Journal Pre-proof

Concomitant consumption of glucose and lactate: A novel batch

production process for CHO cells

I. Martı́nez-Monge, P. Comas, J. Triquell, A. Casablancas, M.

Lecina, C. Paredes, J.J. Cairó

PII: S1369-703X(19)30295-5
DOI: https://doi.org/10.1016/j.bej.2019.107358
Reference: BEJ 107358

To appear in: Biochemical Engineering Journal

Received Date: 16 April 2019

Revised Date: 21 August 2019
Accepted Date: 22 August 2019

Please cite this article as: Martı́nez-Monge I, Comas P, Triquell J, Casablancas A, Lecina M,
Paredes C, Cairó JJ, Concomitant consumption of glucose and lactate: A novel batch
production process for CHO cells, Biochemical Engineering Journal (2019),
doi: https://doi.org/10.1016/j.bej.2019.107358

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Concomitant consumption of glucose and lactate: A novel batch production process

for CHO cells

[I. Martínez-Monge]1,2

[P. Comas]2

[J. Triquell]2

[A. Casablancas]2

[M. Lecina]2,3

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[C. Paredes]2

[J. J. Cairó]2

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1[The -p
Novo Nordisk Foundation Center for Biosustainability, Technical University of

Denmark, DK-2800 Kgs. Lyngby, Denmark]

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2[Department of Chemical, Biological and Environmental Engineering, Autonomous

University of Barcelona, Cerdanyola del Vallès, Spain]

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3[Bioengineering Department, IQS-Universitat Ramon Llull, Barcelona, Spain]

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Corresponding author:

E-mail: ivanmar@biosustain.dtu.dk
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Present address: [I. Martínez-Monge, The Novo Nordisk Foundation Center for
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Biosustainability, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark]

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Highlights:

 CHO cells are able to co-metabolize glucose and lactate during the growth phase.

 Extracellular pH and lactate concentration are the key factors.

 A batch culture in which co-consumption was obtained from the beginning was defined.

 Such behavior yielded to a more efficient substrate consumption.

Abstract

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The rapid consumption of large quantities of glucose in conventional CHO batch processes

yields large quantities of lactate, leading to the inhibition of cell growth. We have observed

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that under certain culture conditions, CHO cells are able to co-metabolize glucose and

lactate, even during the growth phase. -p

Taking advantage of this metabolic behavior, a new batch process has been defined where
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there is a concomitant consumption of glucose and lactate from the beginning of the culture.

This batch process requires the addition of lactate to the culture media as well as keeping
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the pH around 6.8. As a result, an exponential growth phase without lactate production

together with a substantially diminished glucose consumption is achieved.

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Flux Balance Analysis (FBA) confirmed that in glucose consumption/lactate generation,

most of the pyruvate generated through glycolysis is converted to lactate to fulfill the NADH
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regeneration requirements into the cytoplasm, whereas only a 31% of pyruvate enters the

TCA through acetyl-CoA. In contrast, in glucose-lactate concomitant consumption, glucose

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uptake is reduced up to 3-fold, balancing glycolysis and TCA cycle fluxes and thus yielding

to a more efficient substrate consumption. Such metabolic behavior in which glucose and

lactate are simultaneously consumed from the beginning of the culture has never been

reported in CHO cell cultures, and it is promising for the design of new culturing strategies

in batch, fed-batch or perfusion processes.

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Abbreviations: [Use the following text style: Abb1, abbreviation 1; Abb2, abbreviation 2]

𝐪𝐦 , specific consumption/production rate of the metabolite m (nmols·106cells -1·h-1 or

nmols·mgDW-1·h-1)

𝑪𝑶𝟐 , oxygen concentration in the liquid phase (mmols·L-1)

𝑪𝒎,𝟎 , concentration of the metabolite m at time t0 (mmols·L-1)

𝑪𝒎 , concentration of the metabolite m (mmols·L-1)

𝑿𝒗,𝟎 , viable cell concentration at time t0 (106cells·mL-1)

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𝑿𝒗 , viable cell concentration (106cells·mL-1)

𝒌𝒅 , glutamine decomposition rate (h-1)

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𝒓𝑿 , volumetric growth rate (106cells·mL-1·h-1)

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𝒓𝒎 , volumetric consumption/production rate of the metabolite m (mmols·L-1·h-1)

𝝁𝒎𝒂𝒙 , maximum specific growth rate (h-1)

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D.O., relative oxygen concentration in the liquid phase in respect to the air saturation in

equilibrium (%)
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Dt, duplication time of the cells in culture (h)

O.U.R., oxygen uptake rate (mmols O2·L-1·h-1)

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t, time (h)

Xvmax, maximum viable cell density reached in the culture (106cells·mL-1)

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𝒒𝑶𝟐 , specific oxygen consumption rate (nmolO2·106cells-1·h-1) or (nmols·mgDW-1·h-1)

vvm, gas volume per minute and per volume of bioreactor (L·min-1·L-1)
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Keywords: [CHO, Co-metabolism, Flux Balance Analysis, Lactate, Metabolic Shift]

1 Introduction

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A large fraction of proteins for both diagnostic and therapeutic applications are produced

in Chinese Hamster Ovary (CHO) cells [1,2]. Some advantages of CHO cells are 1) their

capability to fold and make human-compatible post-translational modifications on

recombinant proteins, 2) they can be grown in suspension cultures using chemically defined

media maintaining a stable metabolism for long periods in cultivation [3,4], and 3) high titer

of the protein of interest can be reached (about 3-10 g/L) [5].

One of the most important limitations of CHO cells is their inefficient metabolism,

characterized by the consumption of large quantities of glucose and the subsequent

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production of large quantities of lactate, a by-product widely reported to inhibit cell growth

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in cell cultures [6–8]. This phenomenon of generating lactate from glucose, instead of being

completely oxidized to CO2 and H2O, even when oxygen is not limiting, is commonly known

as “the Warburg effect” [9].

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Due to the adverse effects of lactate accumulation on cell growth, significant efforts have

been devoted to reduce its accumulation in mammalian cell cultures. The use of alternative
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carbon sources to glucose, like fructose or galactose [10,11] and media optimization in

terms of amino acids composition [12] have been reported to reduce lactate formation, but
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also resulted in a significant lowering of the cell growth rate. Another alternative for

reducing lactate production consists in keeping low glucose concentrations along the

culture, as it has been demonstrated in continuous operations [13,14]. Moreover, different

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fed-batch strategies limiting glucose concentration have been performed to this end,
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obtaining a significant reduction of lactate formation [15,16].

