gal culture increased
the concentrations of most amino acids. However, the
consumptions of tyrosine, cysteine, leucine, isoleucine,
Fig. 5. Time-course profiles of intracellular energetic metabolites.
(A) ATP; (B) NADH; and (C) NAD+/NADH in culture LG (square
blocks), LG+Gal (shaded blocks), and FB-Gal (black blocks).
J. Microbiol. Biotechnol.
valine, methionine, and aspartate were found to be increased
even after 96 h in FB-Gal culture. In addition to the increased
utilization of these amino acids, it was also interesting to
note the metabolic shift of alanine and serine, which were
produced previously in FB-Gal culture. It was found that
alanine was consumed after 96 h in FB-Gal, while its
concentration was kept steady in both HG and LG+Gal
culture with a higher level in the former. Moreover, while
serine was produced in LG culture at 96 h, considering the
similar concentrations at 96 h in FB-Gal with feeding and in
LG+Gal without feeding, its consumption in FB-Gal culture
could be inferred before 96 h and even more pronounced
after 96 h.
In addition, the intracellular energy states were also
analyzed in HG, LG+Gal, and FB-Gal cultures. Fig. 5 shows
the time-course profiles of indices related to energy state,
including ATP, NADH, and NAD+/NADH. It was found
that intracellular ATP in HG culture decreased dramatically
at 72 h and then continued to decline at a much lower rate.
In contrast, in both LG-Gal and FB-Gal cultures, although
ATP levels also declined at 72 h, it was followed by a
gradual increase, but to a more prominent extent in FB-Gal
culture than that in LG-Gal culture (Fig. 5A). In addition,
the highest level of NADH was observed at 148 h in HG
(Fig. 5B), which was consistent with its lowest ratio of
NAD+/NADH at the same time point (Fig. 5C). In contrast,
a gradual decrease in the level of NADH was noticed in FBGal culture (Fig. 5B), which was accompanied by the
gradual increase in NAD+/NADH (Fig. 5C).
Relationship Between Metabolic Responses and Energy
Regulation
In the present study, three cultures (HG, LG+Gal, and
FB-Gal) were found to show both fast and slow growth
stages. It was revealed that the energy states were different
among these cultures, especially between HG and FB-Gal
cultures. Intriguingly, in HG and FB-Gal cultures, different
metabolic responses of glycolysis, glutaminolysis, and
amino acid metabolism were observed regarding both
extra- and intracellular metabolite profiles. Based on these
results, a comparative analysis of the energy states and
corresponding metabolic responses in HG and FB-Gal
cultures would be able to unravel the relationship between
metabolic responses and energy regulation.
HG culture displayed accumulated NADH level and a
low ratio of NAD+/NADH at 148 h and a drastic decrease
in intracellular ATP at 72 h (Fig. 5). The accumulation of
NADH and decreased ratio of NAD+/NADH indicated the
cells’ inability to maintain the normal energy production
Metabolic Response and Energy Regulation
(i.e., ATP) [5]. Hence, in HG culture, the downregulation of
energy was coincident with the transition of cells from the
fast to slow growth stage, which could be further correlated
with the observed aerobic glycolysis and inefficient utilization
of glutaminolysis during the whole cultivation. In the fast
growth stage, aerobic glycolysis was reflected in the
overflow of extracellular pyruvate and fast production of
lactate. The accumulation of pyruvate is generally recognized
as the consequence of a rapid glycolysis. In addition,
glutaminolysis can also produce pyruvate via the malate
shunt to generate NADPH in most mammalian cells [11].
Hence, the pyruvate accumulation and the resultant lactate
production in HG culture indicated that the majority of
consumed glucose and glutamine inefficiently contributed
to ATP generation via the TCA cycle. As cells in HG culture
went into the slow growth stage, a significant consumption
of pyruvate occurred and the accumulation of lactate
ceased, which suggested that the consumed pyruvate
entered the TCA cycle directly for energy formation and
aerobic glycolysis became less pronounced. However, the
inefficient utilization of glutaminolysis was still present in
HG culture. The dysfunction of glutamine and glutamate
to replenish the TCA cycle was further evidenced by the
metabolism of two amino acids, proline and alanine.
Proline can be synthesized directly from glutamate and
alanine is a sink for the excess nitrogen intake from glutamine.
Based on the observation of the steady accumulation of
proline and high concentration of alanine in HG culture
(Fig. 4), it could be inferred that most of the consumed
glutamine and glutamate contributed to the formation of
non-essential amino acids instead of going through
catabolism as anaplerosis for the TCA cycle.
On the other hand, FB-Gal culture had a decreased NADH
level as cells entered the slow growth stage and a gradually
increasing ratio of NAD+/NADH in the whole cultivation
(Figs. 5B and 5C). Although the intracellular ATP level
after 72 h was lower than that at 48 h, it increased
gradually afterwards and the level remained much higher
than that in HG culture after 72 h (Fig. 5A). These data
implicated that cells in FB-Gal culture upregulated the
energy state during the whole cultivation, which could be
further correlated with alleviated aerobic glycolysis in the
fast growth stage and the replenishment of lactate and
other amino acids into the TCA cycle in the slow growth
stage. In the fast growth stage, none overflow of pyruvate
accompanied by the much lower lactate accumulation was
noticed in FB-Gal culture (Figs. 3A and 3C). As cells went
into the slow growth stage, a notable uptake of lactate was
observed in FB-Gal culture (Fig. 2C). In mammalian cells,
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the conversion between pyruvate and lactate is feasible [8].
