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Pharm. Bioprocess. (2013) 1(1), 19–27

1910.4155/PBP.13.5 © 2013 Future Science Ltd ISSN 2048-9145

Research Article

Process performance of mammalian cell cultures can be strongly impacted by high lactate accumulation, which can be a clone or media-dependent characteristic. In this study, the expression of specific genes was measured in several Chinese hamster ovary cell lines under culture conditions leading to different lactate profiles. A reduced expression of two genes was observed under conditions of high lactate accumulation: AGC1/Aralar1, a member of the malate–aspartate shuttle (MAS) and Timm8a1. Overexpression of either of these two genes in the lactate-producing cell line diminished lactate accumulation. This was achieved by promoting a metabolic switch to lactate consumption after day 6, while maintaining a glycolytic rate similar to the parental cells. On the other hand, the biochemical inhibition of MAS activity increased lactate accumulation. All together, these results indicate MAS as a key factor to promote a shift to lactate consumption in cultivated Chinese hamster ovary cells.

Lactate is one of the most intensively moni-tored waste products in mammalian cell cul-ture. The main concern about lactate accu-mulation in bioreactor cultures is its negative effect on cell growth and productivity [1,2]. Different strategies have been used to control lactate metabolism, such as the replacement of glucose and glutamine with slowly metabo-lized carbon sources [3,4] or the improvement of feeding regimes [5,6]. Mammalian cells have also been engineered to downregulate lactate dehydrogenase expression [7–9] or to overexpress the anaplerotic enzyme pyruvate carboxylase [10–12]. The common goal has been either to reduce lactate accumulation or to foster its consumption late in culture. Indeed, lactate normally accumulates during the exponential growth phase, while a switch to its consumption can occur at the transition to the stationary phase. Often this metabolic shift occurs when glucose is almost depleted or at a low residual concentration [6,13,14]. However, the glucose level in the medium is not the only parameter influencing lactate

metabolism. Indeed, the lactate consumption phase can start before glucose depletion, re-sulting in a simultaneous oxidation of glucose and lactate [15–17]. The mitochondrial oxida-tive capacity seems to play a key role in pro-moting this early lactate consumption [17,18], but the mechanisms underlying the metabolic switch have not been identified.

’Omic approaches are gaining strong in-terest as strategies to specifically identify flux constraints that can be exploited for media and feed optimization [19]. Screening for differen-tially expressed genes, which can be correlated to the metabolic state of the cell, has also been proposed as a tool to better understand lactate metabolism and to provide targets for cell en-gineering [15,20–22]. In this study, the expression of selected genes was compared in cell lines that showed opposite lactate profiles under the same culture conditions.

The criteria used to select genes were their key role in glycolysis and tricarboxylic acid cy-cle progression, or in the glutaminolytic path-way [23]. Mitochondrial carriers of metabolic

High expression of the aspartate–glutamate carrier Aralar1 favors lactate consumption in CHO cell culture

Francesca Zagari1,2, Matthieu Stettler1, Lucia Baldi2, Hervé Broly1, Florian M Wurm2 & Martin Jordan*1

1Merck-Serono S.A., Biotech Process Sciences Group, CH-1804 Corsier-sur-Vevey, Zone Industrielle B, CH-1809 Fenil-sur-Corsier, Switzerland 2École Polytechnique Fédérale de Lausanne (EPFL), Laboratory of Cellular Biotechnology, CH J2-506, Station 6, CH-1015 Lausanne, Switzerland *Author for correspondence: Tel.: +41 21 923 2564 Fax: +41 21 923 2013 E-mail: martin.jordan@ merckgroup.com

Research Article

20 future science groupPharm. Bioprocess. (2013) 1(1)

Zagari, Stettler, Baldi, Broly, Wurm & Jordan

intermediates and components of the translocation machinery were also included [24]. Finally, the expression of the mitochondrial aspartate-gluta-mate carrier AGC1 or Aralar1 was also investigated. Aralar1 is a component of the malate–aspartate shuttle (MAS) that has been extensively studied for its role in nutrient-stimulated insulin secretion in b-cells [25,26]. Interest-ingly, Aralar1/AGC1 overexpression reduced lactate production in these cells, while improving glucose oxi-dation to CO

2 and ATP production

[27]. Therefore, it seemed a promising target for the present study.