Alternatively, several efforts in the field of cell engineering have been made towards

significantly reducing lactate production. Suppression of main sugar membrane

transporters [17], expression of pyruvate carboxylase in the cytoplasm for restoring the link

between glycolysis and TCA [18,19] and down-regulation of lactate dehydrogenase [20,21]

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have been reported. However, while in some specific cases lactate formation was somehow

reduced, it has never been completely suppressed.

Interestingly, it has also been reported that under certain conditions mammalian cells are

able to switch to a different carbon metabolism based on lactate consumption during the

non-growing phases of cell cultures [22,23]. For example, Martínez et al. [24] observed

lactate consumption in CHO cells when glucose was completely depleted during the plateau

phase of the culture [24].

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Even more interesting is the different lactate metabolism that consists in simultaneous

consumption of glucose and lactate. Zagari et al. [25] and Wahrheit et al. [26] observed

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glucose and lactate concomitant consumption in CHO cells at the end of the exponential

growth phase, when glutamine was depleted from medium. Furthermore, glucose and
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lactate co-consumption has also been reported in late stages of fed-batch cultures, but again
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linear cell growth profile was described, indicating a cell growth limitation in this metabolic

phase [22,23,27].
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Li et al. [28] observed that feeding exogenous lactate in CHO cell cultures and replacing CO2

by lactic acid for the pH control led to a reduction of ammonia and pCO2 accumulation. To
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go beyond, Gagnon et al. [29] applied a feed strategy based on pH control in CHO fed-batch

cultures to suppress the lactate accumulation. A metabolic analysis was carried out by Luo
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et al. [30] studying the correlation between copper and the lactate metabolic shift in

chemically defined medium with two different CHO cell lines, indicating that the lactate
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metabolic shift seems to be related to the cells oxidative metabolic capacity.

Recently, we have observed a different glucose and lactate concomitant consumption

metabolic behavior in HEK293 [31,32] and CHO cell cultures. Under certain culture

conditions both cell lines are able to co-metabolize glucose and lactate simultaneously, even

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during the growth phase, resulting in more efficient substrate consumption (more efficient

carbon usage). By using this particular concomitant catabolism of glucose and lactate new

processes can be designed. Furthermore, a deeper knowledge on such metabolism, could

help to design new engineered cell lines with the aim of obtaining more efficient mammalian

cells-based bioprocesses for different applications.

It is well established that metabolic fluxes are key factors in order to study the metabolic

behavior of a given cell line in culture [33,34]. Flux Balance Analysis (FBA) is, by far, the

most popular technique used to determine metabolic fluxes and it has been widely

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employed to predict in silico diverse phenotypes of several biological systems [35,36]. In

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this study, FBA methodology has been applied to analyze the changes on metabolic fluxes

of CHO cells in culture occurring on glucose and lactate concomitant consumption phases
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compared to the metabolic shift from glucose consumption/lactate secretion phases of

conventional processes. The purpose of this work is to design a new batch strategy in order
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to compare it to conventional CHO batches, as a prelude for fed-batch or perfusion cultures.

As far as we know, the implementation of this new culture batch strategy to achieve this
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metabolic behavior and its characterization by FBA, has never been reported in the

literature.
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2 Materials and methods

2.1 Cell line and cell maintenance

The cell line used in this work was FreeStyle™ CHO-S Cells (Invitrogen, Thermo Scientific).

CHO-S cell line was cultured in 125 mL polycarbonate shake-flask (Corning Inc.) with a

working volume of 12 mL incubated in a 5% CO2 air mixture and in a humidified atmosphere

at 37°C (Steri-cult 2000 Incubator, Forma Scientific). Flasks were continuously agitated at

110 rpm on an orbital shaking platform (Stuart SSL110 Incubator, Forma Scientific).

Cultures were passaged every 2 or 3 days at a seeding density of 3.0·105 cells·mL-1.

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2.2 Cell media

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The basal medium used for all experiments and for cell maintenance was CD OptiCHO™

(Gibco, Life Technologies), supplemented with 8 mM of GlutaMAX (Gibco, Life

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Technologies), 50 ppm Antifoam C Emulsion (Sigma Aldrich) and 2 g·L-1 Kolliphor® P 188
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(Sigma Aldrich).

For the experiment in which concomitant glucose and lactate consumption was triggered
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from the onset of the culture, exogenous lactate was added to the initial medium using a

stock solution of 1.6 M sodium L-lactate (Panreac), in MilliQ water and sterile filtered, to
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obtain an initial lactate concentration of 15mM.

2.3 Shake-flasks culturing platform

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250-mL polycarbonate shake-flask (Corning Inc.) with 50-mL of working volume were used

for preliminary experiments. Identical conditions to those described under “cell

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maintenance” were maintained. The seeding density was about 3.0·105 cells·mL-1 and the

sampling volume was about 1 mL.

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2.4 Bioreactor and operational conditions

Bioreactor cell cultures were performed in a Biostat Bplus (Sartorius Stedim Biotech),

equipped with a 2L-cylindrical vessel. Dissolved oxygen concentration was measured with

an optical probe (VisiFerm DO, Hamilton) and maintained at 30% of saturation by a gas mix

air/oxygen unit and an aeration flow fixed at 0.175 vvm. pH was measured with a standard

electrode (EasyFerm Plus, Hamilton) and maintained by using sterile solutions of HCl 0.5M

(Panreac) and NaOH 0.5M (Sigma) on demand. The pH set point for each experiment is

presented in the manuscript. For all experiments, a constant concentration of 5% of CO2 was

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set in the gas-mixing unit and maintained throughout the entire culture.

Temperature was maintained at 37°C. The stirrer was equipped with two marine impellers

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and the stirring speed was set to 100 rpm. The volume of the samples in bioreactor

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experiments was about 5 mL. Duplicates for each culture condition were performed. Due to

the difficulty to obtain similar plots for the duplicates (small differences in inoculum cell
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density, different time points…) Figure 1 shows only one of the experimental duplicates.

The second replicate can be found in Supplementary Data 1. For all conditions, culture
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replicates are comparable.

