Lactate metabolism shift in CHO cell culture: the role of mitochondrial oxidative activity

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RESEARCH PAPER New Biotechnology � Volume 30, Number 2 � January 2013

Lactate metabolism shift in CHO cellculture: the role of mitochondrial oxidativeactivityFrancesca Zagari1,2, Martin Jordan1,, Matthieu Stettler1, Herve Broly1 andFlorian M. Wurm2

1Merck-Serono S.A., Biotech Process Sciences Group, CH-1804 Corsier-sur-Vevey, Switzerland2 Ecole Polytechnique Federale de Lausanne (EPFL), Laboratory of Cellular Biotechnology, CH J2-506, Station 6, CH-1015 Lausanne, Switzerland

Lactate production is monitored in industrial processes as a crucial metabolite for cultured mammalian

cells. Typically lactate is strongly produced during the exponential growth phase, while its net

consumption is frequently observed when cells enter into the stationary phase. Such a metabolic shift is

desirable because it seems to favor optimal process performance. However, this shift is neither generic

nor can it be easily controlled, as the mechanisms modulating lactate production/consumption in cell

culture are still under investigation.

In this study different lactate profiles were observed in a chemically defined medium for the parental

CHO-S cells and a non-recombinant subclone. The initial lactate production phase, which is typical for

fast growing cells, was similar for both cell lines. After glutamine depletion the situation changed: the

parental cell line promptly switched to net lactate consumption, whereas the subclone continued to

produce lactate until glucose was depleted as well.

We speculated that the extra lactate production would be ascribed to a different mitochondrial

oxidative capacity in the subclone. Therefore, the mitochondrial membrane potential and oxygen

consumption were measured for both cell lines. Indeed, a correlation between high lactate production

and a reduced oxidative metabolism was found.

Interestingly, this particular metabolic phenotype was also strongly influenced by the medium

composition: both cell lines underwent a switch to lactate consumption when cultivated in a second

medium, while a third one promoted continuous lactate production even for the parental CHO cells.

Again, the correlation between lactate profile and oxidative metabolism was confirmed, pointing to a

central role of mitochondria on lactate metabolism.

IntroductionChinese hamster ovary (CHO) cells are widely used in industrial

processes for the production of recombinant therapeutic pro-

teins [1]. Optimized chemically defined serum-free media have

been developed for the cultivation of this cell line, ensuring

good process reproducibility. Nevertheless, further media

improvement is still needed to increase yields and decrease

Corresponding author: Jordan, M. ([email protected])

238 www.elsevier.com/locate/nbt 1871-6784/$ - see front m

waste product accumulation, mainly lactate and ammonia,

which has been found to have a negative impact on cell growth

and productivity [2].

Serum-free media are normally rich in glucose and glutamine,

which are needed to support a rapid cell growth often associated

with a partial oxidation of substrates in immortalized cells.

Glucose indeed, instead of being completely oxidized to CO2

and H2O, can be converted into lactate even if enough oxygen is

available; a phenomenon known as ‘Warburg effect’ or aerobic

atter � 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nbt.2012.05.021

mailto:[email protected]

http://dx.doi.org/10.1016/j.nbt.2012.05.021

New Biotechnology �Volume 30, Number 2 � January 2013 RESEARCH PAPER

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glycolysis [3,4]. Aerobic glycolysis promotes rapid glucose con-

sumption and can compensate for the reduced mitochondrial

ATP production [5] in terms of total energy yield. Another

characteristic of fast growing cells is the rapid consumption

of glutamine which is a key source of energy for the TCA cycle.

However, this might imply a truncated cycle with oxidation of

glutamine to the intermediate malic acid, which is then con-

verted into pyruvate, and finally into lactate, in the cytosol [6].

This metabolism, known as glutaminolysis, enables the produc-

tion of NADPH that is needed for anabolic processes; such as

fatty acids synthesis required during cell duplication. Hence,

lactate can be the end product of an incomplete oxidative

metabolism in highly proliferating cells in vitro, where nutrients

are rapidly consumed to fulfill ATP and fatty acids demand.