Lastly, in this work, the role of MAS in the metabolic switch to lactate consumption was investigated in Chi-nese hamster ovary (CHO) cells by both overexpress-ing Aralar1 and by inhibiting the shuttle activity with aminooxyacetic acid (AOAA).

Materials & methods » Cell culture & metabolite measurement

Nonrecombinant CHO-S cells (Life Technologies, Zug, Switzerland) and a subclone of this cell line (re-ferred to in the text as control and subclone, respec-tively) were cultivated in two proprietary chemically defined media (medium 1 and 2) or alternatively in a commercially available chemically defined medium (CD CHO medium, Life Technologies). The initial glutamine concentration was 4.5 mM in all media. Sta-ble clonal cell lines named A and B produced the same recombinant protein and were derived from CHO-GS cells (Life Technologies). These cell lines were grown in medium 1 supplemented with methionine sulph-oximine (Sigma Aldrich GmbH, Buchs, Switzerland). All cultures were performed in batch mode in vented 50 ml shake tubes (TubeSpin® bioreactor 50, TPP AG, Trasadingen, Switzerland) under shaken conditions and controlled atmosphere (Kuhner shaker, 37°C, 5% CO

2,

80% humidity, 320 rpm). The cell density and viability were measured by a Vi-Cell analyzer (Beckman Coulter International S.A., Nyon, Switzerland). Glucose, glu-tamine, lactate, pH and NH

4+ in cell culture superna-

tant were measured using a NOVA Biomedical analyzer (Nova Biomedical GmbH, Rödermark, Germany).

AOAA (Sigma Aldrich GmbH) was added at a final concentration of 0.25 mM on day 0 of culture.

» RNA isolation, cDNA synthesis & real-time PCR assayThe RNeasy Mini kit (Qiagen, Basel, Switzerland) was used for total RNA isolation according to the manu-facturer’s protocol. For each extraction 5 × 106 cells were harvested by centrifugation on different days of culture. The RNA concentration and quality were es-timated spectrophotometrically by measuring the ab-sorbance at 260 and 280 nm. Samples were stored at -20°C until use.

Reverse transcription was performed with the High Capacity RNA-to-cDNA kit (Life Technologies) using 1 µg of total RNA for each sample.

The gene expression array was performed with a custom TaqMan® Array 96-well Plate (Life Technolo-gies) following the manufacturer’s instructions. Murine oligonucleotide primers and probes were used consider-ing the high homology with Chinese hamster [28] since CHO-specific sequences were not available at the time of the experiment. Only the primer sets that gave a reli-able signal were evaluated. Glyceraldehyde-3-phosphate dehydrogenase was used as a reference gene that showed the most stable expression in all tested conditions ac-cording to analysis software (Life Technologies). Data were analyzed following the comparative DDCt method according to the manufacturer’s guidelines.

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Figure 1. Typical growth, glucose and lactate profile of the model cell lines. (A–C) Nonrecombinant Chinese hamster ovary cell lines: control cell line (yellow diamonds) and the subclone (purple squares). (D–F) Recombinant CHO-GS cell lines: clone A (blue circles) and clone B (red triangles). (A & D) Growth, (B & E) glucose and (C & F) lactate profile of the studied cell lines. Each point represents the mean result from two independent cultures with duplicates.

Key Terms

AGC1/Aralar1: Ca2+-activated carrier of the inner mitochondrial membrane catalyzing the exchange of cytoplasmatic glutamate with mitochondrial aspartate.

Malate–aspartate shuttle (MAS): MAS activity, and Aralar1 in particular, have been extensively studied in neuron–glia metabolism and in insulin secretion in b-cells.

Timm8a1: A transporter specific for mitochondrial proteins with a long hydrophilic N-terminus.

Research Article

future science group www.future-science.com 21

High expression of the aspartate–glutamate carrier Aralar1 favors lactate consumption

Independent duplicate cultures of each cell line were used for the test.