2.5 Analytical methods

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Cell number

Cell number was determined by manual counting using a Neubauer hemocytometer and a
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phase contrast microscope (Nikon eclypse, TS100). Cell viability was determined by the

Trypan blue dye exclusion method. Samples were diluted 1:1 mixture of a 0.2% Trypan
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blue (Gibco, Life Technologies) and manually counted.

Metabolites concentration

4.5 mL of sample were centrifuged at 3000 g for 3 min (Spectrafuge) and the supernatant

was filtered through a 0.22-µm filter (Merck Millipore) to remove remaining solid particles

or cell debris. Glucose and lactate concentration were determined using an automatic

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glucose and lactate analyzer (YSI, Yellow Springs Instruments, 2700 Select). Ammonium

was measured with an automatic enzymatic test analyzer (Enology Y15, Biosystems).

Amino acids and GlutaMAX concentrations were determined by HPLC using post-column

derivation method in a PEEK manufactured column with cation-exchange resin (Ultropac,

polystyrene/divinylbenzene sulfonate) 5 µm, 200x4 mm (Biochrm LTd.). Derivatized amino

acids were detected colorimetrically at 570 and 440 nm wavelengths.

2.6 Oxygen uptake rate (OUR)

Oxygen uptake rate (OUR) was determined by applying the dynamic method as previously

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detailed [37]. This method is based on discontinuing the bioreactor airflow inlet for a few

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minutes. During this time, the headspace is flushed with N2 and the decrease in dissolved

oxygen concentration is monitored. By accounting for the oxygen desorption from the liquid
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phase to the gas phase, determined through previous calibration, the respiratory activity of

the cells can be calculated. The specific methodology used in this work is detailed
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somewhere else [38].
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2.7 Specific production and consumption rates calculation

Cell growth rate equation can be derived from the mass balance applied to biomass as
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expressed in Equation 1. The maximum specific growth rate (µmax) was calculated therein

the exponential growth phase from the linearization of this equation (Equation 2).

𝑑𝑋𝑣
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= 𝑟𝑋 = 𝜇𝑚𝑎𝑥 · 𝑋𝑣 [1]
𝑑𝑡

ln(𝑋𝑣 ) = ln(𝑋𝑣,0 ) + 𝜇𝑚𝑎𝑥 · (𝑡 − 𝑡0 ) [2]

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The consumption/production rates for glucose, lactate, GlutaMAX and amino acids (except

glutamine) are expressed by Equation 3. The specific consumption/production rates (qm)

were calculated from the Equation 4, resulting from integrating and rearranging

Equations 1 and 3.

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 𝑑𝐶𝑚
= 𝑟𝑚 = 𝑞𝑚 · 10−3 · 𝑋 [3]
𝑑𝑡

qm · 10−3 · X 0
𝐶𝑚 = 𝐶𝑚,0 + · [𝑒 𝜇𝑚𝑎𝑥·𝑡 − 𝑒 𝜇𝑚𝑎𝑥 ·𝑡0 ] [4]
μ · e𝜇𝑚𝑎𝑥 ·𝑡0

As previously described by [39], glutamine spontaneously decomposes to form ammonia

following first-order kinetics. The consumption rate of glutamine and the production rate

of ammonia are expressed by Equations 5 and 6 respectively, where the decomposition

rate (kd) has been evaluated in similar experimental conditions of this work to obtain a

value of 3.45·10-3 h-1.

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𝑑𝐶𝐺𝑙𝑢𝑡𝑚
= 𝑟𝐺𝑙𝑢𝑡𝑚 = 𝑞𝐺𝑙𝑢𝑡𝑚 · 10−3 · 𝑋 − 𝑘𝑑 · 𝐶𝐺𝑙𝑢𝑡𝑚 [5]
𝑑𝑡

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𝑑𝐶𝑁𝐻4 +
= 𝑟𝑁𝐻4 + = 𝑞𝑁𝐻4 + · 10−3 · 𝑋 + 𝑘𝑑 · 𝐺𝑙𝑢𝑡𝑚 [6]
𝑑𝑡
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The specific consumption rate for glutamine (qGlutm) and specific production rate for

ammonia (qNH4+) were calculated integrating and rearranging Equations 5 and 6 using
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Laplace transform, obtaining the Equations 7 and 8 respectively.

𝑞𝐺𝑙𝑢𝑡𝑚 · 10−3 · 𝑋0
𝐺𝑙𝑢𝑡𝑚 = 𝐺𝑙𝑢𝑡𝑚0 · 𝑒 −𝑘𝑑 ·𝑡 + · [𝑒 𝜇·𝑡 − 𝑒 −𝑘𝑑 ·𝑡 ] [7]
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𝑘𝑑 + 𝜇

𝑞𝐺𝑙𝑢𝑡𝑚 · 10−3 · 𝑋0 𝑋0
𝑁𝐻4 + = 𝑁𝐻4 + 0 + [ − 𝐺𝑙𝑢𝑡𝑚0 ] · [𝑒 −𝑘𝑑 ·𝑡 − 1] +
𝑘𝑑 + 𝜇 𝜇
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𝑞𝐺𝑙𝑢𝑡𝑚 · 10−3 · 𝑘𝑑
· [𝑞𝑁𝐻4 + · 10−3 + ] · [𝑒 𝜇·𝑡 − 1] [8]
0 𝑘𝑑 + 𝜇
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2.7 Metabolic model adaptation

2.7.1 Genome-scale metabolic model

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The CHO metabolic model used in this study was derived from the reduced model obtained

from a generic Mus musculus genome-scale metabolic model [40]. The reduced model was

first developed by Quek et al. [41] and afterwards used by [24]; and is freely available in

Systems Biology Markup Language (SBML) format in the supplementary files of the second

publication. Martínez et al. [24] performed a Flux Analysis in the two glucose and lactate

10
metabolic phases obtained in pH-controlled cell cultures in bioreactor: Phase 1 (glucose

consumption and lactate generation) and Phase 3 (lactate consumption as a sole substrate);

two models are available, one for each metabolic state.