Different approaches have been proposed to keep lactate

production at low level or to promote a switch in its metabolism

in cultivated cells. Cell engineering was used to downregulate

the lactate dehydrogenase gene [7–9] and to overexpress the

enzymes pyruvate carboxylase [10–12] or malate dehydrogenase

[13]. Lactate accumulation was also limited by substituting

glucose and glutamine in the medium with slowly metabolized

carbon sources [14–16] or by applying a sophisticated glucose

feeding regime based on pH measurements [17]. Genomic and

proteomic approaches have been used to characterize CHO cell

lines with distinct lactate profiles, with the ultimate aim of

identifying candidate genes which correlate with the observed

FIGURE 1

Growth and metabolic profile of the control cell line (filled diamond) and the subclonprofiles are comparable in both cell lines, while lactate profiles (d) show distinct tre

while the subclone, after a brief steady state, continues to produce it. Mean � stan

for several independent experiments).

metabolic phenotype [18,19]. Nevertheless, considering the

complexity of carbon metabolism and its regulation [20],

further efforts are needed to fully understand lactate metabo-

lism and subsequently control its accumulation in industrial

processes.

In this study we investigated the carbon metabolism in the

parental CHO-S cell line (referred next as control cell line) and in a

subpopulation obtained during an independent subcloning

experiment (referred next as subclone). Because this subclone

spontaneously produced higher levels of lactate when cultivated

in a proprietary chemically defined medium, it was a good model

to study the effect of the two main carbon sources, glucose and

glutamine, in this context. The mitochondrial oxidative capacity

was also analyzed; in particular the mitochondrial membrane

potential (DCm) and the oxygen consumption were monitored

as key indicators of the energy metabolism. Moreover, the two cell

lines were cultivated in other two media; a distinct proprietary

chemically defined medium and a commercial medium, which

differently influenced lactate metabolism. The objective was to

evaluate if a significant correlation between the oxidative meta-

bolism and the observed lactate profiles could be identified under

different culture conditions.

On the basis of the reported data, we hypothesize that the

mitochondrial oxidative metabolism and/or specific substrate

transporters are responsible for the switch from lactate production

to net consumption.

e (empty square) in medium 1. Cell growth (a), glucose (b) and glutamine (c)nds after day 5. Indeed, the control cell line switches to lactate consumption,

dard deviation from duplicate cultures (note: selected graph is representative

www.elsevier.com/locate/nbt 239


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Materials and methodsCell culture and metabolite measurementCHO-S cells and a non-recombinant subclone of this control cell line

were cultivated in batch mode in Tube Spins (TPP AG, Trasadingen,

Switzerland) in two proprietary chemically defined media (media 1

and 2) or alternatively in a commercially available chemically

defined medium (CD CHO medium, Life Technologies, Zug, Switzer-

land), under shaken condition (Kuhner shaker, 378C, 5% CO2).

Initial glutamine concentration was 4.5 mM in all tested media.

Cell numbers and viability were measured by a Vi-Cell analyzer

(Beckman Coulter International S.A., Nyon, Switzerland).

Glucose, glutamine, lactate, pH and NH4+ in cell culture super-

natant were measured using a NOVA Biomedical analyzer (Nova

Biomedical GmbH, Rodermark, Germany).

Oxygen consumption measurementOxygen consumption was measured using the PreSens Oxoplate

(Precision Sensing GmbH, Regensburg, Germany). Briefly, cells at

FIGURE 2

Viable cells, glucose and lactate profiles of the control cells (a–c) and the subclone (

55.5 mM (empty symbols) and 66.6 mM (dashed lines)). While the control cell lineconcentration (c), the subclone continues to produce lactate until glucose is deplet

the glucose rich media show a higher viable cell number on day 12. Mean � sta

240 www.elsevier.com/locate/nbt

different days of culture were resuspended at a concentration of

106 cells/ml in spent medium and 200 ml was used for the test. The

assay was carried out using a Spectramax Gemini EM fluorometer

(Molecular Devices, Sunnyvale, CA, USA) monitoring fluorescence

emission for 45 min at 378C. Oxygen pO2 values as % of air satura-

tion were calculated after calibration according to the manufac-

turer’s guidelines. Two inhibitors, oligomycin, an inhibitor of the

F0/F1 ATPase, or antimycin A, a respiratory chain inhibitor, were

added as controls to evaluate the residual oxygen consumption as a

result of leaking or non-mitochondrial activity, respectively. Spent

media were also tested to check for possible media components

interference with the assay. Results were normalized by setting the

oxygen consumption of the lactate consuming condition to 100%.