» Generation of stable cell linesSubclone cells were transfected with pCMV6-AC-GFP carrying either the mouse Aralar1 or Timm8a1 ORF se-quences cloned upstream of the GFP-tag (ORIGENE, Rockville, USA). 3 × 106 cells were transfected in medi-um 1, with 2 µg of plasmid DNA. The X-tremeGENE HP DNA Transfection Reagent (Roche, Rotkreuz, Switzerland) was used as a delivery method at a reagent:DNA ratio of 2:1(w/w). 2 days after transfec-tion, the cells were resuspended in medium 1 supple-mented with 800 mg/l G418 (Sigma Aldrich GmbH) for stable selection. After 3 weeks, stable pools were used for the isolation of clones by limiting dilution in 384-well plates. Recovered clones were expanded and then cultivated in shake tubes according to the stand-ard protocol. Three clones were selected for each gene

for futher analysis. Transgene expression was evaluated by GFP detection using a Guava PCA 96 flow cytom-eter (Millipore, Molsheim, France). The subcellular lo-calization of the GFP-tagged protein was evaluated by co-staining with Mitotracker Red (Life technologies), a mitochondrial-specific dye.

Results » Growth & lactate profiles

Our principal cellular model to study lactate metabo-lism consisted of two nonrecombinant CHO cell lines that differed in their respective lactate profiles, whereas the cell growth, glucose (Figure 1A–C) and glutamine consumption were similar over the cultivation period (Zagari et al. 2013) [17]. The difference in lactate me-tabolism was evident only after day 6 of the culture (Figure 1C). Indeed, while the control cell line switched to lactate consumption, the subclone continued to ac-cumulate a higher amount of lactate. Interestingly, the

Table 1. Gene expression fold changes in medium 1 for the lactate producer cell lines versus the lactate consumers on days 6 and 9 of culture.

Gene Subclone versus control Rec. clone B versus A

d6 d9 d6 d9

Hexokinase2 -1.40† 1.46‡ 1.12† -1.14†

Phosphofructokinase liver -1.90§ 1.06† 1.18† -1.54§

Phosphofructokinase muscle 1.08† 1.43†

Pyruvate dehydrogenase -1.35† -1.30† -1.43† -1.35†

Pyruvate dehydrogenase kinase

1.34† -1.01†

Citrate syntase -1.01† 1.17†

Citrate lysase -1.11† 1.08† 1.02† -1.75§

Succinate dehydrogenase -1.60§ 1.46‡ -2.22§ -1.15†

Malate dehydrogenase II -1.17† 1.04† -1.11† -1.22†

Malate dehydrogenase I -1.23† 1.27† -1.16† -1.12†

Malic enzyme cytosolic -1.69§ 1.10† -1.04† -1.23†

Glutamate-oxaloaceate transaminase 2

-1.45† 1.09† 1.14† -1.37†

Aralar1 -2.79§ -2.08§ -3.23§ -2.44§

Mitochondrial dicarboxylate carrier

-1.80§ 1.05† -4.35§ -1.54§

Mitochondrial glutamate carrier

-1.07† 1.16† 1.23† -1.72§

Monocarboxylic avid transporter

-1.65§ 1.42† -1.23† -1.32†

Timm22 -1.54§ -1.11† -1.56§

Timm8a1 -2.45§ -1.76§ -1.40† -1.72§

Timm13 1.02† -1.04† -1.02† -1.19†

Negative values mean that the gene is less expressed under the lactate-producing condition. †Values below the first cut-off. ‡Upregulated genes, which fit the FC≥1.5 or FC≥2 cut off. §Downregulated genes.

Research Article



control cell line consumed lactate and glucose simul-taneously (Figure 1B & C). This behavior indicated that lactate oxidation was not the result of glucose depletion, as for the subclone.

The recombinant clones A and B showed characteris-tics similar to the model cell lines (Figure 1D–F). Despite a viable cell number that was lower, clone A switched to lactate consumption after day 6 of the batch culture and displayed a faster glucose consumption compared with clone B (Figure 1E), which produced lactate until the end of the culture (Figure 1F).

