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Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Influence of HEK293 metabolism on the production of viral

vectors and vaccine
Emma Petiot a , Miroslava Cuperlovic-Culf b , Chun Fang Shen b , Amine Kamen b,c,∗
a
Laboratoire Virologie et Pathologie Humaine (VirPath), EA4610 Université Claude Bernard Lyon 1, 7 rue guillaume paradin, 69008 Lyon, France
b
Vaccine Program National Research Council, 6100 Royalmount Ave, Montreal, QC, Canada H4P 2R2
c
Bioengineering Dpt., McGill University, 817, Sherbrooke West, Montreal, QC, Canada H3A 0C3

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

Article history: Mammalian cell cultures are increasingly used for the production of complex biopharmaceuticals includ-
Received 26 March 2015 ing viral vectors and vaccines. HEK293 is the predominant cell line used for the transient expression of
Received in revised form 20 May 2015 recombinant proteins and a well-established system for the production of viral vectors. Understanding
Accepted 22 May 2015
metabolic requirements for high productivity in HEK293 cells remains an important area of investi-
Available online xxx
gation. Many authors have presented approaches for increased productivity through optimization of
cellular metabolism from two distinct perspectives. One is a non-targeted approach, which is directed
Keywords:
to improving feeding strategies by addition of exhausted or critical substrates and eventually removal
HEK293 cell metabolism
Viral vector and vaccine
of toxic metabolites. Alternatively, a targeted approach has attempted to identify specific targets for
Metabolomics optimization through better understanding of the cellular metabolism under different operating con-
Viral production processes ditions. This review will present both approaches and their successes with regards to improvement of
viral production in HEK293 cells outlining the key relations between HEK293 cell metabolism and viral
vector productivity. Also, we will summarize the current knowledge on HEK293 metabolism indicating
remaining issues to address and problems to resolve to maximize the productivity of viral vectors in
HEK293 cells.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction suitable glycosylation pattern required for viruses efficient replica-

Biopharmaceutical industry have been increasingly investigat-
ing mammalian cell cultures as production platforms for safe,
flexible, fast and cost-effective production of viral vaccines and
viral vectors. HEK293 cells have been used for over 30 years for
production of viral vectors for gene and cell therapy and offer sev-
eral advantages over other cell culture platforms. This cell line
can be easily grown in suspension and in serum-free medium
at cell densities up to 20 million cells/ml (unpublished results).
The development of fast, reproducible and streamlined transfec-
tion protocols for this cell line has increased its use in gene
therapy applications. HEK293 cell is now a cell culture platform
of choice for production of recombinant viral vectors and vac-
cines such as adenovirus or adeno-associated virus [1], retrovirus,
lentivirus, reovirus, influenza, as well as various VLP forms (rabies,
influenza) [2–5]. Additionally, this human origin cell line provides
∗ Corresponding author at: McGill University, Bioengineering, 817 Rue Sher-
brooke West, Mcdonald Engineering Building Roo, Montreal, QC, Canada H3A 0C3.
Tel.: +1 5143985775.
E-mail address: amine.kamen@mcgill.ca (A. Kamen).
tion [6,7].
Nevertheless, number of challenges impede high production
yield of viruses at high-cell density in HEK293 cultures. Remaining
issues include identification of different metabolic requirements
of cells during growth and virus production phases; the “cell den-
sity effects” precluding virus production at high cell densities; the
complex and often undisclosed formulation of cell culture media
that prevent rational design and optimization; and the inherent
complexity of each virus/cell system with specific demands.
All these challenges are related to the concept of “maintaining,
or improving, the cell physiology all along the production process”,
which is strongly linked to the cell metabolic state. Viral replication
induces major changes in the cell physiology and metabolic states
[8]. The cell metabolic efficiency, i.e. the adequate and efficient sup-
ply of metabolic precursors for the synthesis routes, was proven to
impact the viral component synthesis, their assembly, release and
in general the production levels. Consequently, metabolism under-
standing (metabolites profiling) is often a crucial guide to process
optimization.
As cell culture media are often composed of over hundred ele-
ments with a majority at concentrations below detection limits of
routine methodologies, historically, metabolite profiling methods

