Bioreactor Concepts For Cell Culture-Based Viral Vaccine Production

Review
Bioreactor concepts for cell

culture-based viral vaccine
production
Expert Rev. Vaccines Early online, 1–15 (2015)
Expert Review of Vaccines Downloaded from informahealthcare.com by Nyu Medical Center on 07/21/15
Lilı́ Esmeralda Vaccine manufacturing processes are designed to meet present and upcoming challenges
Gallo–Ramı́rez‡1, associated with a growing vaccine market and to include multi-use facilities offering a broad
Alexander Nikolay‡1, portfolio and faster reaction times in case of pandemics and emerging diseases. The final
products, from whole viruses to recombinant viral proteins, are very diverse, making standard
Yvonne Genzel*1 and
process strategies hardly universally applicable. Numerous factors such as cell substrate, virus
Udo Reichl1,2 strain or expression system, medium, cultivation system, cultivation method, and scale need
1
Max Planck Institute for Dynamics of consideration. Reviewing options for efficient and economical production of human vaccines,
Complex Technical Systems, Bioprocess
this paper discusses basic factors relevant for viral antigen production in mammalian cells,
Engineering, Magdeburg; Sandtorstr. 1,
39106 Magdeburg, Germany avian cells and insect cells. In addition, bioreactor concepts, including static systems,
2
Chair for Bioprocess Engineering, single-use systems, stirred tanks and packed-beds are addressed. On this basis, methods
Otto-von-Guericke-Universität towards process intensification, in particular operational strategies, the use of perfusion
For personal use only.
Magdeburg, Universitätsplatz 2,
39106 Magdeburg, Germany
systems for high product yields, and steps to establish continuous processes are introduced.
*Author for correspondence:
Tel.: +49 391 6110 257 KEYWORDS: animal cell culture . bioreactor systems . continuous processes . large-scale vaccine production
Fax: +49 391 6110 203 . process improvement . process intensification . viral vaccines
genzel@mpi-magdeburg.mpg.de
‡
Authors contributed equally Vaccination represents the most effective strategy in the late 1960s, continuous cell lines were
to prevent infectious diseases, and vaccine recognized as suitable hosts for human vaccine
manufacturing is crucial for worldwide disease production, but it was not until 1977 that the
control and eradication. Currently, > 50 cell first production process was licensed [5,6]. Cell
culture-based human viral vaccines are being culture-based vaccine production processes
manufactured (TABLE S1 [supplementary material enable simple infection and harvesting steps in
can be found online at www.informahealthcare. defined environments with closed bioreactor
com/suppl/10.1586/14760584.2015.1067144]), systems ensuring sterility, while further reduc-
and many more are under development. Vacci- ing biosafety risks by automation. Current
nation implies the administration of attenuated plant manufacturing capacity can be scaled up
or inactivated infectious agents (or their compo- to produce millions of vaccine doses, while
nents) delivering antigenic structures that stimu- maintaining cell cultures in controlled cultiva-
late the adaptive immune system in order to tion vessels.
elicit an effective response against specific patho- In this review, we discuss bioreactor con-
gens to prevent future infections. cepts and operational strategies for cell culture-
Since the early 1940s, viral vaccines have based processes focusing on viral vaccines for
been produced in embryonated chicken eggs human use. We first describe different viral
replicating a broad variety of viruses [1]. Cur- vaccine types currently produced, introduce
rently, the egg-based manufacturing method general concepts of cell culture-based viral
provides more than 30 licensed human vac- antigen production and discuss factors that
cines [2–4]. However, the production capacities greatly affect process design. Afterwards, we
of this platform are greatly limited by the address cell cultivation in bioreactors and dis-
availability of fertilized eggs. An alternative cuss operation modes that have enabled the
technology relying on animal cell culture was development and improvement of cell culture-
established in the 1950s using primary cells as based processes for vaccine production in labo-
substrate for virus production. Subsequently, ratory and industrial scales.
informahealthcare.com 10.1586/14760584.2015.1067144 2015 Informa UK Ltd ISSN 1476-0584 1

Review Gallo–Ramı́rez, Nikolay, Genzel & Reichl
Crucial aspect defining the process choice characteristics in cultivation vessels (virus spreading in cell pop-
The choices of the vaccine type, the cell substrate and the pro- ulations, as well as virus release) determining optimal cultiva-
duction process including bioreactor and operation mode are tion conditions, operation modes and harvest strategies. One
crucial for successful manufacturing of human vaccines. All example is the infection dynamics of Modified Vaccinia Ankara
these factors have a significant impact on vaccine quality, as (MVA) virus in continuous cell lines, which remains attached
well as on manufacturing capacity, production volumes, process to the cell membrane after budding and therefore requires
times and product costs. Among many factors that have to be direct cell-to-cell contact to spread infection to neighboring
defined, the following five aspects are of crucial importance. cells [13,14]. Recent adaptation attempts generated successfully a
new genotype of MVA virus that propagates in single sus-
Vaccine demand pended avian cell cultures, facilitating the manufacturing pro-
Vaccine demand varies according to the spread of a virus and cess [15,16]. Some viruses, like rotavirus, influenza virus or
its mutation rate, which may result in new circulating strains Sendai virus, require previous protease treatment to increase
not covered by the available vaccine. For instance, at the begin-
their infectivity in cell culture [17–20]; whereas others have spe-