Mitochondrial membrane potentialMitochondrial membrane potential was measured in 105 intact

cells with 100 nM TMRE (Biotium, Hayward, CA, USA). After

20 min incubation at 378C, the cells were washed twice in PBS

d–f) in medium 1 with different initial glucose levels (44.4 mM (filled symbols),

switches to lactate consumption after day 5 independently from glucoseed (f ). Growth profiles are not impacted (a and d), but for the control cell line

ndard deviation from two independent tests each one done in duplicates.


before flow cytometry analysis using a Guava PCA 96 (Millipore,

Molsheim, France). At this concentration TMRE works in a

quenched behavior, therefore CCCP addition induces an increase

of fluorescence as a result of dye redistribution in the cytosol

[21,22]. The ratio of fluorescence increase, under the same incuba-

tion conditions, was used as an indication of the mitochondrial

membrane potential on different culture days.

FIGURE 3

Metabolic profile of the control cell line (a–d) and the subclone (e–f) in medium 1 aculture: 4 mM (empty symbols) or 6 mM (dashed lines). Glutamine increases almost s

(b,c) compared to control condition (filled symbols). Lactate production is also highe

Glucose (d,h) and growth profile (a,e) are not impacted. Mean � standard deviati

ResultsLactate metabolism in medium 1The control cell line and the subclone showed similar cell growth,

glucose and glutamine consumption profiles when cultivated in

medium 1 (Fig. 1a–c), while lactate profile was clearly different

(Fig. 1d). Indeed, only the control cell line switched to lactate

consumption after day 5, when the glutamine in the medium was

fter glutamine feeding. Two glutamine feeds were added on day 5 (arrows) oftoichiometrically and transiently the lactate production in the control cell line

r in the subclone (f,g) but no switch occurs even when glutamine is depleted.

on from two independent tests.


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FIGURE 4

Mitochondrial oxidative metabolism in medium 1. The mitochondrial

membrane potential (a) and oxygen consumption (b) were measured in

both cell lines at different days of culture. Data are shown for the subclone as% of the control cell line for each day of analysis. The subclone shows a

significant (*P value < 0.05 and **P value < 0.01, calculated with Student’s t-

test) lower membrane potential and oxygen consumption after day 5 of

culture compared to the control cell line. Mean � standard deviation from atleast four independent tests.

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depleted and used glucose and lactate simultaneously. By contrast,

the subclone continued to produce lactate until glucose was

almost depleted as well.

To further investigate this cell specific role of glucose on lactate

profile, the two cell lines were cultivated in media containing

increasing amount of glucose: 44.4 mM, 55.5 mM and 66.6 mM. As

shown in Fig. 2b,c, increasing glucose concentrations did not

impact the metabolic switch to lactate consumption in the control

cells. A steady phase was, however, observed toward the end of the

culture in the case of the highest glucose concentration. By con-

trast, the increasing glucose concentrations delayed the lactate

metabolic switch in the subclone; precisely, from day 10 in the

medium containing 44.4 mM of glucose to day 12 in the 55.5 mM

medium (Fig. 2e,f). Again, this delay correlated with the complete

depletion of glucose. Interestingly, glucose consumption

(Fig. 2b,e) and growth profile (Fig. 2a,d) were similar in all tested

conditions.

The next experiment evaluated the effect of glutamine on the

lactate profile. Two new conditions, addition of 4 mM or 6 mM of

glutamine on day 5, were tested to delay its depletion (Fig. 3b,f).

Results demonstrated that glutamine feeding induced a significant

increase of lactate levels and delayed its consumption in the

control cell line (Fig. 3c). The actual differences were

3.6 � 0.3 mM and 5.4 � 0.2 mM for the 4 mM and 6 mM feed,

respectively, which comes close to a stoichiometric increase, indi-

cating a 1:1 conversion due to the glutaminolytic pathway. The

glutamine feeds also induced a higher lactate production in the

subclone (Fig. 3g), but supplementing 4 mM or 6 mM gave the same

results. Glucose consumption (Fig. 3d,h) and cell growth were not

significantly impacted by the glutamine feeds (Fig. 3a,e).