http://dx.doi.org/10.1016/j.vaccine.2015.05.097
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(2015), http://dx.doi.org/10.1016/j.vaccine.2015.05.097
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were based on the quantification of only major substrates and cell
by-products in the cultures supernatants. Such “first stage” anal-
yses are still often used for identification of cellular needs and
were successful in providing strategies for improvement of HEK293
viral production. Over the past decade the classical approach was
enhanced by high throughput metabolomics analyses using NMR
or MS [9–12]. Thus, larger number of metabolites present at lower
concentrations are quantified providing deeper understanding of
HEK293 cell metabolism.
Metabolite measurements can be analyzed qualitatively,
observing only global metabolic changes between different sys-
tems. Major metabolic changes, including cells’ needs as well
as metabolic waste productions can be determined from quanti-
tative metabolomics, i.e. metabolite concentrations. Quantitative
metabolic analyses can also lead to the development of computa-
tional models of metabolic processes and “metabolic networks”.
Metabolic flux analysis (MFA) allows determination of the degree
of involvement of different pathways in metabolic processes and
cellular functions [13–19]. Through understanding and modelling
of cellular metabolic pathways and network it is possible to engi-
neer and optimize cell metabolism as well as cellular environment.
Examples are presented throughout the text and summarized in
Table 2. Quantitative metabolic data can also be combined with
other omics data for the development of more complete models
of the molecular status of cells. This “polyomics” approach can
overcome traditional limitation of metabolomics which can cur-
rently quantify only a subset of compounds. Such methodologies
and approaches are presented in Fig. 1 and have been extensively
reviewed for HEK293 cells [20–24].
In this review, we will outline current knowledge of HEK293
cell metabolism in virus production leading to various methods
that have been explored in order to increase productivity through
metabolism optimization as well as remaining challenges in utiliz-
ing HEK293 cells for industrial viral production. First we will outline
contributions of metabolomics, fluxomics and polyomics studies
to better understanding of HEK293 metabolism. Subsequently, we
show how the understanding of cell metabolism can be used to
improve production by using different feeding methods and media
optimization.
2. Metabolomics to improve viral productions in HEK293
cell cultures
Metabolomics analyses of several cell lines have shown that viral
infection activates large number of metabolic processes [8,25–28]
unlike the metabolic reduction observed in the case of recombinant
protein production [29]. Infection induced metabolic shift has been
extensively studied and confirmed for diverse cell/virus production
platforms (rubella virus [30–32], cytomegalovirus [25,33], mayaro
virus [34], dengue virus [35], mumps virus [36], newcastle-disease
virus [36], polio virus [37–39], reovirus [40], influenza viruses [41]).
Studies of HEK293 cell infection, with focus on adenovirus produc-
tions, will be described below.
2.1. Metabolomics and fluxomics as fingerprint of the cellular
state under infection
Metabolite profiling of HEK293 cells led to the identification of
specific cellular processes affected by viral production. The first
MFA model for HEK293 cells, presented by Nadeau et al., was fol-
lowed by a series of improved models for comprehensive metabolic
behavior of this cell line under adenovirus infection (Table 1).
Recently, a comprehensive, high quality network of the human cell
metabolism was published [42]. This model (Recon2) attempts to
be an all-inclusive network of all known reactions in human cell
Please cite this article in press as: Petiot E, et al. Influence of HEK293 metabolism on the production of viral vectors and vaccine. Vaccine
(2015), http://dx.doi.org/10.1016/j.vaccine.2015.05.097
(>7000 reactions and 5000 metabolites). However, although it is
simulation-ready and can be used as a generic model for any human
cell system, its large degree of redundancy masks the metabolic
contributions of individual reactions [43]. In order to focus this
model for HEK293 cells analysis, Quek et al. [43] published in 2014
a reduction of Recon2 by removing redundant pathways in the