2.7.2 Biomass equation

Both models available do not contain the biomass equation because the authors maximized

the ATP yield as objective function. Therefore, a biomass equation was developed based on

the literature, as presented in detail in Supplementary Data 2. For this purpose, the

detailed method published by Oliveira et al. [42] was followed. The chemical composition

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of cells in culture was obtained first for the general biomass composition and then for

macromolecular compounds: proteins, DNA, RNA, lipids and carbohydrates [11,43–48]. The

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biomass equation also accounts for the energy requirements (ATP) for polymerization of

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macromolecules (proteins, DNA and RNA), which requires in total 29.18 mmol ATP per

gram of biomass dry weight [46].

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Therefore, a total of 14 reactions were added to the model, including biomass equation and

formation reactions for all metabolites non-included in the base model and required for the
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biomass generation (DNA and RNA macromolecules). A list of all reactions included for the

biomass formation is presented in Table 1. The dry weight for exponentially growing CHO-
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S cells was considered to be 0.36mg·106cells-1·mL-1, being a very similar value to those

previously reported [49].

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2.7.3 Adaptation of the genome-scale metabolic model

For the adapted genome-scale metabolic model used in this study, a combination of the two

CHO available models in Martínez et al. [24] were used. Degradation of methionine, cysteine

and arginine were added to the base model using the pathways included in the generic

11
model published for Mus musculus [40,46]. The biomass formation and GlutaMAX

degradation (GlutaMax -> LAlanine + LGlutamine) were also added, as presented above.

For the phase in which lactate is consumed (Phase 2), two pathways for lactate

metabolization were considered: cytoplasmic and mitochondrial lactate oxidation to

pyruvate. Cytoplasmic lactate transport and metabolization were already included in the

reduced model, but active mitochondrial lactate transport and metabolization had to be

added. While in the generic model from Mus musculus [40,46], as well as, in a recent generic

model derived from Cricetulus griseus (Chinese Hamster) [49] lactate transport through

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mitochondrial membrane was already considered, the mitochondrial lactate

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dehydrogenase was only present in Cricetulus griseus generic model, which derived from

the Chinese Hamster genome.

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The adapted model generated contains 361 internal and 36 external reactions, and it
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includes 395 metabolites. An extensive list of all metabolites and reactions included in the

model are detailed in Supplementary data 3. Reaction fluxes over the metabolic network
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are presented in nmols·mgDW-1·h-1, except for the biomass equation that is represented in

mg·gDW-1·h-1.
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2.8 Flux calculation and model visualization

Parsimonious flux balance analysis approach (p-FBA) was performed using Optflux 3.2.7
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Software [50], a user-friendly computational tool for metabolic engineering applications. p-

FBA is a variant of flux balance analysis that minimize the total material flow to achieve an
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objective, while maintaining the optimal growth [51]. The graphical representation of the

p-FBA was generated using Omix Visualization Software (GmbH&Co.KG), as described in

Droste et al. [52], where only the most significant fluxes for this study were represented.

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As usual in FBA with genomic models, the system was under-determined and there existed

some degrees of freedom. The system had 397 variables (one flux for each reaction) and

395 equations (one for each metabolite), but some equations became linearly dependents,

making the model to have 35 degrees of freedom. Once external measured fluxes were

added as additional constraints, the degrees of freedom of the system were reduced to 10,

therefore the system had a large space of possible solutions in the metabolic network. To

find the optimal state, p-FBA uses the optimization of a certain objective function, in this

case the maximum value of ATP generation. Specific consumption/production rates of

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experimental metabolites measured were added to the model with ±5% admitted deviation.

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3 Results -p
3.1 CHO cells cultures in shake-flasks and bioreactor
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The evolution of macroscopic variables such as viable cell concentration, viability, pH

(bioreactor), glucose and lactate concentration, for the different experiments performed
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with CHO cells are depicted in Figure 1 in the following order: shake-flasks (A), pH-

controlled bioreactor (B), non pH-controlled bioreactor (C) and pH-controlled bioreactor
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to 6.8 with an initial lactate concentration about 15 mM (D). The plots correspond to

replicates #1 for each condition (replicates #2 are shown in Supplementary Data 1).
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Summary compilation of the main cell growth parameters, as well as glucose/lactate

consumption and production specific rates are shown in Table 2. To facilitate the
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comparison of the different experiments, the results are presented together and discussed

by parts going point by point.

As a reference, two different experiments were performed in 250-mL shake-flasks and 2L-

bioreactor with CHO cells. The main difference between both culturing platforms was the

13
presence/absence of pH control; while in shake-flasks was not possible to control the pH,

in the bioreactor the pH was set and kept to 7.2 using an alkali/acid buffer addition.

As observed in the shake-flasks (Figure 1-A), two different glucose-lactate metabolisms can

be clearly distinguished. Glucose and lactate concentration evolution showed an initial

phase of glucose consumption and lactate generation (P1_SF), onwards known as Phase 1,

followed by a metabolic shift to co-consumption of glucose and lactate while remaining in a

growing state (P2_SF), onwards known as Phase 2.

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Nevertheless, cells cultured in a pH-controlled bioreactor (Figure 1-B) did not show

comparable behavior, since the metabolic shift to co-consumption was not observed. Phase

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1 (P1_B1) was also present, where glucose was consumed and large amounts of lactate were

produced; but only when glucose was completely depleted another phase started, onwards
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known as the Phase 3 (also named P3_B1), in which only lactate was consumed with a
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significant growth rate decrease.

The results presented above show that, as a consequence of the environmental conditions
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generated in non pH-controlled (pH free evolution) in shake-flasks, a metabolic behavior in

which glucose and lactate are simultaneously consumed (P2_SF) is possible, while the cells
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remain in a growth state. This metabolic behavior is interesting since higher cell densities

might be reached in culture, and lactate production and accumulation is avoided, a well-
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known cell growth inhibitor.

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In order to confirm the effect of pH on triggering the metabolic shift of CHO cells observed

in shake-flasks, analogous 2-L bioreactor experiments were performed (Figure 1-C),

switching off the pH control, as detailed in Materials and Methods section. The bioreactor’s

CHO cultures are comparable to those carried out in shake-flasks, reproducing both

metabolic phases 1 and 2. The metabolic shift from Phase 1 to Phase 2 coincides with the

14
pH decrease caused by the accumulation of lactic acid in the culture medium. When pH

dropped below 6.8 and lactate accumulation reached a concentration about 12-15 mM the

Phase 2 was triggered. In addition, similar conditions regarding pH and lactate

concentration have been observed in human cells (HEK293) to trigger concomitant glucose

and lactate consumption [31]. In both shake-flasks and bioreactor it exists a short transition

phase between Phase 1 and 2, when the glucose and lactate co-consumption starts. The

amino acids analysis revealed that at the switch point from Phase 1 to Phase 2 all amino

acids were yet present in the culture media (data not shown). For this, the amino acid

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depletion can be discarded as the cause for triggering glucose and lactate co-consumption.

