Biochemical Engineering Journal 114 (2016) 50-61A3

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

A methodology for building a macroscopic FBA-based dynamical

simulator of cell cultures through flux variability analysis
Anne Richelle ∗ , Khadija Mhallem Gziri, Philippe Bogaerts
3BIO-BioControl, Université Libre de Bruxelles, Avenue F. Roosevelt, 50, CP 165/61, 1050, Brussels, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: A dynamical FBA-based simulator of hybridoma cell fed-batch cultures predicting the dynamics of
Received 8 March 2016 biomass growth, substrate consumption (glucose and glutamine), metabolites production (lactate,
Received in revised form 9 May 2016 ammonium and alanine) and associate intracellular metabolism based on a simplified metabolic net-
Accepted 13 June 2016
work is proposed. A preliminary comparison between the range of admissible flux distribution obtained
Available online 15 June 2016
based on all available measurements (the two substrates uptake rates and the three metabolite produc-
tion rates) and based on only part of them (only the two substrates uptake rates) is performed to deal
Keywords:
with the usual problem of system underdetermination in constraint-based modeling context. This com-
Metabolic flux analysis
Flux balance analysis
parative flux variability analysis allows the objective identification of some additional constraints (to
Mathematical modeling be used in the final FBA-based simulator) so as to obtain similar admissible flux intervals in both cases.
Flux variability analysis Moreover, the proposed approach legitimates the cost criterion used for the linear optimization, i.e. cell
Mammalian cells growth maximization. This methodology is validated on experimental data of two fed-batch cultures and
Constraint-based modeling cross validated on a batch culture of hybridoma cells HB-58. The flux distribution results are in agreement
with overflow metabolism description available in literature.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction the mass balance of each intracellular metabolite included in the

Systems biology is a very hard modeling issue due to the inher-
ent complexity of cell’s entities which form the biological systems
studied under this scope. Actually, each cell can be viewed as a non-
linear dynamical system involving many hundreds of reactions that
take place between multitudes of components and interact through
intern regulation mechanisms.
Over the past decades, the increase of available quantitative
data from -omics technologies and related qualitative knowledge
gained at the intracellular level have led to the possibility of
describing and quantifying all these reactions in a unique cell of
a defined line through diverse analysis methods [1]. One of them is
the constraint-based analysis of metabolic network models repre-
senting mathematically all relevant information (e.g. metabolites,
reactions, enzyme capacity, gene interaction, etc.) available about a
specific cell type in structured knowledge bases (i.e. reconstructed
genome-scale metabolic network) [1–3].
The constraint-based modeling approach allows determining
the general characteristics of metabolism dynamics by expressing
∗ Corresponding author.
E-mail addresses: arichell@ulb.ac.be (A. Richelle), philippe.bogaerts@ulb.ac.be
(P. Bogaerts).
metabolic network model which are assumed to be in quasi steady-
state. In doing so, the intracellular metabolism can be described by
a system of linear algebraic equations. Each solution of this system
is associated with a flux distribution representing the stoichio-
metrically feasible phenotypic states of the cell given the network
topology [1].
Nevertheless, metabolic networks involving, most of time, more
reactions than metabolites, the associated system presents, gen-
erally, a multitude of solutions. Different approaches have been
proposed to offset this underdetermination such as the introduc-
tion of additional metabolic constraints representing specific in
and out cell conditions and allowing a more biologically relevant
description of feasible phenotypic states of a cell under given con-
ditions and/or the use of linear optimization techniques (i.e. Flux
Balance Analysis) promoting specific flux with respect to specific
cellular objectives. These approaches require qualitative informa-
tion about the cell under the specified conditions which could be
not reliable enough and/or a priori assumptions that are generally
difficult to legitimate [1,4,5].
In general, some experimental measurements are available,
which are typically the fluxes that connect the intracellular
metabolism and extracellular environment, and they could be used
to solve the metabolic network model (i.e. Metabolic Flux Analysis).