biosynthesis and catabolic peripheral metabolism to emphasize the
flexibilities of the central metabolism. HEK293 updated network is
a functionalized version of Recon2. The simplified model is com-
posed of 357 active reactions and 36 degrees-of-freedom. Results
and conclusions of these studies, together with findings of metabo-
lite profiling studies in the context of viral infection of HEK293 cells,
are presented in Table 1.
2.1.1. Glycolysis & glutaminolysis—Main carbon sources for
HEK293 cells
Most of the metabolomics analysis of HEK293 cells was directed
towards improvement of the energetic efficiency of the cells. Early
studies used ratios between concentrations of major carbon and
energy source and their metabolic by-products for the evaluation
of cell metabolic state. The ratio of glucose and lactate: YLact/Glc indi-
cates proportion of carbon channeled to lactate rather than entering
mitochondria for further ATP synthesize. For HEK293 cells, this
ratio varies between 1.0 and 2.35 [29,44–47]. Lactate can also be
produced from amino acids, through reverse TCA in mitochondria,
which explains how ratio over 2 can be obtained in some cases.
Similarly, the ratio between glutamine consumption and
ammonia production: YNH4/Gln represents the carbon utilized in
glutaminolysis. One molecule of glutamine, when entirely oxidized
into CO2 , produces 27 molecules of ATP while one molecule of glu-
cose can produce maximum of 30 ATP molecules. Either of these
initial molecules can provide precursors for various biosynthetic
pathways particularly in fast cell division, in hypoxic conditions or
in absence of nutrients [48]. These ratios are, however, only indica-
tive of the metabolic energetic efficiency and depend on the cell
growth phases or the feeding strategies [47].
Similarly to other continuous mammalian cell lines, HEK293
cells have a highly active glycolysis [44]. Increased aerobic glycoly-
sis is in part driven by over expression of both glucose transport and
enzymes in glycolytic pathway. Such metabolic pattern unbalances
central and energy metabolism and induces the accumulation of a
strategic metabolic intermediate, the pyruvate. Most of the pyru-
vate in these cells is not used for cellular energy generation through
the TriCarboxylic Acid (TCA) cycle but is converted to a metabolic
by-product, the lactic acid. This is due to (i) increased activity of
responsible enzyme lactate dehydrogenase, (ii) blocked entry into
mitochondria, and (iii) the increase in the cytosolic NADH/NAD+
ratio [49]. As the accumulation of lactate at high concentration
eventually becomes toxic for the cells it is closely monitored in
most cell culture productions.
Different MFA studies of HEK293 cells show that over 50% of
the carbon entering glycolysis goes to lactate production [46,50].
A batch culture of HEK293 cells supplemented with C13 labeled
glucose and glutamine was used by Henry et al. [46] to inves-
tigate carbon repartition in different pathways of HEK293 cell
central metabolism. They found that glucose was utilized mostly
for glycolysis, with smaller (15%) use in PPP for NADPH and ribose
production. Majority of pyruvate (95%) comes from glycolysis and
is converted to lactate in the NAD+ regeneration process (77% of
pyruvate). They also found that glutamine was the main amino acid
consumed by cells, contributing to about 16% of carbon entering
TCA cycle, but interestingly, not significantly to lactate production.
Comparison between the dynamic of glycolysis and glu-
taminolysis in infected and non-infected HEK293 cultures allowed
identification of specific metabolic fingerprints for different states.
In non-infected HEK293 cell cultures, glycolysis and glutaminolysis
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Fig. 1. Metabolites and metabolism can be described and analyzed through: (i) METABOLOMICS: selected subset of metabolites or high throughput unbiased analysis showing
snapshot of concentrations at measuring time. (ii) FLUXOMICS: metabolite kinetics in cells (cellular throughput) through flux analysis. Stoichiometric matrix can include
large number of reactions that can be used for optimization-based analysis such as flux balance analysis. Alternatively, stoichiometric matrix can be limited to a small number
of reactions particularly when applied to isotope-labeling studies showing only a subset of pathways. (iii) POLYOMICS: metabolite measurements can be complemented with
other omics data providing more detailed description of the systems at a given time point.