ning of US immunization campaigns against seasonal influenza, cific demands regarding their host cells, such as polio virus,
up to 100 million vaccine doses are required to be produced in which only proliferates in primate-derived cells [21]; the Mink
a time period as short as 5–6 months [7,8], while influenza vac- enteritis virus (MEV), which only multiplies in mitotic cells [22]
cines in case of a pandemic ideally need to be available within or the influenza virus, which only binds to cells with receptors
a few weeks (which is not possible at the moment using estab- containing sialic acid residues [23].
lished manufacturing technologies). In contrast, vaccines
against, for example, polio or measles require a more or less Host cell characteristics & growth requirements
constant supply of the same vaccine strain for worldwide appli- Different cell substrates possess diverse characteristics that
cation and vaccines against dengue or yellow fever are only make them suitable for specific applications in vaccine pro-
needed in certain regions of the world. Therefore, vaccine duction (see cell substrates used for different vaccine types
demand is not only described by the number of doses pro- in TABLE 1 and in (TABLE S1) for approved vaccines). Therefore,
duced in campaigns, but also by the time window available for the right cell line and optimal process parameter conditions
manufacturing. (e.g., temperature, pH value, dissolved oxygen concentration,
medium composition, etc.) strongly affecting cell growth
Vaccine type and virus replication must be selected, to ensure high viral
Vaccine types are basically defined by the component eliciting yield. In the following section, cell substrates are divided
the immune response. Such antigenic components can be live- into two groups. First, human/higher animal cell substrates
attenuated viruses, inactivated viruses, virus subunits, viral vec- (human, avian and mammalian cell lines), which are mainly
tors or recombinant virus-like particles/proteins. In this work, used for whole virus replication; second, insect cell sub-
we address these five kinds of viral vaccines and summarize strates (lepidopteran and dipteran cell lines), which are
their important characteristics in TABLE 1. From this list, live- mainly used in recombinant antigen and virus-like particle
attenuated vaccines are usually the most immunogenic and thus (VLP) production.
require the lowest concentrations of immunogens per dose [9].
Nevertheless, viruses with a high mutation rate are unsuitable Human & higher animal cell lines
candidates for such a vaccine type as reversion may occur dur- Manufacturing of many cell culture-based human vaccines
ing vaccine production. Another drawback is the potentially employs primary culture of 10- 11-day-old chicken embryo
reduced replication of attenuated virus strains in cell culture, fibroblasts [2,3,24]. Hence, the production capacity depends
which would lower process yields. In contrast, wildtype viruses entirely on the supply of embryonated eggs, which may be
employed for manufacturing of inactivated vaccines typically endangered by outbreaks related to avian pathogens. Primary
lead to higher virus yields. However, handling highly patho- monkey cells (from Chlorocebus aethiops) are also commonly
genic wildtype live viruses may require biosafety level 3 condi- employed for manufacturing of human vaccines. However, in
tions [10,11], and this complicates production significantly. addition to bioethical implications, donor animals may host
Recombinant vaccines allow circumventing these safety require- potential pathogens to humans and must be strictly monitored,
ments, but are often less immunogenic and therefore require increasing process complexity [6].
higher antigen concentrations per dose. (Additional information Continuous cell lines circumvent the previously mentioned
about immune response triggered by different types of vaccines drawbacks related to the use of primary cells and are therefore
can be found in [12]). the preferred substrates in current cell-based manufacturing
processes (TABLE S1) [2–4]. More recent approaches regarding con-
Virus/antigen requirements for growth/expression tinuous cell line development aim on directed generation of
Manufacturing of viral vaccines typically involves the supply of cell lines (so called “designer cell lines”) by specific mutations
eggs or cultivation of animal/insect cells with subsequent infec- to increase cell specific virus yields [25]. Most commercial
tion. The latter step is subject to virus specific replication virus production processes, however, still rely on the Vero,
doi: 10.1586/14760584.2015.1067144 Expert Rev. Vaccines

Bioreactor concepts for cell culture-based viral vaccine production Review
Table 1. Overview of different cell-based viral vaccine types.

Type of Principle Immunogenicity Comments Production
vaccine level platform
Live-attenuated Attenuation during multiple Very high Risks of reversion Mammalian
vaccine passages or under non-physiological Immunocompromised patients may and avian cells
conditions. Whole virus replicates at develop infection
low level in vaccinated patients Often requires cold chain storage
Details of attenuation often unknown
Inactivated Whole live virus is chemically High Risk of incomplete inactivation Mammalian
vaccine inactivated Highly pathogenic live viruses require and avian cells
increased biosafety
Often requires booster/multiple doses
Inactivation may have a negative

impact on antigenic structures
Recombinant Attenuated or recombinant viral High Low biosafety risk as antigen is Mammalian,
vector vaccine vectors express viral antigens in the expressed by non-replicating vectors avian, and
vaccinated person Vector antigens can boost immune insect cells
Chimeric vectors display response
recombinant viral epitopes on their First human vaccines in clinical trials
surface
Recombinant Recombinant expression of viral Medium No infection risk for patients due to Mostly insect
subunit vaccine genes to produce viral proteins or absence of viral genome cells and CHO
virus-like particles Absence of some viral elements may
reduce immunogenicity
Split/subunit Whole live viruses are disrupted by Low No infection risk due to virus split Mammalian
vaccine detergents (split vaccines) and Highly pathogenic live viruses require and avian cells
further purified (subunit vaccines) increased biosafety
Disruption of viruses may negatively
affect the structure of antigens
MDCK, MRC-5 and WI-38 continuous cell lines, which Insect cells
maintain an anchorage-dependent growth. In addition, the The use of insect cell cultures for human recombinant vaccine
human cell lines HEK293 and PER.C6, as well as the avian manufacturing is an upcoming strategy, and so far Cervarix
cell lines AGE1.CR and EB66, are employed at research or (vaccine against certain types of cancer-causing human papillo-
clinical scale for vaccine candidate development [26–31]. They maviruses) and Flublok (influenza vaccine) have been approved
have been successfully adapted to grow in suspension, facilitat- for human use (see TABLE S1). Insect cells can be easily cultivated
ing cultivation of cells and scale-up in vaccine produc- in suspension cultures using serum-free media, representing an
tion [6,26–28,32–34]. Most human and higher animal cells also attractive substrate fulfilling current criteria for vaccine develop-
still require complex media, frequently enriched with fetal calf ment [44–46]. In order to produce vaccines using lepidopteran
serum, for optimum growth and high virus yields. This cell lines (such as Sf9, Sf21, Sf+ and H5), cells are infected
increases not only production costs and batch-to-batch varia- with engineered baculoviruses carrying the heterologous genes
tions, but also involves the risk of introducing adventitious of the desired antigens. In the insect cell-baculovirus expression
agents. Media development enabled the cultivation of system, baculoviruses work as viral vectors for recombinant pro-
HEK293, MDCK and Vero cells in serum-free media achiev- tein expression, while they replicate inside infected insect cells.
ing comparable yields of human influenza virus, equine influ- As a consequence, the formation of new baculovirus particles as
enza virus and rabies virus, respectively, to those obtained in by-product represents an important concern in downstream
serum supplemented media [26,35,36]. Comparing different cell processing of VLP vaccines. In this regard, a vector incapable
lines under this aspect, human PER.C6 cells grown in serum- producing new baculovirus particles has also been developed [47].
free media led to higher cell-specific yields of polio virus Remarkably, this drawback has turned into new applications, as
(types 1, 2 and 3) than Vero cells grown in serum-containing baculoviruses do not represent a risk for human health, and the
medium [28]. Accordingly, an increasing number of cell-based use of such viruses as vector-based vaccines and in gene therapy
vaccine candidates (e.g., against yellow fever, polio, human has also been proved as safe [48–50]. Recombinant vaccine pro-
and avian influenza, dengue and respiratory syncytial virus duction in the dipteran cell line Schneider’s Drosophila mela-
(RSV)) are produced in continuous cell lines [37–43]. nogaster (S2) does not involve viral infection, but the
informahealthcare.com doi: 10.1586/14760584.2015.1067144