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From the results obtained in controlled and non-controlled pH experiments, it could be

stated that CHO cells are capable to both secrete and metabolize lactate depending on the
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external conditions. Moreover, lactate can be consumed either simultaneously with glucose

or as by itself as the main carbon source depending on the medium’s pH and the external
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lactate concentration. However, only the co-consumption of lactate together with glucose

ensures a significant cell growth since when only lactate is consumed very low growth is
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observed.
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The observed pH changes (dropping and rising) in the experiments performed are due to

the lactate uptake/excretion mechanism via the proton-linked monocarboxylate

transporters (MCTs) [53,54]. These high capacity transporters enable the facilitated
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diffusion of lactate together with a proton. This leads to a proton release when lactate is
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being secreted, and a proton income when lactate is being consumed, causing pH profile

change depending on the metabolic phase.

Using this physiological behavior in CHO cells, a new batch culture strategy has been defined

where glucose and lactate are concomitantly consumed from the beginning of the culture

(Figure 1-D). In this strategy, exponential growth occurs at a specific growth rate similar to

15
those obtained in lactate production phases, a fact never reported in CHO cells. To achieve

this, exogenous sodium lactate was added to the media at a concentration around 15 mM

and the pH was kept below 6.80 by the addition of an acid buffer solution from the culture’s

inoculation onwards (imitating the conditions observed when Phase 2 was triggered in non

pH-controlled experiments). Under these conditions, glucose and lactate were

simultaneous consumed (P2_B3) from the beginning of the culture and along the

exponentially growth phase. It is worth noting that the changing conditions from inoculum

medium to lower pH/high lactate concentration in the bioreactor caused the cells to need

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some adaptation time before starting to divide (around 24 hours). This can be avoided using

the same medium to prepare the inoculum. Prior experiments in shake-flasks were

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performed lowering the initial pH, even below 6.8, but cell growth rate was affected and lag

phase was evident in such cultures (data not shown). -p

Regarding the growth parameters presented in Table 2, a higher XVmax was reached for the
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experiments performed in shake-flasks compared with the bioreactor, even in non-

controlled pH bioreactor cultures. This phenomenon might be a consequence of the shear

lP

stress suffered by the cells cultured in bioreactors, caused by the stirring impellers and the

aeration inflow. In addition, a significant reduction of growth rate was estimated concerning
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the switch of the metabolism from Phase 1 to Phase 2 in shake-flasks and non-pH controlled

bioreactor cultures (around 50% of reduction). However, after observing the data obtained
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for the specific growth rate when Phase 2 was triggered from the beginning of the culture

(P2_B3), the relationship between growth rate decrease and change of metabolism is not
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clear. Values near the maximum specific growth rate were obtained also in Phase 2 (when

it was triggered from the beginning), when glucose and lactate were simultaneously

consumed (considering the Phase 1 as a reference).

16
Thus, the decrease in the growth rate observed in Phase 2 in non-controlled pH experiments,

both in shake-flasks and bioreactor (P2_SF/P2_B2), may not be due to the metabolic switch.

The growth rate reduction could be related to the fact that Phase 2 was situated in a second

part of the culture, after the turning point of the growth curve, when the cell growth was

slowed down probably to some other nutrient depletion in the media. Very low growth rate

was obtained in Phase 3, only observed in pH-controlled bioreactor when glucose was

depleted.

It is important to mention the higher glucose consumption and lactate production rates

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estimated along Phase 1 in pH-controlled bioreactor cultures in comparison with the same

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phase in non-controlled pH conditions, where glucose uptake and lactate generation were

reduced about 29% and 31% respectively. In addition, a reduction up to 81% of glucose
-p
consumption was obtained in Phase 2 compared to Phase 1.
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From the results presented, it can be pointed out that pH control affects glucose

consumption and lactate generation in Phase 1, and that both parameters are tightly related
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(as the more glucose is consumed, the more lactate is produced). This demonstrates that pH

plays an important role in the metabolism of glucose and lactate. When lactate is consumed
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together with glucose in Phase 2, a high reduction of glucose consumption is obtained, no

matter if pH is controlled or not. This reduction in glucose uptake, as well as the fact that

lactate is being consumed instead of being produced, indicates that a redistribution of

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metabolic fluxes is occurring in the Phase 2, and that becomes very interesting from the
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production point of view, because cells are fully active.

At this point, we believed that the metabolic versatility presented should be approached

from the metabolic flux analysis point of view, using constrained-based metabolic models

in order to better study the intracellular fluxes redistribution in the different phases; as

presented in the next section.

17
3.2 Metabolic flux balance analysis in the different glucose/lactate metabolism in CHO cells

Once the different behaviors regarding the glucose and lactate metabolism have been

presented, the idea of this section is to study, by means of a Flux Balance Analysis (FBA), the

metabolic behavior obtained when concomitant consumption of glucose and lactate was

obtained from the onset of the culture (Phase 2), and compare it to the obtained in Phase 1

of lactate generation (non-controlled pH experiment).

The consumption of lactate by mammalian cells leads to the question of how lactate reaches

the mitochondrial matrix. The conventional accepted metabolism is based on lactate

of
conversion to pyruvate into the cytosol and thereafter, pyruvate is transported inside the

ro
mitochondria [23,24]. An alternative explanation could be the direct entrance of lactate

through the mitochondrial transporters to be afterwards converted to pyruvate [30,55]. As

-p
previously seen in HEK293 cells [32], both possibilities seem to be feasible when lactate is

consumed by the cells. Therefore, in glucose and lactate co-consumption phase two
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different modelizations were performed and discussed, considering either cytoplasmic (c-

LDH) or mitochondrial (m-LDH) lactate dehydrogenase, as discussed later.