have shown significant flux decrease over cultivation time, espe-
cially when reaching high cell densities. This metabolic fingerprint
was observed for HEK293 cells by different authors under differ-
ent feeding conditions [50,51]. For an adenovirus infected HEK293
culture, intracellular fluxes are increased simultaneously with the
viral synthesis onset until reaching maximum after approximately
33 h post-infection [47,51]. Some authors observed a general aug-
mentation of the fluxes linking glycolysis to TCA cycle, indicating
a more efficient use of the carbon [51], whereas others indicate an
increase in the production of lactate and YLact/Glu ratio from 1.2 to
1.8 [50].
2.1.2. Energy metabolism: TCA cycle and nucleotide pools as
indicative of cell energy state
Very few publications present quantification of TCA cycle
carboxylic acids or nucleotide levels in cell-based production.
However, nucleotide pools could be important factors in cellular
production such as transport processes, macromolecular synthesis
and cell growth [52]. Ratios of nucleotides were used for production
monitoring and control, especially through AEC (Adenylate energy
charge) which represent the energy state of the cells. As an example,
Molinas et al. [52] used the NTP/U ratio (NTP: sum of purine and
pyrimidine nucleotides, U: UDP & UDP GNac) as indicator of cell
physiological state and aging showing that most of the time, NTP/U
ratios increase after 3–4 days of culture, indicating deteriorating
cell conditions.
The energetic state of the cells appears to have a strong impact
on their viral productivity. Nadeau et al. demonstrated that cell
productivity, i.e. number of viruses produced per cell, could be
improved 3 to 4-times by using low protein media. Low protein
media (using proprietary formulation NSFM13) induces more effi-
cient glucose utilization. Flux analysis have shown that in this case
glucose enters TCA cycle more efficiently leading to lower pro-
duction of lactate and higher ATP production (2.5-fold increase
in the TCA cycle flux and 2-fold increase in ATP production) [47].
Flux analysis shows that cells become more oxidative regardless
of the medium during the infection process with adenovirus. Cells
had more efficient glucose consumption, leading to enhanced TCA
fluxes and higher ATP production rate. MFA model of compart-
mentalized cell metabolism in low protein media have shown an
increased pyruvate entrance into the mitochondria and higher exit
of malate from the mitochondria suggesting impairment of malate-
aspartate shuttle in these cells [47].
2.1.3. Metabolic waste: Lactate and ammonia productions
Recently, a metabolic shift from lactate production to lactate
consumption during growth phase has been described for a num-
ber of cell lines (CHO, NS0 cells) [53,54] and oxygenated cancer
cells [55]. Utilization of lactate is primarily influenced by mitochon-
drial oxidative metabolism. Cells consuming lactate were shown to
have 6 times greater energy efficiency (ATP produced per substrate
consumed) than lactate producing cells. This metabolic behavior
seems to be linked to cell status as well as the concentrations of
glutamine and glucose in the medium [56]. HEK293 cells exploited
in viral production context have shown similar metabolic shift from
lactate production to lactate consumption [44,57–60].