Table 2. Comparison of single and multiple harvest strategies applied controlled processes, to improve robust-
in viral vaccine production. ness and to ensure product quality; the
option to employ advanced cultivation
Single harvest Multiple harvests
methods, to maximize process yields by
Virus replication in primary cells (e.g., rabies and Slow lytic viruses (e.g., MVA) controlling cell growth, virus replication
poliovirus) Non-lytic viruses or protein expression through optimized
Virus infection at maximum cell concentration Virus infection at cell inoculation (e.g.,
operating parameters; the possibility to
(e.g., MVA, influenza virus) MEV)
Viruses with a high mutation rate (RNA viruses) Low yield viruses (e.g., live-attenuated increase the production capacity accord-
Viruses that accumulate DIPs at high rates (e.g., viruses, HCV) ing to market demands or requirements
influenza virus, baculovirus) Virus-free systems (e.g., stably for clinical trials and, finally, the poten-
Unstable viruses (e.g., infectious particles for live modified S2 or H5 insect cells) tial to reduce process costs and facility
attenuated vaccines) space. In the next sections, we will dis-
DIPs: Defective interfering particles; HCV: Hepatitis C virus; MEV: Mink enteritis virus; MVA: Modified vaccinia cuss the most common options for vac-
Ankara virus.
cine production in bioreactors and
operation modes, to achieve these goals.
establishment of stable producer cells by cell engineering. Based
on this technology, stably modified H5 cells have recently been Vaccine production in static systems
developed for Japanese encephalitis vaccine production [51]. Over the past few decades, a variety of large-scale devices for
An important drawback of insect cells is the difficultly to static cultivation of adherent cell lines have been
express membrane proteins and secreted glycoproteins at appro- developed (TABLE 3). Most of the cultivation systems applied to
priate levels, which are both relevant antigens for vaccine devel- vaccine production offer restricted inoculation and harvesting
opment (reviewed in [52]). Additionally, insect cells cannot options, with limited monitoring of pH and oxygen and lim-
synthesize the complex glycan structures typical for mammalian ited control of cultivations parameters, such as temperature and
cells. Despite this limitation, several glycosylated vaccine candi- sometimes feeding rates. However, due to the simplicity and
dates produced in insect cells such as those against Chikungu- robustness of such systems some vaccine manufacturing pro-
nya virus, influenza virus, RSV, enterovirus 71 (EV71) and cesses still rely on these cultivation systems (e.g., the human
dengue virus have elicited an antibody response or protection varicella vaccine Varivax produced in MRC-5 cells (Merck)
during virus challenge in animal model systems [53–61]. To date, and the influenza vaccines Celvapan and Vepacel produced in
production processes of human vaccine candidates against the Vero cells (Baxter) [72]). Accordingly, a large amount of exper-
above-mentioned wildtype viruses as well as malaria, avian tise in handling such systems and optimizing corresponding
influenza, measles and Ebola viruses are under development production processes has been accumulated over the years. In
[45,46,58,62–70]. several cases, standardization of processes and modularization
of unit operations has resulted in highly competitive products
Virus/antigen stability regarding costs per vaccine dose, for example for influenza vac-
Single or multiple harvest strategies (TABLE 2) are applied to avoid cines for human use. Such systems (TABLE 3) have also found
virus/antigen degradation in upstream processing, which is their application in the generation of cell seeds for large-scale
mainly caused by the release of cellular proteases after cell lysis production in microcarrier systems or the generation of virus
and low thermostabilities. A simple and quick method adjusted seeds to infect bioreactors. In the following, general properties
to the product properties is therefore crucial. One example is of static systems comprising roller bottles and enlarged multi-
given by hollow-fiber-based perfusion systems enabling selective layer systems including automated solutions will be briefly
separation and further processing of the product-containing discussed.
medium. Operation modes retaining only cells or even viruses
while removing proteases with spent medium are available. If Roller bottles
the virus/antigen is produced at almost constant rates (mostly Uncontrolled roller bottles (RBs) are commonly employed at
for slow/non-lytic viruses and virus-free systems), multiple or low scale or at pre-culture steps to inoculate small bioreactors
continuous harvesting steps can improve the process. After har- with microcarriers. Handling large numbers of RBs involves
vesting, further clarification steps are commonly performed not only a lot of manual (or robotic) work and carries a rela-
batchwise by filtration or by the use of other separation tively high sterility risk, when performing medium exchange,
methods [71]. washing steps, cell harvest and infection, but also requires the
Once the production process has been defined based on the use of dedicated clean rooms and a large capacity of incubators.
abovementioned aspects, the appropriate cultivation systems Therefore, the scale-up of adherent cells based on RBs may
can be chosen. Undoubtedly, the most important advantages of become cost and labor intensive and is then an issue to be con-
vaccine manufacturing in cell culture are those linked directly sidered. In this regard, fully automatized solutions are available
to the use of bioreactors: the use of closed systems including (for instance Cellmate from Tap Biosystems [73] or RollerCell
harvest vessels and pipework; the possibility to establish fully 40 from Synthecon [74]), which allow handling of large

Table 3. Comparison of static cultivation systems in different scales, which are available for cell
culture-derived vaccine production.
Vessel Supplier Area Average cell Working Cells/Volume Control Perfusion
(cm2)† yield† volume (L)† (normalized options option
to T175)‡
T175§ e.g., 175 1.8 107 0.05 1.0 Off-line No
Corning
Roller e.g., 850 8.5 107 0.26 0.9 Off-line No
bottle§ Corning 1750 1.8 108 0.53 0.9
CellSTACK§ Corning 636 6.4 107 0.2 0.9 Off-line No
25,440 2.5 109 7.6 0.9
HYPERflask§ Corning 1720 1.7 108 0.6 0.8 Off-line No

§
HYPERStack Corning 6000 4.0 10 9
1.2 9.3 Off-line No
60,000 4.0 1010 12.0 9.3
Cell Factory¶ Nunc 632 0.2 Off-line No
25,280 8.0
CellCube§ Corning 8500 8.5 108 0.6 3.9 Off-line Yes
85,000 8.5 109 6.0 3.9
BelloCell# CESCO 15,600 5.5 109 0.5 30.6 Off-line Alternate
BioProducts submerging
TideCell# CESCO 320,000 7.0 1010 2 97.2 Off- and Yes
BioProducts 16,000,000 3.5 1012 100 97.2 online control