http://dx.doi.org/10.1016/j.bej.2016.06.017
1369-703X/© 2016 Elsevier B.V. All rights reserved.
 A. Richelle et al. / Biochemical Engineering Journal 114 (2016) 50-61A3
Nomenclature
X Biomass concentration (109 cells/L)
G Glucose concentration in bioreactor (mmol/L)
Gin Glucose concentration in feeding medium (mmol/L)
Q Glutamine concentration in bioreactor (mmol/L)
Qin Glutamine concentration in feeding medium
(mmol/L)
N Ammonium concentration in bioreactor (mmol/L)
L Lactate concentration in bioreactor (mmol/L)
A Alanine concentration in bioreactor (mmol/L)
V Culture volume (L)
F Feeding rate (L/h)
KG Monod constant for glucose uptake (mmol/L)
KQ Monod constant for glutamine uptake (mmol/L)
KIX G Biomass inhibition constant for glucose consump-
tion (109 cells/L)
KIX Q Biomass inhibition constant for glutamine con-
sumption (109 cells/L)
␣ Yield coefficient for biomass growth
mX Specific death rate (1/ h)
␮Gmax Maximum specific uptake rate of glucose
(mmol/109 cells/h)
␮Qmax Maximum specific uptake rate of glutamine
(mmol/109 cells/h)
However, these data are, generally, not in sufficient number and
uncertain to determine a unique flux distribution under defined
extracellular conditions. Additional relevant experimental infor-
mation can also be provided by using 13 C tracers leading to the
13 C MFA technology [6].
Independently of the underdetermination problem, the
metabolic network models, while very useful for explanatory and
descriptive purposes of particular cell’s behavior aspects, are also
too complex to be handled for the development of utilitarian tools
at the process level (e.g. optimization and control of a process plant
exploiting cell behavior) as they have a huge structural complexity.
In this context, the use of a macroscopic modeling approach
allows overcoming these problems while generally providing a
good description of the biological system in a large range of
experimental conditions. These kinds of models have considerably
simpler structural complexity as they directly connect extracellular
substrates and products through the definition of a set of chemi-
cal “macro-reactions” representing the cell metabolism as a whole
but they do not pay much attention to the intracellular metabolism
[7–9].
Some bottom-up approaches have been developed for reduc-
ing metabolic models at the macroscopic scale [10–12]. However,
until now, there are still open problems in the way to handle
the reduction procedure (e.g. the definition of sub-systems rep-
resenting the metabolic pathways which are activated during the
different phases of a culture process and the switching from one
metabolic phase to the other).
In this work, we address the issue of using a top-down approach
to throw a bridge between the macroscopic dynamical modeling
approach and the modeling of intracellular metabolism by using
constraint-based modeling (CBM) theories, and more specifically
the Flux Balance Analysis (FBA). To this end, we present a proce-
dure for the development of a dynamical simulator of hybridoma
HB-58 cells metabolism in fed-batch cultures based on a compar-
ative flux variability analysis (FVA) with respect to the number of
measurements used to solve the system of equations representing
the metabolic network. Moreover, the proposed approach allows
guiding search for additional constraints and legitimating the use
51
of linear optimization so as to overcome the problem related to
system underdetermination in constraint-based modeling context.
In summary, the main objectives and original points of this con-
tribution are
– obtaining a dynamical model for simulating fed-batch cultures
at the macroscopic level
 which does not require the choice and identification of kinetic
model structures, except for the specific uptake rates of the
substrates (glucose and glutamine in our case study);
– which is based on a very simple reaction network whose fluxes
are determined by solving a FBA problem at each time instant;
– which benefits from a very low complexity (only 10 parameters
in our case study);
• legitimating both the objective function and the linear constraints
of the FBA problem thanks to a flux variability analysis comparing
MFA and FBA results.
The first part of this study presents the necessary theoretical
concepts and definitions related to CBM (Section 2) and highlights
the problem of underdetermined systems by introducing the flux
distribution variability in constraint-based modeling context (Sec-
tion 3). The second part of this study is related to the development of
the simulator (Section 4). The experimental field and the metabolic
network used are, respectively, described in Sections 4.1 and 4.2.
Section 4.3 presents the macroscopic approach used to model the
two substrate consumption rates and Section 4.4 defines the FBA
linear programming problem used within the simulator. The FVA
comparative approach used to determine the additional constraints
is presented in Section 4.5 and the validation of the dynamical simu-
lator is performed in Section 4.6. The final part of this paper presents
the conclusions of this study (Section 5).
2. Constraint-based modeling concepts and definitions
Constraint-based modeling has emerged in the mid 1980’s.
This approach is based on the concept that biological systems are
constrained by physico-chemicals laws and by their genetics and
environment. These kinds of models allow describing cell capa-
bilities (e.g. feasible phenotypic states) given different constraints
acting simultaneously on and in the cell under defined conditions
[1,13,14].
The cell metabolism can be represented by a metabolic network
model containing all the relevant biochemical transformations that
take place within the cell. The network model takes the form of a
directed hyper-graph linking metabolites (nodes of the network)
through their transformation rates, called metabolic fluxes (edges
of the network). It can be mathematically converted into a matrix,
called the stoichiometric matrix, whose size depends on the num-
ber of metabolites (matrix rows) and reactions (matrix columns)
included in the metabolic network [14].
Based on the definition of this network, it is possible to
determine the general characteristics of metabolism dynamics
by expressing the mass balance of each intracellular metabolite
included in the network:
dc
= N v − c (1)
dt
where c is the vector of intracellular metabolite concentrations,
v the vector of specific reaction rates (metabolic fluxes), N the
stoichiometric matrix and c is the dilution term accounting for
biomass growth ().
One of the fundamental assumptions related to constraint-
based modeling is the so-called balanced growth condition. It states
that, as the dynamics associated to intracellular metabolites is
52 A. Richelle et al. / Biochemical Engineering Journal 114 (2016) 50-61A3
Fig. 1. Metabolic network for the growth of CHO-320 cells proposed in Provost and Bastin [34] and transposed to hybridoma HB-58 in the context of this work.
(Figure from [34])
much faster than the extracellular ones (e.g. substrate uptakes),
intracellular metabolites are assumed to be in quasi steady-state
(i.e. be consumed and produced in a mass-balanced manner). More-
over, the dilution term c is generally neglected because it does not
significantly affect the metabolite concentration evolution com-
pared to the metabolic fluxes (i.e. term N v in equation (1)). Under
these assumptions, the differential equation (1) can be rewritten as
a system of linear algebraic equations:
Nv = 0
Each solution of this linear system (2) is associated with a
flux distribution (v) representing the stoichiometrically feasible
steady states of the system. It is important to note that gener-
ally there exist a multitude of solutions to this system. Actually,
metabolic networks involving, most of time, more reactions than
metabolites, the associated system of linear equations is generally
underdetermined. Hence, there is a whole area of feasible solu-
tions (space of admissible flux distribution) describing the overall
cell capabilities given the constraints related to the network topol-
ogy and the steady state assumption. The constraint-based analysis
methodology associated to this kind of system definition is called
“Network-based Pathways Analysis” (NPA), such as “Elementary
Fluxes” and “Extreme Pathways” which are used to identify sys-
temic properties of a network. They are based on the convex
analysis theory [15] but are out of scope of this study. For a review
and a comparison of the NPA methods, we refer the reader to Papin
et al. [16].
The solution space can be reduced by introducing additional
constraints (e.g. upper and lower bounds, equality and/or inequal-
ity constraints, Boolean rules) representing specific in and out cell
conditions (e.g. maximum capacity of a reaction flux, nutrients
availabilities, reaction thermodynamics, regulatory mechanisms).
It allows a more biologically relevant description of feasible phe-
notypic states of a cell under given conditions. In this way, most
of metabolic network models take, at least, into account the reac-
tion thermodynamics (by considering that some reactions operate
predominantly in one direction) and the existence of a maximum
capacity for some reaction fluxes. By assuming that all fluxes con-
sidered in a network are positive and present a maximum allowable
(2) operating value, the constraints to be added to Eq. (2) can be written
as a system of linear inequalities:
0 ≤ v ≤ vmax (3)
where vmax is the vector of maximum capacities associated to each
flux included in vector v defined by Eq. (2). In case of reversible
reactions for which no predominant direction is assumed, both the
direct and reverse fluxes should be taken into account still respect-
ing constraints (3).
Another analysis methodology is to solve the metabolic network
model described by (2) and (3) based on a set of experimental
measurements, which are typically the fluxes that connect the
intracellular metabolism and extracellular environment (i.e. sub-
strate uptake rates and extracellular metabolite production rates).
This constraint-based analysis methodology is called in literature
“Metabolic Flux Analysis” (MFA) [3,14,17]. Assuming the availabil-
ity of some measured fluxes (vmes ), the metabolic network model
defined in (2) becomes:
  
N 0 v
=0 (4)
Ne −vmes 1
 A. Richelle et al. / Biochemical Engineering Journal 114 (2016) 50-61A3 53

Fig. 2. Comparison between model prediction (in blue) and the experimental measurements (in red) of glucose and glutamine concentration over time for the two experiments
(direct validation). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Range of admissible flux values satisfying FBA problem (23) and computed by using FVA (24) – Experiment 1.
54 A. Richelle et al. / Biochemical Engineering Journal 114 (2016) 50-61A3

Fig. 4. Range of admissible flux values satisfying FBA problem (23) and computed by using FVA (24) – Experiment 2.