» Gene expression analysisThe expression of the chosen metabolism-related genes was compared between the lactate producer (the sub-clone and recombinant clone B) and the lactate con-sumer cell lines (the control and recombinant clone A) on samples from days 6 and 9 of culture. The aim was to identify genes that showed the same trend of up- or down-regulation in both lactate-producing cell lines. The analysis of samples harvested on day 9 was consid-ered as the most informative since this corresponded to the time point when the lactate profiles differed signifi-cantly. A summary of the expression analysis is given in Table 1 as fold change for the lactate producer cell lines versus the lactate-consumer ones. Twofold change (FC) cut-off values were applied for data analysis: a strin-gent one of │FC≥2│ and a more relaxed one, equal

to │FC≥1.5│. The footnote symbols indicate genes that are significantly downregulated or upregulated in the lactate producers. Values below the │FC≥1.5│ are statistically not relevant.

Among the analyzed genes, the only two genes that fit the cut-off criteria were AGC1/Aralar1 and the translo-case of the inner mitochondrial membrane, Timm8a1. Aralar1 was significantly less expressed on both days of analysis (│FC≥2│, p < 0.005 Student’s t-test), while Timm8a1 fitted the │FC≥1.5│cut-off only on day 9 (p < 0.001 Student’s t-test) in both lactate-producing cell lines (Table 1).

» Media formulation impacts Aralar1 & Timm8a1 expressionThe potential role of Aralar1 and Timm8a1 was fur-ther evaluated by switching the cells from medium 1 to either medium 2 or CD CHO medium. Medium 2 promoted the metabolic switch from lactate pro-duction to consumption after day 5 in the subclone (Figure 2A). Moreover, both glucose and lactate (data not shown) were consumed simultaneously. This was never observed for the subclone in medium 1. In-terestingly, Aralar1 and Timm8a1 were expressed at higher levels in the subclone in medium 2 versus me-dium 1 on day 8 of the culture (Table 2). Conversely, control cells grown in CD CHO medium produced higher lactate concentrations than they did in medi-um 1 (Figure 2B), while glucose and glutamine profiles remained unchanged (data not shown). Accordingly, Aralar1 and Timm8a1 expression in the control cell line was reduced in CD CHO medium compared to medium 1 on day 8 (Table 2).

» Aralar1 & Timm8a1 overexpressionThe results from the gene expression analysis suggested that strong accumulation of lactate could be linked to a low expression of Aralar1 and Timm8a1. To verify this hypothesis, the subclone was stably transfected with ei-ther the Aralar1 or Timm8a1 gene. Both proteins were expressed as GFP-fusion proteins. Subcellular localiza-tion of each of the GFP-tagged proteins was verified by co-staining with the mitochondrial dye Mitotracker Red (Figure 3). Three recombinant clones overexpress-

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Figure 2. Medium 2 and commercial chemically defined medium differently impact lactate profile. (A) Subclone cultivated in medium 1 (yellow squares) and in medium 2 (dashed lines). (B) Control cell line grown in medium 1 (blue diamonds) and in CD CHO (dashed lines). Mean ± standard deviations from three independent cultures with duplicates.

Table 2. Aralar1 and Timm8a1 expression on day 8 in medium 2 and chemically defined Chinese hamster ovary medium are reported as fold changes of the values obtained in medium 1 for the same cell line.

GeneSubclone Control

Medium 2 versus medium 1 CD CHO versus medium 1

Aralar1 +1.56 -6.09

Timm8a1 +2.04 -2.70

CD: Chemically defined; CHO: Chinese hamster ovary.

Research Article



ing either Aralar1 or Timm8a1 were able to switch to lactate consumption after day 6 of culture in me-dium 1 (Figure 4B). In order to evaluate if the lactate consumption was due to a reduction of the glycolytic flux, the specific rates of glucose consumption and lac-tate production were calculated between days 6 and 9 (Figure 4A & B, respectively). Interestingly, all clones showed a glucose consumption rate similar or higher than the untransfected subclones.

» MAS inhibition The transaminase inhibitor AOAA has been frequently used to investigate the role of MAS in various func-tional cell types, even if it is not strictly specific for the inhibition of the MAS complex [29–33].