CelliGen Eppendorf 30,000 2.5 1010 5.0 13.9 Off- and Yes
BLU†† 600,000 5.0 1011 50.0 27.8 online control
iCellis‡‡ Pall 5300 1.5 109 1.0 4.2 Off- and Yes
5,000,000 1.5 1012 70.0 59.5 online control
†
If two values are given, they present the minimum and maximum areas available, together with respective cell yields and working volumes. Within this range, other
areas are equally available.
‡
(Cell yield(vessel x)/working volume(vessel x))/ (Cell yield(T175)/working volume(T175)); the higher the value, the better the volume specific cell yield.
Grey rows: packed-bed reactors.
§
Data given by Corning [76].
¶
Handling of Cell Factory System requires special incubator. Automated systems for seeding, harvesting, and cell detachment are available. Data given by Nunc [77].
#
Calculated from data given by CESCO BioProducts [98].
††
Calculated from data given by Eppendorf; bioreactor pre-loaded with FibraCel microcarriers [99].
‡‡
Data given by Pall [100].
numbers of RBs under sterile conditions, while also reducing hepatitis A virus production [78]. One extensible CellCube unit
the risk of operator handling errors. Using this technology, replaces 50 RBs (1750 cm2) and additionally offers medium
contract manufacturers already offer automated handling of up recirculation and medium exchange for better aeration and sta-
to 1000 RBs per batch (IDT Biologika) [75]. Despite the above- ble pH values. The increased process control by small addi-
mentioned drawbacks, RBs remain a practical and low-cost tional expenditure has clear advantages over other more
option for cell culture at laboratory scale and for large-scale conventional static systems, such as RBs. The CellSTACK
manufacturing of products licensed many years ago or live- (6260 cm2 and 1 l total medium) consists of multilayer
attenuated vaccines with low or local market demand. T-flasks and has been demonstrated at pilot scale for the pro-
duction of HIV pseudovirions via transient transfection of
Multilayer cultivation systems HEK293T cells [79]. One module can be easily expanded by
Multilayer cultivation systems with increased surface-areas show the attachment of three further stacks, which can be handled
a better footprint and easier handling in large-scale production simultaneously. Another alternative is given by Cell Factories
compared to RBs (listed in TABLE 3) [76,77]. Stacked devices are, (25,280 cm2), which has shown a 23-fold yield increase of
for example, the CellCube (Corning), the CellSTACK (Corn- bovine RSV vaccine in the bovine cell line NM57 in compari-
ing) and the Cell Factory (Thermo Scientific), all reducing son to RBs with comparable total surface area [80].
incubator space and the need of manual handling. The large Due to the high cells/volume ratios (TABLE 3), fewer multilayer
surface solution CellCubes (85,000 cm2 and 7.3 l total systems and less working volumes are required for large-scale
medium per batch) was used to cultivate MRC-5 cells for production processes, replacing a large amount of RBs.

However, handling of numerous multilayer systems running in It requires low instrumental and operational intervention and is
parallel, with working volumes of up to 8 l per unit, clearly therefore typically adopted for its ease of implementation and
requires automated handling. This can be provided for Cell process robustness [84]. Its wide adaptation in industry mainly
Factories (Nunc, Thermo Scientific) encompassing automatic derives from relatively good virus yield coefficients and high
filling, emptying and even shaking for cell detachment [81], but nutrient consumption, reducing growth media waste, the major
the investment significantly increases procurement costs. In cost-driving component. However, due to increasing vaccine
comparison to bioreactors, multilayer systems require a lower demands and reduced production costs, process intensification
operator skill level and lower investment costs, so that their through fed-batch or perfusion systems aims on higher cell
implementation at large scale may still constitute an affordable concentrations and increased volumetric virus yields. Less biore-
and competitive option for manufacturers with reduced facility actor runs for certain products give the manufacturer the
complexity. opportunity to extend their vaccine portfolio. The general strat-
egy is the full exploitation of growth media by reducing nutri-
Vaccine production in bioreactors ent limitations during cell growth and virus replication, to
Bioreactors either as glass vessels, stainless steel tanks or single maintain or even increase cell-specific productivities over
use systems have clear advantages over handling static culture extended time periods. In the following section, options for
vessels with limited control possibilities. Many options are process intensification in bioreactors are described for adherent
available to adjust process parameters for improved cell growth and suspension cells.
and virus production and to reduce operational costs as well as
manufacturing time, all critical goals in vaccine manufacturing. Adherent cells on microcarriers
The principal advantage of bioreactors and the reason for their A key step toward large-scale production of vaccines was the
successful implementation in vaccine manufacturing processes is development of microcarrier processes, mainly driven by van
the unsurpassed scale-up advantage. Furthermore, the use of Wezel [85]. Today, adherent cells like Vero, MRC-5 and
controlled bioreactors enables scale-down approaches of estab- WI-38 are grown routinely on microcarriers in quasi-
lished processes, to perform broad optimization studies and the suspension conditions maintained in standard bioreactors with
subsequent implementation of identified parameters at indus- low agitation speed or in wave bioreactors with appropriate
trial scales. The preservation of determined dimensionless quan- rocking-motion [82,86–88]. Microcarrier materials are typically
tities was successfully performed for an inactivated polio porous (macroporous) or non-porous beads made of glass, plas-
vaccine process during the reduction of the production volume tic or dextran. More recently, improvements in virus yield
from 750 to 2.5 l, allowing comparable cell growth and virus compared to conventional static systems have been achieved.
yields at both scales [82]. Another scale-down approach was One example is the propagation of Mink enteritis virus (MEV)
applied to an established manufacturing process for live- in mitotic adherent embryonic feline lung fibroblasts (E-FL)
attenuated polio vaccine with adherent Vero cells on Cytodex-1 cultivated on Cytodex-1 microcarriers. The investigated process
microcarriers [39]. At laboratory scale, poliovirus production was was performed in a wave bioreactor (10 l working volume),
evaluated under different culture temperatures, optimal parame- which replaced a total of 600 RBs (1750 cm2) due to its higher
ters were determined and, based on these laboratory-scale productivity [22]. Another example is given by an optimized
results, the process was subsequently successfully scaled up to rabies virus production process in adherent Vero cells growing
350 l. Such optimization resulted in a modified process applied at Cytodex-1 microcarrier concentrations up to 25 g/L in a
to a new inactivated polio vaccine candidate, which is currently STR (30 l working volume) in perfusion mode. Using a
being evaluated in clinical trials. decanting column and a shear-reduced cell lift impeller, this
Today, a variety of stirred-tank bioreactors (STRs) with well- system allowed for production of 1 million vaccine doses annu-
characterized hydrodynamic properties are available for cell cul- ally under GMP conditions [89]. Examples of recently devel-
ture, enabling seamless transfer of cultivation processes onto oped large-scale application can be found for vaccines against
the several thousand liter scale. In order to maintain most suit- poliomyelitis [39,90] and influenza (H5N1) diseases [91].
able cultivation conditions over such scale, new equipment is Microcarrier cultivations allow the easy separation of media
continuously developed to keep shear forces in large-scale bio- from cells during the process, by reducing agitation speed to
reactors low, while still enabling high oxygen transfer rates [83]. favor bead sedimentation. This facilitates the establishment of
In the following, different bioreactor concepts and operation advanced process operations to improve cell growth and to
modes in cell culture-based vaccine production are discussed. increase virus yields. For instance, media exchange and nutrient
feeding strategies were used to increase concentration of Vero
Vaccine production in batch mode & process cells grown on Cytodex-1 microcarriers by 80%, while polio
intensification options virus type 1, 2 and 3 D-antigen yields were improved by 100,
Bioreactors in cell culture-derived vaccine production are 64 and 76%, respectively, in comparison to batch cultures [90].
mainly operated in discontinuous batch cultivation mode. This Alternatively, volume reduction before virus infection can result
operation mode constitutes the simplest method to grow adher- in an increased virus–cell contact supporting virus adsorption
ent and suspension cell lines, to manufacture a desired product. and therefore help to optimize virus yields [92,93]. Another