Fig. 5. Range of admissible flux values for FBA problem (Eq. (24) – in red) and for reference solution (Eq. (25) −in blue) – Experiment 1. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
 A. Richelle et al. / Biochemical Engineering Journal 114 (2016) 50-61A3 55

Fig. 6. Range of admissible flux values for FBA problem (Eq. (24) – in red) and for reference solution (Eq. (25) – in blue) – Experiment 2. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7. Range of admissible flux values corresponding to FBA problem with the two additional constraints (in red) and for reference solution (in blue) – Experiment 1. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
56 A. Richelle et al. / Biochemical Engineering Journal 114 (2016) 50-61A3
Fig. 8. Range of admissible flux values corresponding to FBA problem with the two additional constraints (in red) and for reference solution (in blue) – Experiment 2. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
where Ne is the stoichiometric matrix associated with the mea-
sured metabolites concentrations (cmes ), which are supposed under
unbalanced conditions contrarily to intracellular ones (c). However,
the available number of experimental measurements is generally
insufficient to offset the underdeterminacy of flux distribution. One
of the most used methodologies to overcome this problem is the
Flux Balance Analysis (FBA), which uses linear optimization of a
cost criterion to promote specific flux vectors within the metabolic
network [18–21]. FBA is based on the hypothesis that cells have
evolved to be optimal with respect to specific cellular objectives
(e.g. biomass growth, ATP yield or productivity). Hence, the flux
distribution (v) corresponding to the stated objective functionZ (i.e.
optimal cellular behavior expressed, most of time, as a linear com-
bination of some fluxes ofv) is obtained by solving the following
linear programming (LP) problem:
N 0 v
max Z s.t. =0
Ne −vmes 1
It is important to underline that if the system (4) is underdeter-
mined, while there is a unique solution of this linear optimization
problem (maximum value of the cost function Z), it generally exists
different equivalent flux distributions (v) leading to this optimum
(alternate optimal solutions). The issue of flux distribution variabil-
ity due to underdetermined system in both MFA and FBA context
will be developed in Section 3.
Unlike MFA, the first goal of FBA is not to determine the flux
distribution associated with a metabolic state observed exper-
imentally (i.e. capacity of a model to reproduce the measured
data). Actually, FBA is mostly used for its capacity to predict
cell phenotypes under hypothetical experimental conditions. In
doing so, this technique was widely used to investigate the
metabolic capacity (e.g. substrate preference, optimal growth and
shift between metabolic states) of a cell according to a defined
objective [14,22,23]. FBA is one of the most promising tools in
cell engineering and biomedical applications as it allows to study
the influence of gene deletion/addition, e.g. prediction knock-out
mutants phenotypes [24], and to identify potential drug target by
evaluating the effect of potential disease treatments [25].
The major drawback of this method is related to the dependence
between the model predictions (i.e. optimal flux distribution) and
the choice of the objective function. Although maximizing biomass
is the most widely used cost criterion, Schuetz et al. [26] clearly
highlight the importance of identifying relevant cellular objectives
with respect to environmental conditions and cell lines considered.
Recently, Schuetz et al. [27] supported this fact by demonstrating
the multidimensionality of optimal cell behavior in microorgan-
isms.
While we will only use MFA and FBA related methodologies in
the context of this work, it must be noted that there exists a mul-
(5) titude of other constraint-based methodologies which have been
widely deployed during these last decades, see Lewis et al. [13] for
an extensive description of all these approaches.
3. Analysis of flux distribution variability in a
constraint-based modeling context
The underdetermination of metabolic network model is a
central problem in constraint-based modeling. Actually, the experi-
mental data, used to solve the system of linear equations associated
to complex metabolic network, are mostly insufficient and uncer-
tain to determine uniquely the associated flux distribution (i.e.
existence of multiple equivalent solutions). This problem can be cir-
cumvented by introducing additional metabolic constraints or by
using linear optimization techniques. However, these procedures
will fail to offset the underdeterminacy if these additional infor-
 A. Richelle et al. / Biochemical Engineering Journal 114 (2016) 50-61A3
Fig. 9. Comparison between simulator predictions (in blue) and experimental measurements with associated confidence intervals (red bars) of Experiments 1 and 2 (direct
validation). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
mation and/or assumptions are not in sufficient number and/or not
reliable enough [4,5].
In this context, the study of Lee et al. [28] was one of the first
to present a procedure allowing a rigorous determination of all the
solutions satisfying an underdetermined linear equation system.
They use a recursive mixed-integer linear programming (MILP)
algorithm to rigorously determine multiple alternate optimal solu-
tions of a FBA problem (i.e. flux distribution satisfying the same
objective function). However, the MILP algorithm is not easy to han-
dle with complex systems and could be computationally expensive
for genome-scale networks [29].
Instead of identifying all possible alternate optimal solutions,
Mahadevan and Schilling [30] have proposed to determine the flux
distribution variability (i.e. range of admissible values for each flux).
They used a series of LP problems to determine the maximum and
minimum values of all the fluxes that will satisfy the same optimal
objective value in FBA context. This approach is currently called
Flux Variability Analysis (FVA) in literature although called “flux-
spectrum” by Llaneras and Picó [31] which applied this strategy
to a MFA problem (i.e. same procedure without objective function
definition). Wiback et al. [32] introduced an equivalent concept,
57
called “alpha-spectrum”, which is related to the definition of the
overall solution space of a linear equation system by using NPA
methods (i.e. range of possible values for each elementary mode
or extreme pathway activity) but it is out of scope of this work.
Note that convex analysis can also be used for determining flux
distribution intervals [33].
For a stated MFA problem, the FVA is related to a simple min-
max LP problem (i.e. each reaction of the network is maximized
and subsequently minimized). It can be written as follows [31]

vi,upper = max vi N 0 v
∀vi , i = 1, . . ., n s.t. =0 (6)
vi,lower = min vi Ne −vmes 1
where n is the number of fluxes in v, vi,upper and vi,lower are respec-
tively the upper and lower values of each flux vi satisfying the
system of linear Eq. (4).
As made in Mahadevan and Schilling [30], this FVA approach can
easily be translated into a two-steps procedure in a FBA context.
First, the optimal value of objective function Z is determined by
solving the LP such as defined in Eq. (5). Secondly, the min-max
LP problem described in (6) is solved by fixing the optimal value
58 A. Richelle et al. / Biochemical Engineering Journal 114 (2016) 50-61A3