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Three p-FBA for the different glucose/lactate metabolisms are depicted in Figures 2 to 4:
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1. FBA1_CHO: Phase 1, non-controlled pH (P1_B2): glucose consumption, lactate

production (Figure 2 for c-LDH).

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2. FBA2_CHO: Phase 2, pH controlled to 6.80 and with the addition of 15 mM of

sodium lactate (P2_B3): glucose and lactate concomitant consumption from the
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beginning of the culture (Figure 3 for c-LDH and Figure 4 for m-LDH).

Experimental consumption/production rates for the different metabolic phases modeled

for CHO cells are summarized in Table 3.

18
3.2.1 Metabolic flux distribution in overflow metabolism of Phase 1 of non-controlled pH

conditions and in Phase 2 obtained from the beginning of the culture in CHO cells

From the intracellular fluxes obtained by means of FBA, it can be observed that in Phase 1

a deregulated metabolism characterized by high glucose consumption and the production

of high amounts of lactate was displayed (Figure 2). On this basis, pyruvate obtained from

glucose was primarily converted into lactate, instead of acetyl-CoA to be further oxidized in

the TCA cycle, obtaining hence the maximum possible amount of energy (only 31% of total

carbon income from glucose was entered into the TCA). This can be understood as an

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“uncoupled” metabolism between glycolysis and TCA, obtaining an important inefficient

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substrate consumption regarding the carbon source.

-p
The high glycolysis flux obtained in Phase 1 (695 nmol·mgDW-1·h-1 from G3P to PEP) leads to

the generation of large amounts of NADH in the cytoplasm that must be regenerated. The
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main mechanism of cells to regenerate NADH is the Malate-Aspartate Shuttle, but indirect

NADH transport into the mitochondria is needed for its regeneration [56]. Therefore, the
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lactate generation in culture indicates somehow a limitation in the NADH transport into the

mitochondria through the Malate-Aspartate Shuttle, and the remaining NADH is then
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regenerated producing lactate via lactate dehydrogenase in the cytosol (c-LDH). Although

it is difficult to determine from the results of the FBA, there could be a bottleneck either on
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some of the reactions of the Malate-Aspartate Shuttle, or in mitochondrial malate

dehydrogenase or in the phosphorylation oxidation pathway. The difference in

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regenerating NADH via Malate-Aspartate Shuttle or via c-LDH relays on the basis that if

NADH is regenerated via the Malate-Aspartate Shuttle, the carbon source is not lost as it is

transported to the TCA via malate, allowing to generate more NADH (malate

dehydrogenase) and, as a consequence, more ATP. On the contrary, if c-LDH is used, both

carbon source and energy are lost, leading to a very wasteful metabolism. In that particular

19
case, a flux of 19 nmol·mgDW-1·h-1 was obtained in the Malate-Aspartate Shuttle (taking the

Glutamate-Aspartate transporter as a reference). However, a flux of 101 nmol·mgDW-1·h-1 in

malate dehydrogenase (reaction that regenerates NADH in the cytoplasm) was obtained

from the mitochondrial citrate export (pathway required to generate lipids for the biomass).

A completely different behavior was obtained in Phase 2 (Figure 3 for c-LDH and Figure 4

for m-LDH). Glycolytic fluxes were more than 3-fold reduced (86 nmol·mgDW-1·h-1 compared

to 383 nmol·mgDW-1·h-1 for the glucose uptake rate) and the carbon income came from both

glucose and lactate. The interesting fact, when comparing both models, is that the TCA fluxes

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were very similar (around 200 nmol·mgDW-1·h-1 on the lower part), and a priori similar

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amounts of energy and thus, similar cell growth rate was obtained. From the total carbon

income (glucose+lactate) in Phase 2 consumed by the cells, the 69% had its origin in the
-p
glucose uptake and the rest 31% in the lactate consumed.
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3.2.2 Alternative LDH pathway for lactate consumption in CHO cells

In order to enrich the discussion of the two hypotheses arisen for lactate consumption
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presented above (c-LDH and m-LDH), and with the results obtained in simultaneous glucose

and lactate consumption from the beginning of the culture, two different models were
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assessed for Phase 2 constraining either cytoplasmic or mitochondrial lactate

dehydrogenase.
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In Phase 1, where high glucose uptake is observed, the results presented evidence the need
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of pyruvate conversion to lactate by the cytoplasmic lactate dehydrogenase (c-LDH), to fulfil

the oxidation of the large amounts of NADH generated by the high glycolytic fluxes. A

completely different situation is obtained in Phase 2, because lactate is consumed instead

of being produced. In this case, the conversion of lactate to pyruvate leads to a NADH

formation, and again some differences were observed depending on whether the reaction

20
is considered to take place in the cytoplasm or into the mitochondria. Comparing both

scenarios (Figure 3 for c-LDH and Figure 4 for m-LDH), the release of NADH in the

cytoplasm leads to a significant activation of the Malate-Aspartate shuttle to regenerate the

NADH (formed both in glycolysis and in the conversion from lactate to pyruvate in the

cytoplasm, 175 nmol·mgDW-1·h-1 for malate dehydrogenase) when c-LDH is considered. In

the second approach, the regeneration of NADH produced through the conversion of lactate

to pyruvate takes place into the mitochondrial matrix in the oxidative phosphorylation

pathway when m-LDH was considered instead. Therefore, the flux of the Malate-Aspartate

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shuttle is considerably reduced, obtaining similar values as in the Phase 1 (99 nmol·mgDW-

1·h-1).

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The results obtained could indicate that the consideration of m-LDH for the lactate
-p
metabolization is, in some way, beneficial for the cell culture development, although the

increase of the Malate-Aspartate Shuttle is completely possible from a biological point of

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view. Despite this, c-LDH for lactate consumption cannot be discarded only with this data,

and more advanced analysis should be performed in order to finally determine if LDH is
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active in the cytoplasm or into the mitochondria in the phase in which lactate is consumed,

as detailed in the Conclusion section.

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ur

4 Conclusions

Three different glucose and lactate metabolism behaviors have been observed and studied
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in CHO cells cultures, captured in the three phases already mentioned: a) Phase 1: glucose

consumption and lactate production (cell growth observed), b) Phase 2: glucose and lactate

simultaneous consumption (cell growth observed), and c) Phase 3: lactate consumption as

a sole carbon source (no cell growth observed).