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Table 1
Metabolomics, fluxomics and polyomics studies performed on HEK293 cell metabolism.

Method Model type Particularities of the study Goal of the study Reference

Fluxomics—MFA 34 fluxes model: catabolic Assumptions of pseudo-steady Identification of nutrients [51]

pathways of glucose,
glutamine, 17 amino
acids + TCA cycle
Fluxomics—MFA 43 fluxes model: catabolic
pathways of glucose,
glutamine, 17 amino
acids + TCA
cycle + pentose-phosphate
path-
way + Compartmentalization
between mitochondria
and cytosol
Fluxomics—MFA 30 fluxes model: central
carbon metabolism, amino
acid metabolism,
nonessential amino acids
biosynthesis
Fluxomics—MFA 16 fluxes model: catabolic
pathways of glucose
glutamine and 19 other AA
+compartmentalization of
pyruvate, malate and
AccoA + AA entering TCA
cycle grouped with AcCoA,
Oxaloacetate and malate
Metabolomics + Transcriptomic
+ Fluxomics (MFA) 342 fluxes model with 42
undetermined: 347
reactions and transporters
required for biosynthesis
and energy metabolism, 60
metabolic rates
Metabolomics + Transcriptomis Central carbon metabolic
pathways (carbohydrate
metabolism, energy
metabolism, amino acid
metabolism, pentose
phosphate
pathway) + glycosylation,
vitamin & co-factor,
polyamines, nucleotide,
gluthation/detox and lipid
metabolisms
Fluxomics—MFA Reduced Recon 2 Network:
357 active reactions and 36
degrees-of-freedom
state & constant cell volume
Some reactions are reversible
Assumptions of pseudo-steady
state and constant cell volume
Total ATP production
calculated from both ATP
producing and consuming
reactions
The general equation for
biomass included glucose and
all amino acids
Use of labeled glucose and
glutamine (1-, 2-, 6-U-13 C
glucose and U-13 C glutamine
traces supplemented to glucose
and glutamine-free medium)
Focus on exponential growth
phase under non-limiting
glucose and glutamine
conditions, a usual state for
cells producing virus
Recombinant protein
production. Model based on a
reduction of human genome
scale model. Model constrained
by Flux variability analysis
Combination of a metabolomic
and transcriptomic analyses
Model constrained by Flux
variability analysis. MFA
performed on growth phases
indicating steady-state
metabolic behavior. 8
reduction phases of original
Recon 2 model
that are used to fulfill cells’
energy needs leading to
better feeding strategy
under adenovirus infection
Determination of the effect [47]
of low protein medium on
the adenoviral vector
production. Analysis
included the development
of model of biochemical
reactions. Low-protein
medium resulted in up to 4
fold increase in adenoviral
vector production
Optimization of medium [50]
composition for higher cell
concentration and virus
production, to reduce
nutrient concentrations for
continuous cell growth and
to minimize production of
lactate and ammonia
Determination of the [46]
amounts of glucose carbon
through the Pentose
Phosphate (PPP) pathway,
incorporated into the Krebs
cycle for energy production
or lipid biosynthesis, or
used for cytosolic and
mitochondrial malic
enzyme fluxes
Comparison between a [29]
stable, recombinant
protein producing cells and
non-producer HEK293 cells
Comparison of a high- and [5]
low-enveloped virus
producer 293 FLEX cell
lines and producer 293
FLEX to non-producer
HEK293 cells
Identification of metabolic [28]
changes in HEK293 cells
following depletion of
l-alanyl-l-glutamine
dipeptide

Although ammonia can be produced by catabolism of any amino
acid, it is mostly derived from the deamination of glutamine. Addi-
tion of glutamine to culture medium results in drastic increase
(5-fold) of ammonia released by the cells [44]. High levels of
ammonia concentrations are known to induce number of toxic
effects such as inhibition of cell growth or enzyme reactions [61],
changes in intracellular pH [61], and induction of apoptosis [62].
2.1.4. Amino acid metabolism—The balance of cellular
metabolism
The majority of amino acids (AA) are consumed by HEK293
cells in serum-free medium, except for alanine, glycine, proline and
cysteine which are generally accumulated [63,64]. Similarly to
other cell lines, glutamine is the most consumed AA in HEK293 cell
culture, followed by serine, asparagine/aspartate, leucine, valine
and arginine [46,51,65]. AA consumption contributes to the genera-
tion of toxic by-products, especially lactic acid and ammonia. Pham
et al. demontrated that the ratio of YLact/Glc and YNH4/Gln increases
with increased consumption of amino acid and the addition of pep-
tones [63].
Nadeau et al. confirmed that AA actively participate to HEK293
cell energetic metabolism [51]. Flux analysis demonstrated that
cells can adapt to a low glutamine medium by increasing flux
of essential AA in the TCA cycle. Indeed, AA have several entry