Fig. 10. Comparison between flux distribution determined with FBA problem (34) (in red) and the range of admissible flux values for reference solution (in blue) – Experiment
1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

of the original objective through the introduction of an additional culture process and are defined with the following differential
equality constraint within the linear equation system. equations:
dG F  
= −vG X + Gin − G (7)
dt V
dQ F  
= −vQ X + Q in − Q (8)
dt V
4. Dynamical simulator of hybridoma cell metabolism in
dN F
fed-batch cultures = vN X − N (9)
dt V
The purpose of this study is to present the development of a dA F
= vA X − A (10)
dynamical simulator of hybridoma cell metabolism in fed-batch dt V
culture based on a FVA. In addition to the initial conditions char- dL F
acterizing the process (substrates and products concentrations and = vL X − L (11)
dt V
culture volume at the start of the process), the input of the simula-
tor consists of the culture medium feeding rate. Two macroscopic dX F
= vX X − mX X − X (12)
models (extended Monod equations) allow, respectively, the repro- dt V
duction of glucose and glutamine consumption rates based on the where vG and vQ are modeled by using the extended Monod formal-
simulated values of the corresponding extracellular concentrations ism (see section 4.3) and vN ,vA , vL and vX will be determined with
(Section 4.3). On the basis of a metabolic network representing FBA (see Section 4.2.), F is the feeding rate, V the culture volume,
the intracellular metabolism of hybridoma cells [34] (Section 4.2), Gin and Q in the glucose and glutamine concentrations in the feed-
the simulator allows predicting the dynamics associated with cell ing medium and is the specific death rate. As highlighted in Section
growth, and the metabolite production (ammonium, alanine and 3, although optimal objective of FBA problem is unique, there exist
lactate) by connecting these latters with associated fluxes involved different flux distributions leading equally to this optimal solution.
in the metabolic network and by computing their values over time To circumvent this problem, a preliminary FVA was performed in
with FBA (Section 4.4). order to assess the space of feasible flux distribution given the two
Assuming that reactions take place in a perfectly mixed liquid input fluxes (glucose and glutamine consumption rates). This lat-
phase, mass balance performed on each of the five metabolites ter was compared to a reference solution: the space of feasible flux
considered (glucose, glutamine, ammonium, alanine and lactate) distribution obtained from a MFA problem computed by using all
describe the evolution of their concentrations over the fed-batch the available external fluxes (glucose and glutamine uptake rates;
 A. Richelle et al. / Biochemical Engineering Journal 114 (2016) 50-61A3
Fig. 11. Comparison between flux distribution determined with FBA problem (34) (in red) and the range of admissible flux values for reference solution (in blue) – Experiment
2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
lactate, ammonium and alanine production rates). This strategy
allows the objective identification of some additional constraints
in order to reduce the space of feasible flux distribution associated
with the two simulated input fluxes to the one of the reference
solution. Moreover, this strategy allows legitimating the choice of
the objective cost function for the FBA problem definition used in
the simulator.
4.1. Experimental field
This work uses experimental data from two fed-batch (Exper-
iments 1 and 2) and one batch (Experiment 3) cultures using
hybridoma cell line HB-58 (American Culture Collection – ATCC)
performed at the State Key Laboratory of Bioreactor Engineering,
East China University of Science and Technology (ECUST), Shanghai.
Both fed-batch experiments were performed within 1 L bioreactors
(37 ◦ C, 50% DO, pH 7, stirring rate 120 rpm) in fed-batch mode with
constant feeding strategy (0.1L/day) after an initial batch phase
varying from 35 to 64 h. The cultures are fed with glucose (15 mM)
and glutamine (9 mM). The batch experiment was performed in
a 2 L bubble free bioreactor (37 ◦ C, 40% DO, pH 7.1, stirring rate
120 rpm) initiated with 25 mM glucose and 5.5 mM glutamine.
All experiments were performed from the same initial medium
(0.2 mM Asp, 0.1 mM Glu, 0.05 mM Asn, 0.43 mM His, 0.25 mM
Gly, 0.8 mM Thr, 1 mM Arg, 0.05 mM Ala, 0.4 mM Tyr, 0.3 mM
Met, 1 mM Val, 0.15 mM Trp, 0.4 mM Phe, 0.8 mM Ile, 1.2 mM Leu,
1 mM Lys) and supplemented with the same additives (2 mM Na-
Pyruvate, 100uM ETA, 23.1 nM Na-Selenite, 10 mg/L Transferrin,
10 mg/L Insulin 10 mg/L, 100 mg/L BSA, 2.44 g/L NaHCO3). Sam-
59
ples (10 mL) were taken about every 12 h for measuring viable cell
density and glucose, glutamine, lactate, ammonium and alanine
concentrations. The measurement variation coefficients (standard
deviation of the measurement errors divided by the correspond-
ing values) are defined such as to be in agreement with the used
analytical measurement protocols (10% for glucose and lactate and
5% for biomass, glutamine, ammonium and alanine). More details
about the material and experiments can be found in Amribt et al.
[7].
4.2. Metabolic network
The metabolic network considered in this work is the one pre-
sented in Provost et al. [34] for CHO-320 cells metabolism during
growth phase and is presented in Fig. 1. In this work, we assume
that it could be transposed for hybridoma cells without transforma-
tion as it only includes the main pathways associated with glucose
and glutamine bioconversion (i.e., the glycolysis, the glutaminoly-
sis, the TCA cycle and the nucleotide synthesis) which are present in
both organisms. In this study, all the fluxes are assumed to be pos-
itive, meaning that the direct reaction is predominant with respect
to the inverse one.
Based on this network, the fluxes vG vQ ,vN ,vA and vL (i.e. con-
sumption rates of glucose and glutamine and production rates of
ammonium, lactate and alanine) are connected with associated
metabolic fluxes and introduced in Eqs. (7)–(11):
dG F  
= −v1 X + Gin − G (13)
dt V
60 A. Richelle et al. / Biochemical Engineering Journal 114 (2016) 50-61A3

Fig. 12. Comparison between simulator predictions (in blue) and experimental measurements with associated confidence intervals (red bars) of Experiment 3 (cross
validation). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

dQ F   Q KIX Q
= − (v16 + v17 + 2v18 ) X + Q in − G (14) vQ = ␮Qmax (20)
dt V Q + KQ KIX Q + X
dN F The parameters associated to vG and vQ are identified by solving
= (v15 + v16 ) X − N (15)
dt V the dynamic Eqs. (7) and (8) in combination with (19) and (20) with
dA F Matlab’s ODE solver ode15 s and using the Nelder-Mead simplex
= v7 X − A (16) method (function fminsearch) in order to minimize a least squares
dt V
criterion (sum of squared differences between model predictions
dL F and experimental measurements):
= v6 X − L (17)
dt V
As made in Llaneras and Picó [31], the formation rates of purine