The inhibitor was used in this study to evaluate the role of the MAS in the metabolic shift to lactate con-sumption. Control cells treated with 0.25 mM AOAA showed a higher lactate accumulation than untreated cells. Moreover, this difference persisted until glucose depletion (Figure 5B). Despite this, glucose and gluta-mine consumption were not impacted by the inhibitor (Figure 5C & D). The treated cells had a slightly extended viability, although the growth peak was reached on the same day as the untreated cells (Figure 5A).

DiscussionIn this study, the expression of a selected number of genes was evaluated in different cell lines characterized by a different capacity of consuming lactate (Figure 1). The main aim was to identify promising targets for host cell engineering that could improve lactate metabolism in CHO cells.

Two genes were identified as less expressed in condi-tions of high lactate production: Aralar1 and Timm8a1 (Table 1).

The aspartate–glutamate carrier Aralar1 is a Ca2+-activated transporter localized in the inner mitochon-drial membrane and a crucial component of the MAS [26,27,34]. The function of this shuttle is to regenerate the cytosolic NAD+ pool, which is necessary to maintain the glycolytic flux [25,35]. Alternatively, NADH could also be recycled through pyruvate reduction into lac-tate, catalyzed by the lactate dehydrogenase enzyme. Therefore, MAS activity can influence the metabolic fate of pyruvate and reduce its conversion to lactate, as well as promote the switch to lactate consumption (Figure 6). The oxoglutarate carrier was not included in the study since most of the publications on MAS activ-ity are focused on AGC1. Indeed, AGC1 promotes the only irreversible step in the MAS [34].

The second identified gene that seems to be involved in lactate metabolism is Timm8a1, a translocase of the inner mitochondrial membrane that, together with its

Figure 3. Subcellular localization of Aralar1-GFP and Timm8a1-GFP in the transfected clones. (A) Representative of Timm8a1 clones and (B) of the Aralar1 clones. GFP-tagged protein (green), Mitotracker red (red) and merge (orange).

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Figure 4. Effect of Aralar1 and Timm8a1 overexpression in the subclone. (A) Glucose-specific consumption and (B) lactate production are calculated between days 6 and 9. The untransfected subclone is considered as control. Mean ± standard deviations from three independent cultures with duplicates.

Research Article



partner TIMM13, promotes the correct localization of some mitochondrial proteins [36]. Aralar1 has been reported to be one of Timm8a1’s substrates [24,37,38]. Interestingly, Korke et al. reported a 1.5-fold up regu-lation of TIMM13A in a hybridoma cell culture that underwent the metabolic shift to lactate consump-

tion [22]. However, the impact of this gene expression on lactate metabolism was not investigated in the cited work.

It is noteworthy that the correlation between the expression of Aralar1 and Timm8a1 and the lactate profile was also observed in other chemically defined media that modified the lactate profile of the subclone (Figure 2A) or of the control cells (Figure 2B). These results ruled out an irreversible downregulation of Aralar1 and Timm8a1 in the lactate producers. Rather, they indicated that the media composition can strongly influence the expression of these two genes.

Finally, Aralar1 and Timm8a1 were overexpressed in the subclone. Interestingly, the recombinant clones overexpressing either gene switched to lactate consump-tion around day 6 of culture. It is noteworthy that the specific glucose consumption rate was similar (or even higher for one Timm8a1 clone) to the untransfected sub-clone (Figure 4A & B). This result clearly indicates that the clones’ ability to consume lactate was not due to a slow-down of the glycolytic flux, which consequently forced the oxidation of lactate as an alternative source of energy. Instead, Aralar1 overexpression most likely allowed a more efficient regeneration of the NADH derived from lactate consumption, which hence favored the simultane-ous oxidation of glucose and lactate (Figure 6).