Since the uptake of pyruvate was found instead of
accumulation (Fig. 3A), it could be possible that the
consumed lactate was exploited to substitute pyruvate and
enter the TCA cycle for energy production. In addition, the
greater consumption of some amino acids and the metabolic
shift of produced serine and alanine in the slow growth
stage were also found in FB-Gal culture (Fig. 4). Besides its
contribution in the synthesis of antibody (representing the
largest molar fraction in anti-human CD20 monoclonal
antibody), serine can be converted to pyruvate for the
subsequent entry into the TCA cycle. The relative steady
concentration of glutamate in FB-Gal culture (Fig. 2E) also
suggested that the consumed alanine might be metabolized
via the transamination pathway, in which pyruvate is
generated from alanine for entry into the TCA cycle. As the
basic building blocks for proteins, amino acids play key
roles in the antibody production process. However, amino
acids can also contribute to catabolism for energy generation
via the TCA cycle. Thus, the more efficient utilization of
amino acids accompanied by anaplerosis of some produced
amino acids in FB-Gal culture enhanced the energy
generation through the efficient replenishment to the TCA
cycle.
Among all the monitored metabolic responses, some
were found directly correlated with energy regulation.
Specifically, in the slow growth stage, a switch from
production to consumption of lactate was noted in both
LG+Gal and FB-Gal cultures. Feeding of amino acids and
vitamins in FB-Gal culture caused even more pronounced
lactate consumption. In contrast, in HG culture, although
the rapid accumulation of lactate in the fast growth stage
stopped quickly as cells entered the slow growth stage,
none consumption of lactate was detected. In the meantime,
the ATP profile in LG, FB-Gal, and HG cultures varied
along with the shift in lactate metabolism, suggesting that
energy regulation could be correlated with lactate metabolism.
Although the shift of lactate metabolism had been described
in similar cultures, in which glucose was either delivered at
a very low concentration or was substituted with other
hexoses [3, 7], its relationship with energy regulation has
not yet been established. Consistently with the lactate
metabolism, the intracellular concentrations of pyruvate
and lactate in LG+Gal and FB-Gal cultures were less
fluctuated than those in HG culture during the whole
cultivation, which suggested that more stable intracellular
pools of pyruvate and lactate might also be associated with
more efficient energy regulation in protein-producing cell
cultures.
November 2013 ⎪ Vol. 23 ⎪ No. 11
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Sun et al.
Influence of Energy Regulation on Cell Growth and
Productivity
Although the energy regulation was found to be different
between HG and FB-Gal cultures, the only difference in
these two culture conditions was their condition of energy
source supply. In FB-Gal culture, glucose was supplemented
at a low level (10 mM) and galactose was supplemented as
a second carbon source. Since the glucose transporters in
mammalian cells usually have a much lower affinity for
other hexoses [20], the supply of energy source with other
hexoses is not as sufficient as that with glucose. As shown
in this study, energy-efficient metabolic responses leading
to more efficient energy regulation were induced under
this condition. In contrast, when surplus glucose was
supplied, aerobic glycolysis and inefficient utilization of
glutaminolysis could be observed during the whole
cultivation and thereby led to downregulated energy
metabolism. However, the reason why the manipulation of
energy source supply could induce distinct metabolic
responses and resultant energy regulation was not explored
in the present study. Nevertheless, these results were
consistent with other reported studies. HepG2 cells were
found to increase the respiration rate to maintain ATP level
in galactose media [29]. It was also reported that the
defective mitochondrial function of cancer cells could be
considerably enhanced by altering the substrate [23]. In the
present study, we were more concerned whether the energy
regulation had a direct influence on cell growth and
productivity. This can be of great significance for process
development and optimization to control the metabolic
responses towards more efficient energy regulation via
effective strategies.
Despite the distinct energy states, the profiles of cell
growth in HG and FB-Gal cultures were similar in both the
fast and slow growth stages, suggesting that energy
regulation had less influence on cell growth. However, if
glucose was restricted to a low concentration or glycolysis
and glutaminolysis were inhibited, cell proliferation would
be affected owing to the deficient nutrients. In the present
study, prolonged growth profiles were noted in HG, LG,
and FB-Gal cultures, whereas transient growth profiles
were in both LG and LG+Pyr cultures. In LG culture, cells
consumed even more pyruvate and glutamate than those in
FB-Gal culture, both of which could directly enter the TCA
cycle for efficient energy generation, suggesting that the
energy metabolism in LG culture might be even more
efficient in the fast growth stage than that in FB-Gal
culture. However, when running out of glucose after 48 h,
the only carbon source in LG culture, cells could not
J. Microbiol. Biotechnol.
survive any longer with deficient energy source. In LG+Pyr
culture, the cell specific growth rate was even 21.7% lower
than that in LG culture in the fast growth stage (Table 2).