N

Yij  − Ymes,ij Wij−1 Yij  − Ymes,ij

T
J = (21)
and pyrimidine (v17 and v18 ) are assumed to be equal (v17 = v18 )
i=1
and to equally contribute to the biomass formation. Hence, Eq. (12)
becomes:  of stoichiometric and kinetic
where  is the vector parameters
 to be
dX F identified ( T = ␮Gmax ␮Qmax KG KQ KIX G KIX Q ), YijT  = Gij Qij is
= ˛(v17 + v18 )X − mX X − X (18) the vector of simulated variables calculated from mass balances
dt V
th th
where ˛ is a yield coefficient for biomass growth.
(7)–(8) combined with
T
 (19) and (20) at the i time instant of the j
experiment, Ymes,ij = Gmes ij Qmes ij is the vector of corresponding
measurements. Wij is a weighting matrix defined as:
4.3. Macroscopic modeling of substrates consumption rates
Wij = diag( Gmes,ij 2 ,  Qmes,ij 2 ) (22)
Taking inspiration of kinetic structures identified with a system-
atic methodology for the same experimental data [35], the specific where ␴2
are the variances of the corresponding measurements
consumption rates of glucose and glutamine are described as: errors.
Note that the biomass term (X) of Eqs. (7), (8), (19) and (20) is
G KIX G approximated by using linear interpolation of biomass measure-
vG = ␮Gmax (19)
G + KG KIX G + X ments (Xmes ).
 A. Richelle et al. / Biochemical Engineering Journal 114 (2016) 50-61A3 61

Table 1 Results presented in Figs. 3 and 4 clearly highlight the prob-

Parameter values (dim = 6) identified with all experiments.
lem of system underdetermination. Only four fluxes (v13 , v14 , v17
Range for initialization Identified values Units and v18 ) are uniquely determined in both experiments and many
␮Gmax 0.5–5 1.0131 mmol (109 cell h)−1 other ones present very large variability. Specifically, all fluxes (v6 ,
␮Qmax 0.1–1 0.1246 mmol (109 cell h)−1 v7 , v15 and v16 ) required to solve (13) − (18) involve a zero solu-
KG 0.01–10 1.2567 mmol L−1 tion in their admissible values leading to the potential impossibility
KQ 0.01–5 0.1767 mmol L−1 to reproduce metabolite production. To overcome this problem,
KIX G 1–20 0.4621 109 cells L−1
some additional constraints need to be added in order to reduce
KIX Q 1–10 2.0177 109 cells L−1
the range of feasible values associated with these metabolite pro-
duction fluxes.
To circumvent local minima and convergence problems with
the optimization algorithm used for minimizing (21), a multistart 4.5. Constraints identification approach using FVA
strategy was considered for the initialization of the parameter val-
ues. 100 uniformly distributed pseudo-random values over a given The proposed constraints identification approach allows inves-
range (Table 1) were used as multistart strategy for the initializa- tigating and legitimating the choice of these latters based on FVA
tion of the algorithm. The identified parameter values (based on the results of a MFA problem computed by using as inputs all the
2 experiments) are presented in Table 1 and the direct validation extracellular measurements available (substrates consumption and
in Fig. 2. metabolites production), called reference solution. The LP problem
associated with the reference solution can be stated as follows:
4.4. FBA problem statement
∀tk , ∀i = 1, . . .n
Based on the definition of the specific consumption rates of
glucose and glutamine (Eqs. (19) and (20)), the different fluxes Nv = 0
(v1 ,v6 , v7 , v15 , v16 , v17 and v18 ) introduced in Eqs. (13)–(18) can vi,upper tk = max vi tk v≥0
be determined at each time instant by solving a FBA problem s.t. N v ≤ 1 + e v (25)
e v mes tk
vi,lower tk = min vi tk
which maximizes biomass production through the synthesis of
Ne v ≥ 1 − ev vmes tk
nucleotides by v14 = v17 + v18 and stated as follows:
Nv = 0 where N ∈ R18X24 is the stoichiometric matrix of balanced
metabolites, Ne ∈ R5X24 is the stoichiometric matrix of
v≥0
∀tk , vopt t = max v14 tk s.t. N v ≤ 1 + e v (23) unbalanced metabolites considered for the reference solution (glu-
14 k e v mes tk T

cose, glutamine, lactate, ammonium and alanine) and vmes tk =
Ne v ≥ 1 − ev vmes tk vG tk vQ tk vL tk vN tk vA tk . vG and vQ are computed as before
with Eqs. (19) and (20) and the set of identified parameters
where N ∈ R18X24 is the stoichiometric matrix of balanced (Table 1). The three other reaction rates (vL , vN and vA ) are deter-
metabolites (the 18th row corresponds to the additional assump- mined from the combination of smoothing splines (function spaps
tion v17 = v18 ), Ne ∈ R2X24 is the stoichiometric matrix of in Matlab) applied to the corresponding concentration measure-
unbalanced metabolites (glucose and glutamine), vmes are con- ments, the derivatives of these splines (function fnder in Matlab)
sumption rates of glucose and glutamine (vmes T tk = vG tk vQ tk ) and the mass balance system (9)–(11). Figs. 5 and 6 present the
computed with Eqs. (19)–(20) and the set of identified parameters comparison between FVA resulting from (24) (FBA problem – in
(Table 1) and evaluated at each time measurement tk and ev is the red) and (25) (MFA problem – in blue) for Experiments 1 and 2,
coefficient variation associated with experimental measurements respectively.
as performed in Llaneras and Picó [31] to consider the intrinsic Note that the FBA objective function which aims at maximizing
uncertainty of measurements (called « interval representation of biomass production has been legitimated on this case study [36] by
fluxes »). This variation coefficient is fixed at 15% which is the lowest comparing the range of admissible flux values satisfying the refer-
value such that LP (23) admits a feasible solution at each measure- ence MFA problem defined by (25) with those satisfying the same
ment time tk for the two experiments considered (meaning that the MFA problem but using the constraints defined in (23), i.e. only
constraints can be fulfilled at each time instant). the measurements of the substrate (glucose and glutamine) uptake
The range of feasible flux distribution vopt (tk ) given the two fluxes. Indeed, in this comparison (not shown here), it has been
input fluxes used (vG and vQ ) satisfying the LP problem of (23) can observed that the maximum value in the range of v14 = v17 + v18 ,
be computed in a second step with FVA approach such as presented when using only the constraints defined in (23), was very close to
in Section 3: the mean value of v14 when using all the constraints defined in (25),
∀tk , ∀i = 1, . . .n at least in the fed-batch phase. This legitimates the fact that v14 (and
hence biomass production) should be maximized in FBA.
Nv = 0 The comparison presented in Figs. 5 and 6 allows the objective
identification of additional constraints such as the solution space
v≥0
vi,upper tk = max vi tk associated to FBA problem is “shrinked” to the one of reference
s.t. Ne v ≤ 1 + ev vmes tk (24) solution, ensuring a good prediction of metabolite production rates
vi,lower tk = min vi tk by the simulator. In doing so, two different additional constraints
Ne v ≥ 1 − ev vmes tk
v14 tk = vopt
14 k
t are identified in order to compensate the lack of information about
vL and vN by decreasing the flux variability of v6 and forcing v16 to
where vi,upper (tk )and vi,lower (tk ) represent respectively the maxi- present a “non-zero” solution, respectively.
mum and minimum admissible values of each flux vi satisfying the The constraint associated to lactate production rate(v6 ) is for-
LP problem (23) and corresponding to the maximal objective func- mulated by fixing an upper bound on the input flux of Krebs cycle
opt
tion value (v14 (tk )) at each time measurement tk . Figs. 3 and 4 (v8 ):
present respectively the FVA results (solutions of Eq. (24)) for
Experiments 1 and 2. v8 (tk ) ≤ v8max (26)
61A1 A. Richelle et al. / Biochemical Engineering Journal 114 (2016) 50-61A3