approach comprises a cell dilution step during virus production facilitating inoculum preparations. Obviously, high cell concen-
(a so-called volume expanded fed-batch) to increase virus titers, trations require intensive bioreactor-volume exchanges to guar-
as described for Parapoxvirus ovis production by factor 40 in antee sufficient nutrient supply of cells and to remove waste
bovine kidney cells growing on Cytodex-3 microcarriers [94]. metabolites such as lactate. While porous surfaces, packing den-
A general drawback of microcarrier-based processes, as men- sity and bioreactor volume exchange rate are rarely limiting fac-
tioned earlier, is the need for high cell numbers to inoculate tors for achieving maximum cell concentrations in such
vessels with microcarrier concentrations at increasing volumes. systems, mass transfer rates into macropores, that is, the volu-
In addition, the use of serum-free (SF) media often results in metric oxygen transfer coefficient (kLa), often limit cell
poor cell attachment [95], so recombinant adhesion factors are growth [101]. Nevertheless, the iCELLis system mentioned above
usually supplemented to facilitate cell binding. Another con- enabled confluent cell growth on a surface of about 133 m2
straint of bead-based cultivations can emerge, when the recov- replacing 760 RBs (each 1750 cm2) or one 85 l bioreactor
ery of intracellular viruses requires cellular disruption or when with 3 g/L Cytodex-1 microcarriers with respect to similar
macroporous carriers entrap cells and make them difficult to growth surfaces [102].
access [96]. In addition, microcarriers are comparatively expen- Macroporous supports have also been used to demonstrate
sive and typically not recycled after use. Nevertheless, new their potential use in viral vaccine manufacturing. In
microcarriers are still introduced to the market. For instance, a laboratory-scale experiments, adherent MRC-5 cells grown in
new low-cost microcarrier material made out of plant-derived an iCELLis Nano packed-bed (850 cm2, 200 ml fixed-bed,
polysaccharides enabled to achieve rabies virus titers in Vero 1000 ml medium reservoir) produced hepatitis A and Chikun-
cells similar to those obtained in Cytodex-1 microcarrier cul- gunya viruses at yields that were nearly twofold higher than in
ture in a stirred bioreactor, with a sixfold lower cost [97]. As RBs, while media consumption was partially reduced [103]. Fur-
discussed previously however, the main drawback of microcar- ther, Vero cells were cultivated on Fibra-Cel discs in perfusion
rier cultures remains the difficulty to scale-up processes. These mode performing multiple harvests to produce vaccines against
are, in particular, technical challenges involving harvest of con- rabies. Higher cell concentrations as well as virus titers were
fluent cells and efficient bead-to-bead transfer. Few cells like an obtained compared to 500 mL spinner cultures using Cytodex-
adherent bovine kidney cell (BK KL3A) allow direct cell expan- 1 microcarriers [104]. Additionally, other packed-bed systems
sion, where cells attach to freshly added microcarriers in the with newly developed micro- or macropores have become avail-
next process scale [94]. In most cases, more sophisticated proc- able, such as the BioNOC II polyester carriers from Cesco
essing steps are required to avoid cell damage during trypsiniza- (e.g., case studies on EV71 and rabies virus produced in Vero
tion and to obtain high plating efficiency at the next passage. cells) or the AmProtein Current Perfusion Bioreactor with
Another aspect to consider is the intensification of polymer fiber paper carriers for influenza virus production [105].
microcarrier-based processes. Process intensification based on Besides the biological compatibility to maintain cell cultures
higher microcarrier concentrations to increase surface areas and virus replication, disposable bed bioreactors deliver pre-
requires higher cell numbers for inoculation which, in turn, validated and pre-characterized cultivation vessels with easy and
results in higher demands of culture seeds in the process train. flexible handling, saving hands-on time. Multiple packed-bed
In addition, a higher power input may be required to keep bioreactors allow on-line monitoring of culture parameters such
microcarriers in suspension, which increases the shear stress. as pH, dissolved oxygen, temperature and, more importantly,
Furthermore, microcarrier concentrations cannot be arbitrarily cell growth or progress of infection through permittivity sensors
increased as friction between beads can lead to cell for viable cell concentration determination [106]. However, diffi-
abrasion [92]. culties related to the harvest of viable cells from rough surfaces
However, certain viruses relevant for vaccine production can and porous matrixes exclude packed-bed reactors from seed
only be produced at acceptable yields in adherent cell lines and train purposes [107]. Overall, while cultivation of adherent cells
quasi-suspension culture on microcarriers remains as a viable is broadly established and often a must for achieving product
cultivation option for vaccine manufacturing. yields required for economic production of vaccines, the devel-
opment of suspension cell lines with high cell-specific virus
Adherent cells in packed-bed bioreactor production would be preferred for most large-scale
An alternative system to microcarrier suspension cultures is the applications.
use of disposable fixed-bed systems or packed-bed systems,
which protect adherent cells against mechanical stress. Such cul- Batch cultivation of suspension cells
tivation vessels typically rely on highly porous polyester micro- Conventionally, suspension cell lines are generated through an
fiber carriers or discs delivering very large surface adaptation process, where adherent cells lose their anchorage
matrices (TABLE 3) [98–100]. For example, the iCELLis 1000 dependence by occasional mutation, so that the new cell lineage
provides surface areas of up to 1000 m2 in a 25 l fixed-bed starts to proliferate freely in medium. At small volumes of up
volume perfused with 70 l medium stored in an additional ves- to 500 ml, suspension cells are usually cultivated in shake
sel. As cells grow and expand on such matrices, very high cell flasks, whereas wave bioreactors or STRs are handy options for
concentrations can be reached from low initial cell numbers, large scale. The main advantage of suspension cultures is their