Consistently, the consumption rates of both glucose and
glutamine were found lower than those in LG culture.
Since glycolysis and glutaminolysis are the main pathways
in mammalian cells for supplying intermediates such as
ribose, purine, and NADPH for macromolecular synthesis
[19], the growth inhibition in LG+Pyr culture might result
from the inadequate nutrient supply with dysfunction of
glycolysis and glutaminolysis.
On the other hand, the cell productivity profiles in HG
and FB-Gal cultures were different in the slow growth
stage, which suggested that energy regulation might have a
direct influence on cell productivity. When cells were under
the condition of abundant energy source (HG), although the
deregulation of energy metabolism seemed less pronounced
in the slow growth stage, the inefficient replenishment to
the TCA cycle for energy generation still caused the
downregulated energy state. Instead, when cells were under
the limited condition of energy source with the feeding of
amino acids and vitamins (FB-Gal), the upregulated energy
state was achieved through the metabolic shift of lactate
and steady intracellular pools of pyruvate and lactate in
the slow growth stage. In the meantime, the antibody
production rate decreased with time in HG culture, whereas
it remained constant in FB-Gal culture. The correlation
between protein production rate and energy metabolism
has been implicated in several previous studies. Kuwae
et al. [14] found that the limited availability of energy
coincided with the decreased production of recombinant
human antithrombin by CHO cells. A positive correlation
between the oxidative phosphorylation rate and antibody
productivity was also found in the production of the
recombinant MUC1 fusion protein [15]. In the present
study, it was demonstrated that inefficient energy regulation
in the slow growth stage would cause the decrease of cell
productivity. In addition to the formation of cellular
proteins for normal needs, protein-producing cells bear an
extra burden to synthesize a large quantity of recombinant
protein products. Since protein production is an extremely
energy-intensive process in which the energetic demand of
cells is rather high [26], the energy requirement for
production could not be omitted although cell growth
became decelerated. In the present study, the decreased
uptake of glucose was noted in the slow growth stage in
HG culture, which implied that the energy obtained via
glucose phosphorylation would be decreased in this stage.
Furthermore, the inefficient replenishment to the TCA cycle
Metabolic Response and Energy Regulation
for energy generation further caused the downregulated
energy states in HG culture. Therefore, the decreased
productivity in HG culture might result from the failure to
satisfy the energy requirements for antibody production.
However, different from that in HG culture, the intracellular
ATP in LG+Gal culture increased gradually after 72 h.
Nevertheless, the antibody production rate still declined
after 72 as that in HG culture, which suggested an
alternative mechanism responsible for the decreased cell
productivity in LG+Gal culture. As a matter of fact, it was
found that most amino acids, including cysteine, leucine,
isoleucine, valine, histidine, and aspartate, were at lower
concentrations than those in HG culture. Although the
intracellular ATP was at a higher level in LG+Gal culture
than in HG culture, the inadequate precursors for protein
synthesis might have caused the decrease in productivity
in LG+Gal culture, suggesting that insufficient nutrient
supply could also result in inefficient protein production.
On the other hand, in FB-Gal culture, the feeding of amino
acids and vitamins at 72 h might attenuate the deficiency of
certain amino acids as encountered in LG+Gal culture.
Moreover, it was found that more lactate and amino acids
were utilized as replenishment for the TCA cycle, which
stimulated more efficient energy regulation than LG+Gal
culture. The probable reason for the influence of additional
feeding on cell metabolism was not explored extensively.
Nevertheless, it has been reported that some fed-batch
cultures with more balanced nutrients or metal ions can
bring about more efficient energy regulation when cells
seemed to have more utilization of amino acids as well as a
phenotype of lactate consumption [16, 17]. Considering
that both energy regulation and nutrient supply have
direct influence on cell productivity, it is expected that a
fed-batch culture with increased productivity would be
established via further optimizing the feeding medium and
feeding strategy to better balance the energy and nutrient
requirements.
In conclusion, comparative analyses of the metabolic
responses in cultures with distinct energy states revealed
that the shift from production to consumption of lactate
and steady intracellular pools of pyruvate and lactate were
closely correlated with efficient energy regulation. In
addition, the efficient utilization of amino acids as the
replenishment into the TCA cycle was also associated with
upregulated energy metabolism. By comparing both the
cell growth and productivity in different cultures, it was
revealed that the energy regulation had less influence on
cell growth. When cells stepped into the slow growth stage,
1596
the inefficient energy regulation would cause the decrease
in cell productivity, indicating that it is important to meet
energetic needs in the cultivation of protein-producing cells.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (No. 21106045, No. 21206040), the
National High Technology Research and Development
Program of China (863 Program) (No. 2012AA02A303), the
National Science and Technology Major Project (No.
2013ZX10004003-003-003), and Shanghai Training Program
for College Young Teacher (No. hlg11011).
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