21
The different metabolic phases observed were mainly related to two cell culture

parameters: the pH and the lactate concentration in the culture broth. When pH was

controlled in bioreactor cultures, Phase 1 appeared at the beginning of the culture, and once

when glucose was completely depleted Phase 3 was observed instead (characterized by

lactate consumption as the sole carbon and energy source). In contrast, when pH was

uncontrolled in bioreactors, Phase 1 was again obtained but when pH dropped below 6.80,

due to lactic acid secretion and accumulation, the Phase 2 was triggered, while the cells were

still growing in both situations.

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The most important point of this article is the fact that Phase 2 can be triggered at will by

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adding lactate to the initial media and setting the pH below 6.80. As far as we know, glucose

and lactate simultaneous consumption obtained from the beginning of the culture in
-p
bioreactor has not been reported yet in the literature in CHO cells, and it is proposed as a

new alternative for CHO cells batch cultures.

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From the different experiments performed with CHO cells, it can be concluded that the
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lactate transport system is highly influenced by both the extracellular lactate concentration

and pH, as lactate transport is performed via proton symporter. The authors believe that it
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would be of great interest to further explore the relationship between pH and lactate

concentration (both intracellular and extracellular) that switch from lactate generation to

consumption. We propose to develop a thermodynamic model of the lactate transport to

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define the dependency effects of both lactate concentration and pH to trigger co-
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consumption of lactate and glucose.

The fact that lactate is a widely known growth inhibitor [7], together with the point that

lactate generation is a wasteful metabolism since the majority of carbon source (glucose) is

spent to generate a non-desired by-product, motivate to further study this metabolic

behavior. This will lead to better understand the cell metabolic redistribution due to the

22
environmental conditions, to confirm the different hypothesis performed as a previous step

to be applied in bioprocess optimization.

The uncoupled metabolism of glycolysis and TCA in Phase 1 was evidenced by the much

higher fluxes of glycolysis compared to the TCA fluxes. This leads to the generation of higher

amounts of pyruvate that cannot be transported into the mitochondria, resulting in the

generation of lactate. In contrast, in Phase 2 both glycolysis and TCA were somehow coupled,

as all the pyruvate generated both from glycolysis and lactate was directly transported into

the mitochondria to generate ATP. A limitation in the capacity of the Malate-Aspartate

of
Shuttle to regenerate NADH could be the reason of lactate formation in Phase 1 to complete

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the regeneration all the NADH produced through glycolysis.

The possibility of lactate entrance directly to the mitochondria to be converted into

-p
pyruvate (m-LDH) versus the conventional accepted cytosolic lactate dehydrogenase (c-
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LDH) has been shown to be feasible through the intracellular fluxes obtained. The

consequence of releasing the NADH directly into the mitochondria through m-LDH is the
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decrease of the flux of the Malate-Aspartate Shuttle, which could be beneficial for the cells.

Anyway, none of both possibilities can be discarded and future work should be performed
na

in order to clear it up. The use of a GPF-tag in LDH in order to localize this enzyme either in

cytoplasm or in mitochondria, and the application of more advanced flux techniques (13C

substrate labelling) to determine the real fluxes in the Malate-Aspartate Shuttle could help
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to enlighten the exact metabolic pathway.

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The present work explores an easy and promising alternative to reduce the lactate

generation in culture by means of modifying the conventional culture strategies and

conditions. The glucose and lactate co-metabolization resulted in a better-balanced cell

metabolism, as can be seen from the metabolic fluxes calculated. Moreover, the generation

and secretion of lactate is totally reversed, so the main drawback of processes based on

23
mammalian cell cultures was also overcome, opening the door to use this strategy in batch

and also fed-batch or perfusion strategies in CHO bioprocesses.

Acknowledgement

The present work has been supported by the grant FI-DGR (Generalitat de Catalunya,

Catalonia, Spain).

The authors would like to thank The Novo Nordisk Foundation and the two NNF Grant

numbers: NNF10CC1016517 and NNF14OC0009473.

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Conflict of interest

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The authors declare no financial or commercial conflict of interest.

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lP
na
ur
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24
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Table 1 List of reactions added to the CHO base model that account for the biomass

formation.

Reaction ID Function Stoichiometry

BIOM_AA Protein 0.0909 LAlanine + 0.0505 LArginine +0.0505 LAspartate + 0.0404
formation LAsparagine + 0.0202 LCysteine + 0.0505 LGlutamine + 0.0606
LGlutamate + 0.0808 Glycine + 0.0202 LHistidine + 0.0505
LIsoleucine + 0.0808 LLeucine + 0.0808 LLysine + 0.0202
LMethionine + 0.0303 LPhenylalanine + 0.0505 LProline + 0.0606
LSerine + 0.0606 LThreonine + 0.0101 LTryptophan + 0.0303
LTyrosine + 0.0606 LValine + 4.306 ATP + 3.306 H2O -> PROT +
4.306 ADP + 4.306 Orthophosphate
BIOM_dAMP DNA (dAMP) dATP + 2 H2O -> dAMP + 2 Orthophosphate
formation
BIOM_dCMP DNA (dCMP) dCTP + 2 H2O -> dCMP + 2 Orthophosphate
formation
BIOM_dGMP DNA (dGMP) dGTP + 2 H2O -> dGMP + 2 Orthophosphate

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formation
BIOM_dTMP DNA (dTMP) dTTP + 2 H2O -> dTMP + 2 Orthophosphate
formation
BIOM_AMP RNA (AMP) ATP + 2 H2O -> AMP + 2 Orthophosphate

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formation
BIOM_CMP RNA (CMP) CTP+ 2 H2O -> CMP + 2 Orthophosphate
formation
BIOM_GMP RNA (GMP) GTP +2 H2O -> GMP + 2 Orthophosphate

BIOM_UMP

BIOM_DNA
formation
RNA (TMP)
formation
DNA formation
-p
UTP + 2 H2O -> UMP + 2 Orthophosphate

0.3 dAMP + 0.2 dCMP + 0.2 dGMP + 0.3 dTMP + 1.372 ATP + 1.372
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H2O -> DNA + 1.372 ADP + 1.372 Orthophosphate
BIOM_RNA RNA formation 0.18 AMP + 0.30 CMP + 0.34 GMP + 0.18 UMP + 0.4 ATP + 0.4 H2O -
> RNA + 0.4 ADP + 0.4 Orthophosphate
BIOM_LIP Lipids formation 0.1315 Cholesterol + 0.5006 Phosphatidylcholine + 0.1898
lP

Phosphatidylethanolamine + 0.0688 1PhosphatidylDmyoinositol +

0.0189 Phosphatidylserine + 0.0096 Phosphatidylglycerol + 0.0204
Cardiolipin + 0.0605 Sphingomyelin -> LIP
BIOM_CARBO Carbohydrates Amylose -> CAR
formation
BIOM_T Biomass 6.990 PROT + 0.050 DNA + 0.1910 RNA + 0.144 LIP + 0.280 CAR
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formation -> BIOMASS

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Table 2 Summary of culture parameters and glucose/lactate consumption/production

rates related to cell physiology calculated from the experiments performed with CHO cells.