easy expansion by simple volume increase, enabling the full concentrations increased threefold resulting in higher volumet-
exploitation of bioreactor capacities of up to 20,000 l, as dem- ric yields of the recombinant dengue NS1 protein [114]. Similar
onstrated for influenza vaccine production with PER. improvement was shown with a 30% increased recombinant
C6 cells [108]. Furthermore, like quasi-suspension culture with expression yield of the reticulocyte binding protein PfRh5 as
microcarriers, suspension cultures are amenable for process antigen for malaria vaccine in S2 cells [64], and 2.3-fold increase
automation as well as for easy regulation and control of opti- in volumetric yields of recombinant influenza vaccines pro-
mized conditions, resulting in being the current choice for duced in Sf+ cells [115]. Second, perfusion systems can be used
most large-scale biomanufacturing processes [109]. feeding fresh medium, while withdrawing spent medium to
Despite the adaptation to growth in suspension culture, cells achieve higher cell concentrations and extended run times
can keep their susceptibility and permissiveness to allow effi- beyond typical upper limits of batch and fed-batch processes.
cient virus replication. This was demonstrated for a relevant Newer perfusion systems are based on external hollow-fiber
large-scale MDCK cell line adapted to suspension that yielded modules with certain molecular weight cut-offs, retaining sus-
similar cell-specific influenza virus titers in comparison to the pension cells, and optionally viruses, in the cultivation vessel.
parental adherent cell line [6]. Infection studies with influenza These cell separation devices mainly work in cross flow mode
A/PR/8/34 H1N1 in a 1 l bioreactor showed that the viruses with either constant, pulsed or alternating flow directions
spread efficiently over the whole cell population, even at a very avoiding membrane fouling and promoting disaggregation of
low multiplicity of infection of 10–5 (based on TCID50). The cell clumps due to increased shear forces. The exchange of
first approved cell culture-based influenza vaccines, Optaflu medium prevents the depletion of nutrients and removes inhib-
and Flucelvax (Novartis), produced in a proprietary MDCK itory metabolites or proteins, while cells concentrate in the cul-
suspension cells, are based on such findings. Both vaccines tivation broth. This may help to avoid reduced cell-specific
could show similar efficiency and safety in comparison to tradi- virus yields observed in batch processes at high cell concentra-
tional egg-based vaccines [32,110]. tions for which the mechanism is not yet completely under-
Cultivation of EB66 and AGE1.CR.pIX avian cells in 200 l stood (so-called “high cell density effect”) [116]. Overcoming
disposable stirred tanks to produce MVA vector-based vaccine this issue with perfusion systems, increasing volumetric yields
candidates against tuberculosis and Ebola, respectively, are lead to reduced working volumes by multiple factors of up to
recent examples of the input of suspension cultures in current 100, without decreasing product quantity [117]. While current
process development [14,31]. Cell adaptation to suspension cell concentrations of batch processes in commercial vaccine
growth is not every time as straightforward and successful as production range between 2 and 20 106 cells/ml, perfusion
previously described. More recent studies have indicated that systems target up to 8 107 cells/ml with run times of
during adaptation, certain cell lines may also change their 90 days and longer [118]. Record-breaking appears recent pub-
expression of surface receptors, which then affects their suscep- lished PER.C6 cell concentrations of 3.6 108 cells/ml in
tibility to viral infection [38]. Moreover, not all human, avian alternating tangential flow perfusion systems (ATF, Refine)
or animal cell lines can be adapted to robust growth in suspen- within 14 days [119]. This benefit gives the option to operate a
sion retaining permissiveness to viral infection [107]. In this single perfusion bioreactor as constant seed reactor for multiple
regard, insect cell cultures constitute a robust platform and are batch processes in parallel or even time-shifted (hybrid process-
easily maintainable as suspension cells in conventional bioreac- ing) [120]. Through this implementation, whole manufacturing
tors producing a broad range of recombinant vaccines chains can be shortened, as already demonstrated for antibody
(reviewed in [111,112]). This was demonstrated for the approved expression processes employing CHO cells. The direct inocula-
recombinant influenza vaccine Flublok (Protein Sciences Cor- tion from highly concentrated perfusion bioreactors into
poration) produced in SF+ insect cells at 2500 l working vol- production vessels reduced working volumes and numbers of
ume. Due to the high scalability and efficient HA expression at pre-culture steps saving several days [121]. This strategy can be
adequate volumetric yields (detail unknown), large-scale readily applied in processes for vaccine production. In addition,
production has been recently projected to 15,000 l, without continuous media exchanges enabling stable and high cell con-
anticipated compromises in final yields or product quality [113]. centrations over an extended time period can be highly advan-
tageous for recombinant vaccine production in virus-free
Process intensification in suspension cultures systems, such as those based on S2 and H5 stably modified
There are several strategies to optimize and modify batch culti- insect cell lines.
vations maintaining suspension cells in laboratory scale. In comparison to batch or fed-batch systems, prolonged cul-
Although new technologies are intensively investigated, most tivation times may involve relatively elaborate and time-
intensification processes could not yet find broad application in consuming licensing procedures, for example, demonstration of
commercial production. The simplest strategy for process opti- long-term passage stability, definition of a new lot and batch
mization is, first, the fed-batch mode proving a simple feed system. However, some companies like Crucell Holland con-
strategy to improve cell growth, cell viability and life time of cluded that these difficulties are well worth the effort and
cells resulting in higher virus yields. This was successfully implemented high cell density cultivation processes for inacti-
employed in insect cell cultures where, for instance, Sf9 cell vated polio production using PER.C6 suspension cells [41,122,123].