The introduction of this constraint is justified by the existence of Table 2

Identified parameter values (dim = 10).
a maximum catabolic transformation rate of glucose through res-
piratory pathway (Krebs cycle) in hybridoma cells such as in many Initialization values Identified values Units
other living organisms. Indeed, when the glucose consumption rate Gmax 1.0131 1.1388 mmol (109 cell h)−1
is greater than the maximum allowable respiratory flux (v8max ), the Qmax 0.1246 0.1236 mmol (109 cell h)−1
glucose excess is directed into the pathway leading to lactate pro- KG 1.2567 1.4479 mmol L−1
duction. This phenomenon is called overflow metabolism [7,37]. KQ 0.1767 0.0945 mmolNL−1
KIX G 0.4621 0.4802 109 cells L−1
The identification of v8max is done by checking its influence on flux
KIX Q 2.0177 1.7281 109 cells L−
variability of v6 for different values in a trials and errors procedure. ␣ 2.3449 2.7887
In doing so, v8max is fixed at 0.15 mmol (109 cells h)−1 . mX 0.0384 0.0462 h−1
The second constraint is the one associated with the lack of v8max 0.15 0.0535 mmol (109 cell h)−1
a 0.14 0.1409
information about the ammonium production rate(v15 + v16 ). It is
formulated by introducing an inequality between the glucose con-
sumption rate vG and v16 in order to ensure a non-zero solution for
dX opt F
this latter: = ˛v14 X − mX X − X (33)
dt V
v16 (tk ) ≥ a.vG (tk ) (27) opt
where v6,7,14,15,16 are determined by solving the following FBA
where a is a proportionality coefficient. problem:
The proportionality between glucose consumption rate (vG ) and Nv = 0
a part of the glutamine consumption rate (vQ = v16 + v17 + 2v18 ) is
v≥0
justified by the hypothesis of coordinated uptakes of these carbon
and nitrogen sources under the experimental conditions consid- Ne v ≤ 1 + ev vmes tk
∀tk , v opt
tk = max v14 tk s.t.
Ne v ≥ 1 − ev vmes tk
(34)
ered [38,39]. Moreover, this inequality ensures that all glutamine
fed to the cell (vQ ) is not only used for nucleotides synthesis through v8 tk ≤ v8max
v17 + 2v18 . By empirically testing different a values, it was deter- v16 tk ≥ a.vG tk
mined that 0.14 corresponds to the best reproduction of v16 in both
experiments. where vopt (tk ) is a flux distribution vector optimal with respect to
Figs. 7 and 8 present the comparison between FVA of FBA prob- v14 and satisfying, at each time measurement tk , the linear
 equation
lem with the two additional constraints (Eqs. (24)–(27) in red) and system described by the right term of (34), vmesT tk = vG tk vQ tk
FVA of reference solution (MFA problem, Eq. (25) – in blue) for is computed with Eqs. (19) and (20) and the set of identified param-
Experiments 1 and 2, respectively. eters (Table 1) and the variation coefficient ev is fixed at 0.15.
The introduction of a maximum capacity on the input flux of All parameters values of the simulator model have been iden-
Krebs cycle (v8max ) allows to shrink the range of admissible flux tified previously except the parameters associated with biomass
values of FBA with additional constraints to the one of reference growth and death (˛ and mX ). To this end, the dynamic Eq. (33)
solution, ensuring that the lactate production will be well pre- is solved independently of the rest of the model (ode15s) and the
dicted by the simulator. As expected, the flux v16 is no more equal parameter values are identified with the simplex method (fmin-
to zero and is compliant with the one obtained in reference solu- search) based on a least squares criterion such as made for substrate
tion. Moreover, it is interesting to note that, in Figs. 5 and 6 (FBA consumption model in Section 4.3 but using only the biomass con-
problem without additional constraints), the flux v14 is well esti- centration as experimental measurements. The parameter values
mated only after 100 h of cultures (i.e. during fed-batch phase) for identified are presented in the column “Initialization values” of
both experiments while it is always included within (or very close Table 2.
to) the confidence interval of reference solution when additional The last step of the simulator development procedure con-
constraints are taken into account (Figs. 7 and 8). This is due to the sists in using the parameter values identified for the substrate
introduction of inequality related to v16 which strongly decreases uptake rate models and for the additional constraints as initial
the overestimation of v14 in first phase of culture. This fact legiti- values for a global optimization routine using fminsearch opti-
mates both choices of objective function and additional constraints. mization algorithm minimizing the sum of squared differences
between simulator outputs and experimental measurements.
4.6. Global parameter identification and validation of the This can be expressed by the general formalism used for Eq. (21)
but where  T = ␮Gmax ␮Qmax KG KQ KIX GKIX Q˛mX av8max ,
simulator 
YijT  = Gij Qij Nij Lij Aij Xij and T
Ymes,ij =