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points in the TCA cycle via deamination or transamination reac-
tions [46]. Consequently, AA are often used by mammalian cells to
balance energy needs. Henry’s model of HEK293 cell metabolism
[46] also demonstrated that a majority of AA entering the TCA cycle
ultimately get used for lipid biosynthesis. In addition to AA, lipid
biosynthesis can be also driven by glucose [33,66].
Under adenovirus infection, Nadeau et al. also observed that
HEK293 cells favor AA other than glutamine as carbon sources [51].
Metabolic pattern of AA consumption and production depends on
culture media, culture feeding modes and also viral infection. As an
example, in the case of adenovirus production asparagine is essen-
tial for the formation of virions, and the highest flux observed for AA
consumption is for asparagine/aspartate [51]. On the contrary, for
influenza production, highest flux is observed for proline in batch
and serine and leucine in continuous feeding mode [64].
2.1.5. Integrated pathway analysis
Extensive gene expression analysis showed major differences
between a viral producing and non-producing HEK293 cell, par-
ticularly in the expression of metabolic genes [5]. Several major
pathways were recruited in the virus production including amino
acid and carbohydrate catabolism, energy, lipid, nucleotide and glu-
tathione metabolism, pentose phosphate pathway and polyamines
biosynthesis. Genes possibly limiting viral production include
enzymes from cholesterol and glutathione biosynthesis and the
regulatory system of polyamine biosynthesis. Genes involved in
lipid and cholesterol biosynthesis were down-regulated and lipid
uptake genes were up-regulated during virus production. Opti-
mization of culture medium following these results achieved 6-fold
increase in productivity in 293 FLEX cells. Particularly benefi-
cial was the manipulation of concentrations of nucleotides and
polyamines in the medium [5]. Dietmair et al. [29] presented an
analysis of changes in metabolite use during a recombinant pro-
tein production by HEK293. Combined analysis of gene expression,
metabolite concentrations and fluxes has shown for example the
complexity of metabolic regulation of phosphofructokinase (PFK),
one of the regulators of energy and fatty acid metabolism. Further
analyses of metabolites, genes and proteins would lead to more
information about the changes in producer cell lines and possible
routes for increased productivity and viability.
3. Feeding strategies and medium modification for viral
production improvements
Viral productivity of a cell depends, amongst other factors, on
the cell density at infection [58,67]. A “Cell density effect” (CDE)
defined as “a decrease in virus per cell productivity appearing con-
comitantly with the increase in cell concentration at infection” has
been observed in HEK293 and other cellular systems [68,69]. As
indicated in Table 2, CDE is generally observed for HEK293 cells
between 1 and 2 × 106 cells/ml [1,61,70], whereas cell densities of
8–10 × 106 cells/ml can be achieved in batch cultures [64]. CDE is
virus dependent with specific adenovirus production decreasing at
densities higher than 2 × 106 cell/ml [1,70,71], while for influenza
virus, high productivity is maintained up to 4 × 106 cells/ml in batch
cultures [58,64]. Consequently, most of the transfection/infection
steps are operated at cell concentrations below 4 × 106 cell/ml.
Cell metabolic activity, nutrient limitation or by-products accu-
mulation was explored as possibly responsible for the CDE in
viral production. Cell density has been shown to affect the energy
metabolism [72], activation of p53 pathway [73] and de novo syn-
thesis of sphingolipids [74]. Additionally viral productivity depends
on other factors, including cell substrate, cell cycle, culture time at
the infection, multiplicity of infection, culture medium and feeding
mode [61,75–77].
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5
3.1. Culture and production media
Medium composition can greatly influence viral productivity of
a cell line [78–80]. Additionally, an industrial application of cell
cultures requires use of serum-free media, further limiting viral
productivity. Optimal medium composition for cell growth, often
does not provide nutrients needed for viral particle production. For
example, Cervera et al. tested five commercial serum-free media
for HIV1-Gag Virus-like particle (VLP) production in HEK293 cells
[60]. The efficiency of VLP productions was highly dependent on
the medium composition with only two out of five supporting a
detectable production. On the other hand, Shen et al. have been
developing for several years an in-house medium that can sustain
both HEK293 cell growth and production of variety of viral vectors
with most success in production of adenoviruses [80,81].
Nevertheless, in the case of HEK293 cells cultivated in a batch
mode, none of the available commercial, or thus-far published in-
house media, can support high viral productivity at cell densities
higher than 2 × 106 cell/ml. Consequently, in order to overcome the
CDE and low viral production yields, alternative feeding strategies
are explored.
3.2. Feeding strategies
3.2.1. Continuous feeding
The best, albeit costly, strategy is the continuous feeding. By
continuously feeding cells with appropriate amount of critical sub-
strates, this approach reduces excess carbon used in standard
culture media (glucose at 4–6 g/l and glutamine at 2–4 mM). At
the same time potentially toxic metabolic waste (i.e. lactate and
ammonia) is removed thereby providing conditions similar to cell
environment in living organs. Continuous feeding approach has
been successfully applied to HEK293 suspension cell cultures for
production of different viruses providing 8.8-, 3.5- and 6-fold
increase, for adenovirus, influenza virus and lentivirus production,
respectively. Interestingly, for influenza produced in perfusion,
cells showed increased glycolytic activity before and after influenza
infection, consistent with the increase in glycolysis observed in
HEK293 upon adenovirus infection. One important aspect of con-
tinuous feeding is the concomitant removal of labile viral products
such as lentiviruses from the cell culture vessel. Hence, perfusion
is not only used for production at high cell density but also for
continuous harvest of viral product. Ansorge et al. reported sus-
tained lentivirus perfusion production with viral titers between
107 and 108 IVP/ml using feeding rates of 1–2 vol/day [82]. Sev-
eral authors showed the use of perfusion for retrovirus production
(reviewed in [83]). Consequently, continuous feeding appears to
be the best “metabolic” option for reaching high productive cell
concentrations.
3.2.2. Fed-batch feeding
The second approach explored for increasing cell density and
productivity is the batch supplementation with concentrated
medium or fed-batch method. Supplementation of critical nutri-
ents during the production maintains sufficient levels of critical
substrates and has been successful at reaching higher cell densities
than batch mode. Fed-batch strategies are often oriented towards
supplementation of depleted glucose and amino acids. Fed-batch
strategy requires development of specific supplements through
extensive screening of components and design of expriments to
reach significant improvements.
Nevertheless, feb-batch approach has provided some inter-
esting indications for improvement of HEK293 viral production
processes. First, although serum-free media are desired in viral pro-
duction, addition of foetal bovine serum (FBS) or calf serum (CS)
is a well-known strategy to increase viral yields [66,78,80,84]. It
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Table 2
Examples of viral production performed in HEK293 suspension cells. All these productions were performed in serum free culture conditions except* .