Analysis of mitochondrial metabolismData, hitherto reported, demonstrate that glutamine can modu-

late lactate production. Once glutamine was depleted, lactate was

readily consumed in the control cell line, most likely to compen-

sate for the reduced glutamine entry into the TCA cycle. This did

not occur in the subclone, thus the subclone might not be able to

fully compensate the mitochondrial oxidative metabolism after

glutamine depletion. To verify if mitochondria metabolic activity

was reduced in the subclone, the mitochondrial membrane

potential (DCm) and oxygen consumption were measured in the

two cell lines.

Changes of DCm were monitored at different stages of cell

growth and data are reported for the subclone as percent of the

control cell line. As shown in Fig. 4a, the mitochondrial membrane

potential in the subclone was comparable to the control cell line

during the first 5 days, while it decreased significantly and reached

around 80% of the control on day 7 and 60% on day 9 (P

value < 0.05 and <0.01, respectively).

Oxygen consumption in the subclone also significantly declined

after the first five days of culture, reaching about 60% of the control

on days 7 and 9 (Fig. 4b, P value <0.01 and 0.05, respectively).

Oligomycin or Antimycin A addition inhibited oxygen consump-

tion to the same extents in both cell lines (data not shown).

Media composition effects on lactate consumptionTo further clarify whether the lactate profile is determined by the

intrinsic cell line properties or if it could be influenced by the


medium composition as well, the two cell lines were cultivated in

medium 2 and in the commercially available CD CHO medium.

Interestingly, in medium 2 (Fig. 5a–c) the subclone showed the

same profile as the control cell line with a switch to lactate

consumption after glutamine depletion on day 5 (Fig. 5a). Accord-

ingly, the DCm (Fig. 5b) and oxygen consumption (Fig. 5c) of the

subclone were comparable to the control cell line, indicating an

improved mitochondrial oxidative metabolism.

By contrast, CD CHO medium triggered a higher lactate pro-

duction and even prevented the metabolic switch in the control

cell line (Fig. 5d). Accordingly, the mitochondrial membrane

potential and oxygen consumption showed a significant decrease

on day 7 compared to the same cell line cultivated in medium 1

(Fig. 5e,f).

DiscussionIn this study two cell lines with distinct lactate profiles were

cultivated in different media to identify a possible correlation

between altered oxidative capacity of mitochondria and the

observed switch of lactate metabolism.

The reported data clearly indicate that glutamine has an impor-

tant role in modulating the initial lactate production in both cell

lines, while glucose has a different impact depending on the cell


FIGURE 5

Influence of media composition on lactate profile and mitochondrial oxidative metabolism: control cells (filled diamonds) versus the subclone (empty squares) in

medium 2 (a–c) and medium 1 (filled diamonds) versus CD CHO medium (dashed line) for the control cells (d–f). The subclone switches to lactate consumption (a)in the medium 2 and the mitochondrial membrane potential (b) and oxygen consumption (c) are also similar to the control cell line (data are plotted only for the

subclone relative to the control set at 100%). By contrast, control cells show a higher lactate production with no metabolic switch in CD CHO medium (d). DCm (e)

and oxygen consumption (f ) in CD CHO are significantly decreased on day 7 (***P value < 0.005) compared to the values obtained for the same cell line in medium

1. Mean � standard deviation from three independent tests with duplicates.

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line and medium tested. In particular, in medium 1 lactate is

accumulated during the first five days of culture for both cell lines,

while a switch to net consumption occurs afterwards only in the

control cells (Fig. 1d). This metabolic shift is concomitant with a

strong reduction of the residual glutamine concentration in the

medium (Fig. 1c), while glucose is still present at relatively high

level (Fig. 1b). Interestingly, glutamine feeding on day 5 induces a

temporary and almost stoichiometric increase in lactate produc-

tion in the control cell line (Fig. 3b,c), which eventually switches

to net lactate consumption at the time-point when the addition-

ally provided glutamine has been consumed. On the basis of these

data, we hypothesized that during the first five days of culture,

lactate production is significantly influenced by the glutaminoly-

tic pathway. This metabolism has been well established in cancer

cells [23] and it is also reported in CHO cells cultures [24]. Glu-

taminolysis involves the efflux of malate out of the mitochondria

and its conversion to pyruvate, and finally into lactate, with the

production of NADPH. Most probably, our fast growing cells

undergo the same metabolic pathway to fulfill their NADPH need.