On the other hand, MAS inhibition with AOAA prevented the metabolic switch in the CHO cell line. Lactate accumulation was, therefore, the alternative sys-tem to maintain the NAD/NADH equilibrium in the

NADHNADH

NADNAD

Glucose

Pyruvate

Lactate

Oxoloacetate

aKG

Malate

NADH

NAD

Oxoloacetate

aKG

Asp Asp

Glu Glu

Cytosol

OM IMS IM

Mitochondrial matrix

Malate

AGC1/Aralar1

Timm8a1

Oxo-glutaratemalatecarrier

AGC1/Aralar1

1

23

1

2

Figure 6. Model relating Aralar1 & Timm8a1 to lactate metabolism. Enzymes are numbered in red: 1 = cytosolic and mitochondrial aspartate aminotransferase; 2 = cytosolic and mitochondrial malate dehydrogenase: 3 = lactate dehydrogenase. Timm8a1 allows Aralar1 translocation into the inner mitochondrial membrane. IM: Inner mitochondrial membrane; IMS: Intermembrane space; OM: Outer mitochondrial membrane.

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Figure 5. Impact of aminooxyacetic acid on lactate metabolism. Control cells were cultivated in the absence (purple diamonds) and presence of (yellow diamonds with dashed lines) 0.25 mM of aminooxyacetate. (A) Cell growth, (B) lactate, (C) glucose and (D) glutamine profiles. Mean ± standard deviations from two independent cultures with duplicates.

Research Article



treated cells. Moreover the resulting metabolic pheno-type was similar to the one previously reported for the subclone [17].

The lactate profile observed in the expressing clones and in the control cell line can be explained by the hy-pothesis that during the first 5 days of culture, both glu-cose and glutamine contribute to lactate accumulation. After glutamine depletion, a switch to lactate consump-tion can occur if Aralar1 expression is sufficiently high to allow efficient NADH recycling. In this case, both lactate and the glycolysis-derived pyruvate could replen-ish the tricarboxylic acid cycle after glutamine depletion and sustain mitochondrial oxidative activity [17].

It must be mentioned that Timm8a1 allows the translocation of proteins other than Aralar1. Timm23, for instance, is also a substrate of the Timm8a1/Timm13 complex and it mediates the transport of pro-teins targeted to the matrix or inner membrane [24]. Therefore, the effect on lactate metabolism observed for the Timm8a1 clones cannot only be ascribed to the interaction with Aralar1.

Taken together, the reported data indicate that the MAS can represent an important bottleneck in lactate oxidation. Aralar1, in particular, seems to be a good candidate for the generation of a host cell line able to consume lactate, although its effect on recombinant protein productivity remaining to be investigated.

Future perspectiveThis study finds a good correlation between the abil-ity of a cell line to switch to lactate consumption and

MAS activity. Interestingly, this correlation could be confirmed for all the cases studied: it did not mat-ter whether the switch was cell line or rather media-dependent. The latter observation means that the media composition influences gene expression. Thus, further studies might identify the media components involved in lactate metabolism providing valid in-formation for media design. In addition, it can be speculated that a more efficient oxidation of substrates would provide more energy for protein production. So the overexpression of genes such as AGC1/Aralar1 should have a positive effect on protein yield. Finally, in the coming years, the identification of additional cellular bottlenecks by metabolic and genomic analy-sis will contribute to a more comprehensive under-standing and a continued improvement of industrial processes.

AcknowledgementsThe authors are grateful to M Kobr for scientific support and helpful comments and to D Hacker for revising the manuscript.

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involve-ment with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, hono-raria, stock ownership or options, expert t estimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Executive summary

Gene expression analysis » A higher AGC1/Aralar1 and Timm8a1 expression has been observed in cell lines able to switch from lactate

production to consumption in the presence of glucose. » The observed correlation was confirmed by cultivating the cells in media that differently impacted lactate

metabolism.AGC1/Aralar1 & Timm8a1 overexpression » The overexpression of AGC1/Aralar1 or Timm8a1 in a lactate-producing cell line promoted the switch to lactate

consumption after day 6 of culture. » The glycolytic rate in the recombinant clones was similar to the parental cell line.

Malate–aspartate shuttle inhibition » The biochemical inhibition of the malate–aspartate shuttle in a lactate-consuming cell line resulted in a strong

lactate accumulation. » A link between malate–aspartate shuttle activity and the metabolic switch from lactate production to

consumption is postulated.

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