The final dynamical simulator of hybridoma cell metabolism is Gmes Qmes Nmes Wij =
ij ij ij Lmes ij Ames ij Xmes ij and
described by Eqs. (28)–(34):
diag( Gmes,ij 2 ,  Qmes,ij 2 ,  Nmes,ij 2 ,  Lmes,ij 2 ,  Ames,ij 2 ,  Xmes,ij 2 ).
dG G KIX G F   The system of dynamic Eqs. (28)–(33) is solved with ode15s and
= −␮Gmax X+ Gin − G (28) the LP problem (34) with linprog function.
dt G + KG KIX G + X V
In order to evaluate the goodness of the fit of measured values
dQ Q KIX Q F   against simulated ones, the correlation coefficient R2 (35) is com-
= −␮Qmax X+ Q in − G (29)
dt Q + KQ KIX Q + X V puted for each simulated variable with direct (Experiments 1 and
 opt opt  2) and cross validation (Experiment 3) of the model:
dN F
= v15 + v16 X − N (30)
n
Yij  − Ymes,ij Wij−1 Yij  − Ymes,ij
dt V T
j=1
R2 = 1 −
n T
(35)
dA opt F Yij  − Ymes,ij Wij−1 Yij  − Ymes,ij
= v7 X − A (31) j=1
dt V
 
dL opt F where , YijT  , Ymes,ij
T T
and Wij are defined as above. Ymes,ij is the
= v6 X − L (32)
dt V mean of corresponding measurements.
 A. Richelle et al. / Biochemical Engineering Journal 114 (2016) 50-61A3 61A2

Fig. 13. Flux distribution determined with FBA problem (34) – Experiment 3.

Table 3 the time, underdetermined systems. Approaches using additional

Correlation coefficients of each simulated variables for direct (Experiments 1 and 2)
metabolic constraints or linear optimization techniques have been
and cross validation results (Experiment 3).
proposed to offset this underdetermination. However, these latters
Direct validation Cross validation often require a priori assumptions that are difficult to legitimate. To
R2 G 0.8984 0.9035 this day, the way to overcome objectively this problem remains an
R2 Q 0.9198 0.9131 open question.
R2 N 0.9807 0.9921 In this study, we present a methodology for designing a dynam-
R2 A 0.9844 0.9965
ical FBA-based simulator through flux variability analysis. It is
R2 L 0.9710 0.9934
R2 X 0.9745 0.8817 applied to hybridoma cell fed-batch cultures and allows predicting
the dynamics associated with cell growth, and metabolite produc-
tion (ammonium, alanine and lactate) on the basis of a simple
The identification and correlation results are presented in metabolic network representing the intracellular metabolism of
Tables 2 and 3, respectively. The direct validation of simulator out- hybridoma cells [34] and two macroscopic models describing sub-
puts is presented in Fig. 9 and the associated flux distributions for strate consumption dynamics (glucose and glutamine).
Experiments 1 and 2 in Figs. 10 and 11. Fig. 12 (simulator out- We propose using comparative flux variability analysis to guide
puts) and Fig. 13 (flux distribution) present a cross validation of the constraint choices related to the development of the simula-
the identified model on Experiment 3. tor. To this end, a comparison between the ranges of admissible
The proposed simulator gives good predictions for all metabo- flux distribution (determined from linear programs) obtained
lites considered, both in direct (Fig. 9) and cross validation (Fig. 12). when the metabolic network model is solved using all available
This goodness of fit is also attested by the regression coefficients measurements (input and output fluxes) and using only part of
which are all greater or equal to 0.88. The flux distribution deter- them (only input fluxes) is performed. The proposed strategy
mined with FBA (34) is included within (or very close to) the allowed the objective identification of two additional constraints
confidence interval defined by the range of admissible flux val- which both represent existing biological phenomena (glucose over-
ues of the reference solution. Moreover, the identified parameter flow metabolism and coordination between nitrogen and carbon
values are in the same range of the initialization values. Interest- sources uptake rates). Moreover, this procedure has allowed legit-
ingly, the identified value for v8max is very close to the value of imating the use of biomass growth as cost function for linear
0.05 mmol (109 cells h)−1 found in literature references [7,40]. optimization. Finally, the simulator has been validated on exper-
imental data of two fed-batch cultures and cross validated on a
5. Conclusions batch culture of hybridoma cells HB-58. It presents good predic-
tions for biomass growth, substrate consumption and metabolite
Constraint-based modeling is a powerful tool for represent- production, even though its final structural complexity is quite low
ing intracellular metabolism of living organisms. Nevertheless, (10 parameters).
the metabolic network models used in this context are, most of
61A3 A. Richelle et al. / Biochemical Engineering Journal 114 (2016) 50-61A3