In this set-up, applying ATF modules to 500 l stirred bioreac- possible virus vaccine generation in continuous processes from
tors allowed achieving similar cell numbers to those reachable a biological point of view and the possibility to reach a steady
in 10,000 l non-perfused bioreactors, representing a 20-fold state in any infected culture. The effect on virus antigenicity or
decrease of production volumes for vaccine capacities [41]. virulence due to mutation accumulation during extended peri-
Laboratory-scale experiments gave further examples for its suc- ods of viral replication remains as concern (reviewed in [129]).
cessful process intensification of AGE1.CR and human CAP Technical challenges in process design and operation also lead
cells infected with various influenza A virus strains that reached to difficulties in scalability, process robustness and process vali-
similar cell-specific virus yields in comparison to batch pro- dation. In addition, regulatory acceptance is not clear, as autho-
cesses [124,125]. Of particular interest is the constant cell-specific rization requirements have not been defined so far.
virus yield maintained for AGE1.CR cells at concentrations as Overcoming technical challenges, laboratory-scale investiga-
high as 48 106 cells/ml, overcoming high cell density issues tions with a continuous process for influenza A virus produc-
due to permanent media exchange [124]. Also insect cells tion using a two-stage stirred-tank bioreactor system were
responded positively onto perfusion systems. S2 insect cell cul- undertaken [130]. AGE1.CR cells grew constantly in a first bio-
tivations have been optimized through perfusion systems, reactor and were fed into a second bioreactor for influenza A
resulting in a 12-fold increase in the expression yield of the virus infection and propagation. The harvest was removed con-
recombinant PfRh5 protein during a 9-day perfusion run in tinuously over a time period of 17 days. However, in the virus
comparison to its fed-batch process [64]. production bioreactor, neither the cell concentration nor the
However, perfusion systems have certainly also critical disad- virus titers reached a steady state, but fluctuated periodically (in
vantages explaining their rare commercial application. Besides inverse correlation) during cultivation time. This could be
limited passage numbers of approved cell lines, and higher explained by accumulation of defective interfering particles
complexities resulting in increased sterility risks (which is con- (DIPs) in the bioreactor, leading to a drop in infectious viral
trollable with today’s standards), perfusion systems cannot be titer. As a result, the cell-specific virus yield was reduced in
performed at optimal medium exchange rates yet. Complex comparison to batch processes. DIP accumulation was also
biological systems, like herein discussed cells, their impact on observed in a continuous process of the insect cell line
stress conditions and their variability are not fully understood Se301 infecting with baculovirus which, finally, resulted in a
and complicate media development (basal growth and perfu- fast decrease of recombinant protein expression and its com-
sion medium), reflecting best nutrient concentrations and fac- plete cessation after 12–18 days post infection [131]. DIP accu-
tors. This leads, in comparison to batch cultivations, to higher mulation in the insect cell baculovirus expression system is
and therefore less efficient medium consumption. widely described and has been related to the loss of large seg-
ments of the viral genome including foreign genes.
Vaccine production in continuous mode These examples demonstrate clearly that regardless potential
Continuous bioreactor operations aim to improve manufactur- changes concerning vaccine immunogenicity or safety associ-
ing by increasing process efficiency and plant utilization, and ated to virus mutation, virus instabilities (DIP formation)
to implement automated process control, while maintaining and changes in virus replication dynamics can cancel out pos-
flexibility and product quality [117,126]. Conversely to batch sible benefits of continuous cultivation strategies and prevent
mode processes, the concept of continuous manufacturing the application of this method for vaccine manufacturing at
describes an ongoing flow of material in and out of the biore- any scale. Whether this is true for all viruses, which are con-
actor, altogether aiming at a constant harvest of virus without sidered for vaccination, remains an open subject of investiga-
restarting the system. This strategy requires less manual operation. Nevertheless, hybrid processing methods combining
tion during the process and avoids down-times for vessel continuous (cell production bioreactor equipped with perfu-
cleaning, maintenance, calibration of sensors and sterilization sion modules) and batch operation units (virus production
(last-mentioned in cases of STRs). In addition, continuous pro- bioreactor) are feasible and have already improved viral vac-
cesses are expected to keep by-product concentrations (e.g., cine manufacturing [123].
proteases) at negligible concentrations, and to handle labile
products rapidly [127], as most viruses lose their infectivity at Use of disposable bioreactors in vaccine production
higher temperatures and proteins forfeit their functionality. Single-use bioreactors (SUBs) are in the focus of commercial
Since several years, regulatory agencies such as the US FDA vaccine production since cultivation studies have shown compa-
encourage the establishment of cell-based continuous rable cell growth kinetics and virus/recombinant protein yields
manufacturing processes, which has been the subject of serious for bioreactors made out of disposable plastic compared to
attempts for recombinant protein production in CHO stainless steel tanks [64,132–135]. For its application, there are sev-
cells [117,126–128]. Whether similar concepts can be transferred to eral chemical and physical criteria which have to be fulfilled.
cell-based virus manufacturing remains to be clarified as muta- Clearly, polymeric multilayer films and welds in the plastic bag
tion rates of cells and viruses restricted their temporal use to should be free of any extractables or leachables to be compliant
currently 20 and 5 passages, respectively (including master with pharmacopoeia standards [136] and should not bind
seed). Further, and most importantly, is the uncertainty of medium components or products. In particular, safety

measurements regarding prevention of bag bursting are Expert commentary

required, not only because of product loss and spillage, but Many new methods for viral vaccine production have been
also to comply with safety issues regarding (highly) infectious developed over the last few years. Improvements in cell lines,
material. Further, the supply chain of cultivation bags has to be viruses, culture media, bioreactor technologies and cultivation
guaranteed and alternative suppliers are indispensable for a strategies have significantly extended the toolbox for vaccine
licensed process. production. In the foreseeable future, conventional batch pro-
SUBs are, in theory, relatively simple to install and univer- duction systems will be complemented with fed-batch and per-
sally applicable by plug and play connections. They are fusion systems to increase space-time-yields in manufacturing
replaced within shortest time after the process, saving cleaning of vaccines for human use. Especially, the availability of new
and sterilization steps while reducing cross-contamination suspension cell lines and the development of disposable cultiva-
risks [137]. However, even though disposables are delivered with tion methods will result in an additional boost toward process
qualification tests, it does not eliminate the need for process intensification. Poorly replicating viruses, viral vectors or VLPs
now have new potential to become viable vaccine candidates
validation studies. Nevertheless, the faster turnaround between

batches in combination with often lower initial investment with these improvements. Regarding large-scale production, cell
compared to fixed asset stainless steel facilities are an attractive concentrations exceeding 1 108 cells/ml, which maintain
option to increase the production efficiency [138,139]. In addi- high cell-specific virus yields, appear obtainable. Further
tion, new production facilities relying on disposables can be increases in titers may be achieved by multiple harvest strategies
commissioned fast as the complexity involved in planning, or expanded volume fed-batch processes. Being more efficient
installation and validation of production facilities is reduced in cell-specific productivity should allow for smaller cultivation
greatly. For vaccine production, this could have significant units or a steady increase of vaccine doses for existing produc-
advantages in case of pandemics or the need to build up pro- tion plants. So far, however, the potential of many approaches
duction capacities in emerging or developing countries [139,140]. has been demonstrated at laboratory scale only, and the future
Accordingly, single-use solutions can be currently ordered will show, which concepts can be transferred successfully to
custom-made with different disposable sensors, diverse sparging large-scale operation. To cope with an ever increasing demand
systems and various mechanical agitation systems (top- or of efficacious and safe vaccines for a growing world population,
bottom-mounted impeller, rocker, orbital shaker of up to 200 l considerable efforts in research and development as well as in
working volume) [141]. Disposable bioreactors are currently large-scale manufacturing are still required.
available at working volumes up to 500 l for rocking systems
and 2000 l for stirred tank systems [142–145]. Large-scale vaccine Five-year view
production often employs even larger volumes, so producing As the global human vaccine market is expected to grow in the
larger SUBs with identical configurations and proportions is next 5 years with an annual rate of 10–15% [147], there is a
challenging, but would circumvent the use of multiple single- demand for continued process development and process inten-
use systems in parallel. However, more systems increase the risk sification. Newly established cell lines, new bioreactor concepts
of leaky seams or breakage of single plastic units but also avoid and advanced cultivation strategies will be required to make
the discard of large batches. The direct transfer from stainless use of the full potential in this field for the upcoming years. In
steel tanks to SUBs typically diminishes over increasing vol- addition, a deeper biological understanding of cell growth,
umes and then requires laboratory-scale investigations from the virus-host cell interaction and virus replication dynamics is
scratch. required, and media development should be intensified to reach
An example of a vaccine facility fully equipped with dispos- higher cell concentrations, while maintaining cell-specific yields.
able technology is the Novavax pilot plant in Rockville, US. In the long run, fed-batch and perfusion systems, for some
The plant, with 75 million doses capacity, was equipped with application even continuous processes, should be established,
200 l wave bioreactors (GE Healthcare) to produce an insect provided that new vaccine candidates successfully pass all clini-
cell-based H5N1 influenza vaccine for evaluation in Phase I cal hurdles. This will change the current vaccine supply system:
and II clinical trials. In addition, a 1000 l SUB (Xcellerex, GE simplicity and flexibility of new production plants already
Healthcare) was employed for scale-up toward commercial addresses newly industrialized countries like India and China,
manufacturing. The company claimed a threefold plant size who will significantly increase their production efforts to supply
reduction and a 20-fold facility cost reduction in comparison local people and other regions with still limited access. This
to egg-based or mammalian cell culture-based influenza vaccine trend will expand toward developing and emerging countries
manufacturing [68,139]. The same disposable technology has utilizing small-scale facilities that are positioned in a completely
been recently implemented, to develop a vaccine candidate flexible way to support self-supply and independency. There-
against Zaire Ebola virus for clinical trials [146]. Another indus- fore, highly adjustable multi-product plants are conceivable,
trial process employing either 200 or 50 l SUB has been most probably mobile, operating dispersed in regions according
described by ProBioGen for the manufacture of a MVA to demands. In higher developed industrial countries, this plat-
vectored-based tuberculosis vaccine candidate produced in form would allow fast reaction time, low-volume vaccine pro-
AGE1.CR.pIX suspension cells [14]. duction and simplified supply of clinical material.