Viral production type Cell density at infection Feeding strategy Improvement rate References

Adenovirus 2 × 106 cell/ml Batch [1,70,71]

1 × 106 cell/ml
Adenovirus 8 × 106 cell/ml Perfusion: 0.2 and 1.2 vol/day 4.3-fold vs batch [57,71,89]
11.8 × 106 cell/ml 8.8-fold vs batch
Adenovirus 1 × 106 cell/ml Medium exchange [50]
Adenovirus 0.5 × 106 cell/ml Fed-Batch with medium 1.6-fold vs batch [78]
supplement: Cell boost 5 5.4-fold vs batch
Foetal bovine serum*
Influenza 4 × 106 cell/ml Batch [58,64]
Influenza 6 × 106 cell/ml Perfusion: 0.5 vol/day 3.5-fold vs batch [64]
Lentivirus 8 × 106 cell/ml Perfusion 2 vol/day +medium 26-fold vs batch + perfusion [82]
supplement Cell Boost 5 started at 48 h
post-transfection
HIV gag VLPs 2–3 × 106 cell/ml Medium exchange + Lipid 7-fold vs batch [79]
mixture supplement
Retroviral vectors 4.5 × 10 cell/ml
5
Fed-Batch with medium [90,91]
supplement: pluronic, lipid
mixture
Foetal bovine serum albumin*