The proposed approach is a contribution in the objective search calculability and the balanceability of conversion rates, Biotechnol. Bioeng. 43
for additional constraints and adequate cost function that can be (1) (1994) 3–10.
[18] J.M. Savinell, B.Ø. Palsson, Network analysis of intermediary metabolism
used to determine fluxes correctly in a FBA context based on the using linear optimization. I. Development of mathematical formalism, J.
knowledge of admissible solutions for the corresponding MFA- Theor. Biol. 154 (4) (1992) 421–454.
based representation. It will be interesting to check at which extend [19] J.M. Savinell, B.Ø. Palsson, Network analysis of intermediary metabolism
using linear optimization.II. Interpretation of hybridoma cell metabolism, J.
this procedure could be applied to a more complex metabolic model Theor. Biol. 154 (4) (1992) 455–473.
such as the one presented in Zamorano et al. [33]. On the contrary, [20] J.D. Orth, I. Thiele, B.Ø. Palsson, What is flux balance analysis? Nat. Biotechnol.
the possibility of building an FBA-based simulator using a simpler 28 (2010) 245–248.
[21] A. Varma, B.Ø. Palsson, Metabolic flux balancing: basic concepts, scientific and
metabolic model will also be analyzed.
practical use, Nat. Biotechnol. 12 (1994) 994–998.
[22] B.Ø. Palsson, Systems Biology: Properties of Reconstructed Networks, first ed.,
Acknowledgements Cambridge University Press, New York, 2006.
[23] N.D. Price, J.A. Papin, C.H. Schilling, B.Ø. Palsson, Genome-scale microbial in
silico models: the constraints-based approach, Trends Biotechnol. 21 (4)
We thank Dr. Hongxing Niu for providing the experimental data. (2003) 162–169.
This work has been supported by FEDER (European Union and Wal- [24] D. Segre, D. Vitkup, G.M. Church, Analysis of optimality in natural and
perturbed metabolic networks, Proc. Natl. Acad. Sci. 99 (2002) 15112–15117.
lonia Region) in the Hainaut-Biomed program (OCPAM project).
[25] I. Thiele, N.D. Price, T.D. Vo, B.Ø. Palsson, Candidate metabolic network states
in human mitochondria. Impact of diabetes, ischemia, and diet, J. Biol. Chem.
References 280 (2005) 11683–11695.
[26] R. Schuetz, L. Kuepfer, U. Sauer, Systematic evaluation of objective functions
for predicting intracellular fluxes in Escherichia coli, Mol. Syst. Biol. 3 (2007)
[1] A. Bordbar, J.M. Monk, Z.A. King, B.Ø. Palsson, Constraint-based models
119.
predict metabolic and associated cellular functions, Nat. Rev. Genet. 15 (2014)
[27] R. Schuetz, N. Zamboni, M. Zampieri, M. Heinemann, U. Sauer,
107–120.
Multidimensional optimality of microbial metabolism, Science 336 (2012)
[2] A.M. Feist, M.J. Herrgård, I. Thiele, J.L. Reed, B.Ø. Palsson, Reconstruction of
601.
biochemical networks in microorganisms, Nat. Rev. Microbiol. 7 (2009)
[28] S. Lee, C. Phalakornkule, M.M. Domach, I.E. Grossmann, Recursive MILP model
129–143.
for finding all the alternate optima in LP models for metabolic networks,
[3] G. Stephanopoulos, A.A. Aristidou, J. Nielsen, Metabolic Engineering:
Comput. Chem. Eng. 24 (2000) 711–716.
Principles and Methdologies, Elsevier Science, 1998.
[29] J.L. Reed, B.Ø. Palsson, Genome-scale in silico models of E. coli have multiple
[4] H.P.J. Bonarius, G. Schmid, J. Tramper, Flux analysis of underdetermined
equivalent phenotypic states: assessment of correlated reaction subsets that
metabolic networks: the quest for the missing constraints, Trends Biotechnol.
comprise network states, Genome Res. 14 (2004) 1797–1805.
15 (8) (1997) 308–314.
[30] R. Mahadevan, C.H. Schilling, The effects of alternate optimal solutions in
[5] S. Klamt, S. Schuster, E.D. Gilles, Calculability analysis in underdetermined
constraint-based genome-scale metabolic models, Metabol. Eng. 5 (2003)
metabolic networks illustrated by a model of the central metabolism in
264–276.
purple nonsulfur bacteria, Biotechnol. Bioeng. 77 (7) (2002) 734–751.
[31] F. Llaneras, J. Picó, An interval approach for dealing with distributions and
[6] J. Niklas, E. Heinzle, Metabolic flux analysis in systems biology of mammalian
elementary modes activity patterns, J. Theor. Biol. 246 (2) (2007) 290–308.
cells, Adv. Biochem. Eng.: Biotechnol. 127 (2012) 109–132.
[32] S.J. Wiback, R. Mahadevan, B.Ø. Palsson, Reconstructing metabolic flux vectors
[7] Z. Amribt, H. Niu, Ph. Bogaerts, Macroscopic modelling of overflow
from extreme pathways: defining the alpha-spectrum, J. Theor. Biol. 224 (3)
metabolism and model based optimization of hybridoma cell fed-batch
(2003) 313–324.
cultures, Biochem. Eng. J. 70 (2013) 196–209.
[33] F. Zamorano, A. Vande Wouwer, G. Bastin, A detailed metabolic flux analysis
[8] G. Bastin, D. Dochain, On-line Estimation and Adaptative Control of
of an undertermined network of CHO cells, J. Biotechnol. 150 (4) (2010)
Bioreactors, first ed., Elsevier Science, Oxford, 1990.
497–508.
[9] B. Ben Yahia, L. Malphettes, E. Heinzle, Macroscopic modeling of mammalian
[34] A. Provost, G. Bastin, S.N. Agathos, Y.J. Schneider, Metabolic design of
cell growth and metabolism, Appl. Microbiol. Biotechnol. 99 (2015)
macroscopic bioreaction models: application to Chinese hamster ovary cells,
7009–7024.
Bioprocess Biosys. Eng. 29 (5–6) (2006) 349–366.
[10] J.E. Haag, A. Vande Wouwer, Ph. Bogaerts, Systematic procedure for the
[35] A. Richelle, Ph. Bogaerts, Systematic methodology for bioprocess model
reduction of complex biological reaction pathways and the generation of
identification based on generalized kinetic functions, Biochem. Eng. J. 100
macroscopic equivalents, Chem. Eng. Sci. 60 (2005) 459–465.
(15) (2015) 41–49.
[11] H. Niu, Z. Amribt, P. Fickers, W. Tan, Ph. Bogaerts, Metabolic pathway analysis
[36] Ph. Bogaerts, K. Mhallem Gziri, A. Richelle, From MFA to FBA: Legitimating
and reduction for mammalian cell cultures – towards macroscopic modeling,
objective function and linear constraints, in: Proceedings of the 11th IFAC
Chem. Eng. Sci. 102 (2013) 461–473.
Symposium on Dynamics and Control of Process Systems, Including
[12] A. Provost, G. Bastin, Dynamic metabolic modelling under the balanced
Biosystems (DYCOPS-CAB 2016), Trondheim (Norway), 6–8/6/2016, 2016.
growth condition, J. Process Control 14 (2004) 717–728.
[37] M. Doverskog, J. Ljunggren, L. Öhman, L. Häggström, Physiology of cultured
[13] N.E. Lewis, H. Nagarajan, B.Ø. Palsson, Constraining the metabolic
animal cells, J. Biotechnol. 59 (1997) 103–115.
genotype–phenotype relationship using a phylogeny of in silico methods, Nat.
[38] Y.-H. Jeong, S.S. Wang, Role of glutamine in hybridoma cell culture: effect on
Rev. Microbiol. 10 (2012) 291–305.
cell growth, antibody production, and cell metabolism, Enzyme Microb.
[14] F. Llaneras, J. Picó, Stoichiometric modelling of the cell metabolism, J. Biosci.
Technol. 17 (1) (1995) 47–55.
Bioeng. 1 (2008) 1–12.
[39] J. Wahrheit, A. Nicolae, E. Heinzle, Dynamics of growth and metabolism
[15] R. Rockafellar, Convex Analysis, Princeton University Press, Princeton, New
controlled by glutamine availability in Chinese hamster ovary cells, Appl.
Jersey, 1970.
Microbiol. Biotechnol. 98 (2014) 1771–1783.
[16] J.A. Papin, J. Stelling, N.D. Price, S. Klamt, S. Schuster, B.Ø. Palsson, Comparison
[40] S. Dhir, K.J. Morrow, R.R. Rhinehart, T. Wiesner, Dynamic optimization of
of network-based pathway analysis methods, Trends Biotechnol. 22 (8)
hybridoma growth in a fed-batch bioreactor, Biotechnol. Bioeng. 67 (2) (2000)
(2004) 400–405.
197–205.
[17] R.T. Van der Heijden, B. Romein, J.J. Heijnen, C. Hellinga, K.C. Luyben, Linear
constraint relations in biochemical reaction systems: I. Classification of the