Technological advances in vaccine manufacturing will support Financial & competing interests disclosure
the implementation and expansion into new options, such as LE Gallo-Ramı´rez received financial support from CONACYT Mexico as
the use of viral vectors for gene therapy or personalized medi- an international postdoctoral fellow (Grant 208266). The authors have
cine. Finally, highly productive single-use bioreactors to manu- no other relevant affiliations or financial involvement with any organiza-
facture specific vaccines for patient groups can support new tion or entity with a financial interest in or financial conflict with the
high-value drug manufacturing processes against orphan subject matter or materials discussed in the manuscript apart from those
diseases. disclosed.
Key issues
. Prior to establishment of a vaccine production process, specific aspects have to be considered to choose the appropriate cultivation
technology and operation mode. These are, inter alia, the choice of the vaccine type, a full understanding of cell substrate and virus
propagation requirements, as well as the projected number of required vaccine doses to ensure economic and technical viability.
. Despite their limitations in process monitoring and control, static cultivation systems still represent an economic platform for seed
preparation or even production of human vaccines. Mechanized support and automatic handling allow large-scale application of roller
bottles and multilayer systems.
. Bioreactors allow vaccine production under fully controlled and monitored conditions, ensuring high batch-to-batch consistency, and
processes sterility. In addition, the risk of operator errors can be reduced due to automation. Conventional stainless steel stirred tanks as
well as single-use bioreactors have a great scale-up potential, which is crucial to meet increasing vaccine demands.
. Cell culture-based vaccine production employing bioreactors has expanded manufacturing capacities in terms of larger volumes, shorter
response time, lower costs and higher process control, while ensuring product quality.
. Microcarrier technology has enabled large-scale cultivation of adherent cells in quasi-suspension conditions and facilitates media
exchange as well as certain infection strategies. However, process scale-up demands laborious bead-to-bead transfers, while process
intensification is primarily restricted to volume expansion.

. Packed-bed bioreactors representing a cost–efficient platform typically enable cell seeding at low concentrations but restrict process
monitoring. Single-use systems constitute a flexible option, whereas oxygen input is often the limiting factor for porous materials.
. Suspension culture reduces process complexity, facilitates large-scale processes and offers various options for process intensification.
Therefore, it will remain the preferred cultivation method. However, a larger portfolio of high-yield virus producing suspension cell lines
that grow in serum-free media would be desirable.
. Perfusion systems allow to achieve cell densities higher than 1 108 cells/ml which results in significant volume reductions in seed and
production bioreactors. The use of this technology in commercial vaccine manufacturing should result in considerable cost savings.
. Continuous vaccine manufacturing shows defective inferring particles accumulation, which, so far, limits viral vaccine yields. In addition,
high mutation rates of cells and viruses might restrict its use to certain limited passage numbers. However, establishment of continuous
bioprocesses for cultivation of cells expressing recombinant vaccines may be a viable option.
. Disposable technology has expanded bioprocess potential and flexibility by reduced investment cost, shorter validation times and saved
facility space. Single-use bioreactors can contribute to fulfill present and upcoming demands in vaccine production and represent
therefore a seminal alternative to traditional stainless steel tanks.
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Review

Bioreactor concepts for cell

informahealthcare.com 10.1586/14760584.2015.1067144 2015 Informa UK Ltd ISSN 1476-0584 1

their infectivity in cell culture [17–20]; whereas others have spe-

doi: 10.1586/14760584.2015.1067144 Expert Rev. Vaccines

Table 1. Overview of different cell-based viral vaccine types.

Inactivation may have a negative

informahealthcare.com doi: 10.1586/14760584.2015.1067144

doi: 10.1586/14760584.2015.1067144 Expert Rev. Vaccines

HYPERflask§ Corning 1720 1.7 108 0.6 0.8 Off-line No

BioProducts 16,000,000 3.5 1012 100 97.2 online control

informahealthcare.com doi: 10.1586/14760584.2015.1067144

doi: 10.1586/14760584.2015.1067144 Expert Rev. Vaccines

informahealthcare.com doi: 10.1586/14760584.2015.1067144

doi: 10.1586/14760584.2015.1067144 Expert Rev. Vaccines

informahealthcare.com doi: 10.1586/14760584.2015.1067144

measurements regarding prevention of bag bursting are Expert commentary

validation studies. Nevertheless, the faster turnaround between

doi: 10.1586/14760584.2015.1067144 Expert Rev. Vaccines

intensification is primarily restricted to volume expansion.

informahealthcare.com doi: 10.1586/14760584.2015.1067144

14. Jordan I, Woods N, Whale G, Sanding V. 4975-82 J Virol Methods 2013;193(1):28-41

doi: 10.1586/14760584.2015.1067144 Expert Rev. Vaccines

(RSV) F nanoparticle vaccine induces like particle (VLP) vaccine: homologous

51. Yamaji H, Nakamura M, Kuwahara M, and monkeys. Vaccine 2010;28(15):2705-15 tapbiosystems.com/tap/applications/

informahealthcare.com doi: 10.1586/14760584.2015.1067144

108. Sanofi Pasteur initiates phase II trial of cell

doi: 10.1586/14760584.2015.1067144 Expert Rev. Vaccines

BioProcess International 2011;9(10):18-22 Cytotechnology 1997;24(2):89-98 Influenza vaccines; Geneva, Switzerland:

initiate CHO cell culture manufacturing vectors with enhanced stability in

informahealthcare.com doi: 10.1586/14760584.2015.1067144

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