is now evident that lipids usually contained in serum have to be
supplemented for successful viral production in serum-free media,
especially for production of enveloped viruses such as lentivirus
and retrovirus [85]. The addition of a lipid mixture, recombinant
insulin and transferrin in a medium exchange step prior to trans-
fection resulted in a 7-fold increase in virus-like particle [60].
Peptones or extracts from rice or yeasts were also extensively
tested. Peptone addition to serum-free culture medium directly
impacted the HEK293 cell metabolism with dramatic change in
the production and consumption of AA [63]. Addition of rice
hydrolysate allowed overcoming the CDE, improving an adenovi-
rus production yield by 3-fold [75]. In the same study, a pool of
different supplements (lipids and their precursors, bases, nucleo-
sides, vitamins, energy metabolites and rice hydrolysates) allowed
over 10-fold increase in viral production. These examples clearly
demonstrate the relationship between the CDE experienced in viral
production, cell feeding and metabolic state however without pro-
viding any insight into molecular mechanisms.
3.2.3. Medium substrate substitution
Substitution of carbon sources has also been evaluated in order
to reduce production of toxic by-products, primarily lactate and
ammonia. Reduced glutamine levels led to decreased ammonia
concentration (below 1 mM) reducing its toxicity [44,45]. Media
defined in the literature as non-ammoniagenic, have substitution of
glutamine by other AA (glutamate, asparagine), alanine–glutamine
dipeptide (Glutamax® ) or tricarboxylic acids (pyruvate, oxoglu-
tarate), all having either none, or lower, degradation to ammonia.
This strategy has been applied successfully to a number of differ-
ent cell lines (Vero cells and MDCK cells [86,87]) and for HEK293
cells [61]. Glutamate was evaluated for the production of adenovi-
rus [61], and such non-ammoniagenic medium strategy resulted
in a 5-fold increase in adenovirus productivity. Utilization of
glutamate-based medium maintains intracellular pH (pHi) at 7.3 for
10 days of culture, whereas classical glutamine containing medium
resulted in a decrease in pHi down to 6.2 after only 3 days of culture.
Ferreira et al. also demonstrated that adenovirus infection severely
affects pHi with a drop of 1 pH unit at minimum. This drop is more
pronounced when the cells are infected at cell densities higher than
1 × 106 cell/ml [88].
3.3. Cell engineering for metabolic efficiency
Various cell line engineering strategies have been developed,
largely trying to overcome problems of lactate and ammonia
accumulation. One of the major modifications was two decades
ago with the introduction of glutamine synthetase to CHO and
myeloma cells allowing their growth in glutamine-free media.
In HEK293 cells, cell line engineering focused on reducing the
lactic acid production. Elias et al. and Henry et al. described
the use of yeast pyruvate carboxylase overexpression to restore
the link between glycolysis and TCA cycle [57,59], reducing the
amount of produced lactate and ammonia (2-fold decrease for
both). This approach led to a 2-fold increase in cell density, and
a 33% increase in the target recombinant protein production [57].
Although these approaches have only been tested for protein
production, they might be beneficial for improvement in viral
production.
4. Conclusions
High cell density, high yield, large-scale viral vectors and vac-
cines production processes using HEK293 cell or other cell culture
platforms are hindered by following:
• Production of most of the viral vectors and replication compe-
tent viruses is a biphasic process with cell growth and a virus
production phase. These phases generally require different cul-
ture conditions, particularly in terms of nutrient needs; making
large-scale production optimization difficult.
• Production of different virus types is often associated with
specific metabolic demands which in turn require particular opti-
mization of the cell culturing conditions.
• High cell productivity needed for industrial cell-based applica-
tions can be achieved by either improving the virus per cell
productivity, or by increasing the total number of productive
cells. Increase of the number of productive cells by infecting at
high cell densities, i.e. later in the growth process, is impeded
by the “cell density effect”. Therefore, at this time viral produc-
tion processes are constrained to lower cell densities (approx.
1–4 × 106 cell/ml), even though HEK293 cells could grow at much
higher densities (approx. 20 × 106 cell/ml).
• The complexity of the cell culture media complicates understand-
ing of nutrient needs and the effects of media on viral productions.
Most of the media currently used have over a hundred compo-
nents, some at very low concentrations (below 1 mM), however
only a small subset of less than 30 compounds are routinely mon-
itored (e.g. glucose, amino acids, ammonia, some organic acids as
lactate, and some vitamins).

Please cite this article in press as: Petiot E, et al. Influence of HEK293 metabolism on the production of viral vectors and vaccine. Vaccine
(2015), http://dx.doi.org/10.1016/j.vaccine.2015.05.097
G Model
JVAC-16544; No. of Pages 8 ARTICLE IN PRESS
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All of these challenges are associated with maintaining, or
improving, the cell physiology during the production process,
which is directly linked to the cells’ metabolic state. Consequently,
better understanding of the metabolism is crucial for further
improvement in the production process. Further investigation and
optimization of metabolism will likely lead the development of
HEK293 suspension cell cultures and their establishment as a pow-
erful system for virus production.
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