Afterward, once glutamine is depleted, lactate is consumed to

replenish the TCA cycle.

On the contrary, the subclone has a lactate profile which

deviates from the control cell line after day 5 (Fig. 1). Indeed,

lactate production persists for a longer period. Eventually, net



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lactate consumption occurs as well, but only later in culture, when

both glutamine and glucose are almost depleted (Fig. 1b,c). This

behavior is more similar to examples reported in literature where

the metabolic shift is induced by a reduction of glucose availability

[16,17,24]. To study the impact of glucose on lactate production,

three increasing concentrations were tested in medium 1. While

the higher carbon availability has no impact on the control cell

line (Fig. 2b,c), in the subclone a delay of lactate metabolism shift

can be observed (Fig. 2e,f). Glutamine feeding also increases lactate

production in the subclone (Fig. 3f,g). However, this increase is not

proportional to the feed concentration, as observed in the control

cell line, and no metabolic switch occurs, due to glycolysis con-

tribution to lactate accumulation.

Considering these data, it is probable that the higher lactate

production in the subclone can be linked to an impairment of the

mitochondrial metabolic activity which occurs in medium 1 and is

detectable only when glutamine is consumed. Indeed, from this

time point onward, the subclone uses mainly glucose as energy

source. This leads to an extra production of lactate and a conse-

quent reduction of intermediates entry into the TCA cycle, com-

pared to the control cell line.

To confirm our hypothesis we also measured other parameters

reflecting the level of oxidative metabolism in the mitochondria:

the mitochondrial membrane potential (DCm) and the oxygen

consumption. DCm depends on reducing equivalents availability

in the mitochondria, which can be either produced in situ or

transported through the NADH/NAD shuttles. Oxygen consump-

tion is also dependent on the membrane potential, as it is the final

acceptor of the mitochondrial electron chain [25]; therefore it was

used to further confirm our findings.

Data shown in Fig. 4a,b indicate a significant decrease of DCm

and oxygen consumption on days 7 and 9 in the subclone com-

pared to the control, when cultivated in medium 1. Interestingly

this reduction is concomitant with the divergence in lactate

metabolism between the two cell lines. This behavior confirms

our hypothesis of a decreased mitochondrial oxidative capacity in


the subclone as a result of the extra lactate production and the

consequent reduction of TCA cycle fuelling. Indeed, in medium 2,

both the mitochondrial membrane potential and oxygen con-

sumption in the subclone are similar to the control cell line

(Fig. 5b,c), and this also correlates with the improved lactate

profile (Fig. 5a). On the contrary, in the CD CHO medium the

control cells accumulate higher amount of lactate compared to

medium 1 (Fig. 5d). Accordingly, DCm and oxygen consumption

are reduced on day 7 (Fig. 5e,f) compared to the same cell line in

medium 1.

Considering these data, we speculate that the mitochondrial

oxidative capacity could be used as a marker of an impaired lactate

metabolism in CHO cells, either to select clones, as already pro-

posed by Hinterk et al. [26], or in media optimization to identify

key metabolites which can boost oxidative metabolism.

In conclusion, this study shows that a combination of cell

characteristics and media composition can affect lactate metabo-

lism; however, in both cases an impairment of the mitochondrial

oxidative metabolism can be identified. Therefore, mitochondria

play a key role in regulating the switch to lactate consumption, in

agreement with what was recently reported by Luo et al. [27] even

if in their system the high lactate production during the initial

exponential phase was linked to glycolytic metabolism.

We hypothesize that lactate entry into the TCA cycle could be

influenced by mitochondrial transport systems or enzymes, whose

regulation is somehow influenced by unidentified media compo-

nents. Further studies are needed to clarify this point; in particular

the analysis of expression levels of key mitochondrial proteins

could help to identify specific targets for future CHO cells engi-

neering to improve the understanding and control of lactate

metabolism.

AcknowledgementsThe authors are grateful to Dr. David Hacker and Dr. Lucia Baldi for

the valuable discussions.

This work has been financed by Merck-Serono S.A.

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