Bioreactor
Bioreactor
Bioreactor pH-controlled to
Shake-Flasks Non pH-
pH-controlled 6.8 + 15mM
controlled
NaC3H5O3

P1_SF P2_SF P1_B1 P2_B1 P1_B2 P3_B2 P2_B3

Phase
Phase 1 Phase 2 Phase 1 Phase 2 Phase 1 Phase 3 Phase 2
Xvmax
6
11.8 8.5 8.9 9.8
(10 cells/mL)

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µmax (h-1) 0.049 0.026 0.039 0.020 0.044 0.013 0.036
Dt (h) 14 27 18 35 16 53 19

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qglucose
-323.3 -46.3 -394.5 -71.8 -562.3 0.0 -82.0
(nmols/mgDW·h)
qlactate
(nmols/mgDW·h)
602.6 -71.4 555.3 -p
-80.4 807.3 -52.6 -79.8
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Table 3: Summary of input/output experimental fluxes of the different metabolic phases

modelled for CHO cells. All data are expressed in nmol·mgDW-1·h-1 except biomass mg·gDW-

1·h-1. Real glutamine consumption: GlutaMax + Glutamine. Real alanine generation:

GlutaMax + Alanine.

Phase 1 Phase 2
P1_B2 P2_B3
Alanine 105.960 ± 5.298 58.860 ± 2.943
Arginine -25.890 ± 1.295 -15.926 ± 0.796
Asparagine -10.686 ± 0.534 -9.737 ± 0.487
Aspartate -24.980 ± 1.249 -4.810 ± 0.240

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Biomass 39.300 ± 1.965 35.800 ± 1.790
Cysteine -8.823 ± 0.441 -5.101 ± 0.255
Glucose -394.530 ± 19.727 -82.050 ± 4.103

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Glutamate -12.460 ± 0.623 -15.450 ± 0.772
GlutaMax -93.295 ± 4.665 -47.890 ± 2.394
Glutamine 47.401 ± 2.37 7.170 ± 0.359
Glycine
Histidine
7.343 ± 0.367
-6.022 ± 0.301
-p3.778 ± 0.189
-5.487 ± 0.274
Isoleucine -24.040 ± 1.202 -14.120 ± 0.706
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Lactate 555.287 ± 27.764 -79.770 ± 3.989
Leucine -33.613 ± 1.681 -22.094 ± 1.105
Lysine -27.665 ± 1.383 -20.233 ± 1.012
lP

NH4+ 51.147 ± 2.557 30.694 ± 1.535

Oxygen -583.083 ± 29.154 -554.440 ± 27.722
Phenylalanine -12.840 ± 0.642 -5.926 ± 0.296
Proline -25.797 ± 1.290 -10.487 ± 0.524
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Serine -55.000 ± 2.750 -35.000 ± 1.750

Threonine -24.395 ± 1.220 -20.516 ± 1.026
Tryptophan -7.484 ± 0.374 -3.446 ± 0.172
Tyrosine -8.140 ± 0.407 -4.946 ± 0.247
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Valine -25.733 ± 1.287 -16.120 ± 0.806

Real glutamine
-45.894 ± 5.233 -40.720 ± 2.421
consumption
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Real alanine
12.665 ± 7.059 10.970 ± 3.794
generation

35
Figure 1: Batch cultures of CHO cells in (A) a 250-mL shake-flasks without pH control; (B)

2-L bioreactor under pH-controlled conditions at pH 7.2 by means of 5% CO2 sparging, alkali

or acid buffer addition on demand; (C) 2-L bioreactor with free pH evolution (a constant

concentration of 5% CO2 was used in the gas phase); (D) 2-L bioreactor in pH-controlled at

6.8 by means of 5% CO2 sparging and acid addition, adding 15 mM of sodium lactate to the

initial media. Evolution profiles for cell density ( ), viability ( ), glucose concentration

( ), lactate concentration ( ) and pH ( ). The plots correspond to replicates #1 of

duplicates performed for each condition (replicates #2 are shown in Supplementary Data

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1).

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Figure 2: Scheme of the main metabolic fluxes calculated for CHO cells in Phase 1 in non-
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controlled pH culture (glucose consumption and lactate production phase; phase P1_B2).

Arrows indicate the direction of the flux and their width the magnitude of fluxes (the exact

value is detailed close to the arrows). The box represents the mitochondrion. All the fluxes

are expressed in nmol·mgDW-1·h-1.

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Figure 3: Scheme of the main metabolic fluxes calculated for CHO cells in Phase 2 when pH

was controlled to 6.80 and 15 mM of sodium lactate was added. Cytoplasmic Lactate
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dehydrogenase (c-LDH) was considered (glucose and lactate concomitant consumption

phase; phase P2_B3 c-LDH). Arrows indicate the direction of the flux and their width the
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magnitude of fluxes (the exact value is detailed close to the arrows). The box represents the

mitochondrion. All the fluxes are expressed in nmol·mgDW-1·h-1.

37
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Figure 4: Scheme of the main metabolic fluxes calculated for CHO cells in Phase 2 when pH

was controlled to 6.80 and 15 mM of sodium lactate was added. Mitochondrial lactate
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dehydrogenase (m-LDH) was considered (glucose and lactate concomitant consumption

phase; phase P2_B3 m-LDH). Arrows indicate the direction of the flux and their width the
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magnitude of fluxes (the exact value is detailed close to the arrows). The box represents the

mitochondrion. All the fluxes are expressed in nmol·mgDW-1·h-1.

38
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