B i o p r o c e s s Disposable Bioreactors

Disposable Bioreactors
in Cell Culture-Based
Upstream Processing
Regine Eibl and Dieter Eibl

D
uring the last 10 years, cost of therapeutic agents (recombinant disposable bioreactor types, the
pressures and the changing proteins, antibodies, secondary category of mechanically driven
requirements for bioreactors metabolites) and the production of bioreactors that overcomes the
in the modern viruses for gene therapies as well as limitations of long-term established
pharmaceutical industry have resulted veterinary and human vaccines (1,2). static systems (restricted surface for
in the increased use of disposable The key element of every cell expansion and relatively low cell
bioreactors in both R&D and disposable bioreactor is its presterile density with low total cell output)
manufacturing. Numerous studies cultivation container, which is made has gained in importance in recent
have demonstrated their efficiency in of FDA-approved plastics and is only years (1). In this article, we focus on
cell culture-based upstream intended for single-use. The common shaken, stirred and wave-
processing at small- and middle- cultivation containers (consisting of a mixed systems. Particular attention
volume scales. As shown in Figure 1, multiwell plate, a tube, a f lask, a is paid to the CultiFlask 50
disposable bioreactors with culture cartridge or a bag) vary in design, disposable bioreactor, the
volumes between 10 mL and 2 m3 are polymer type, scale, instrumentation, SuperSpinner D 1000 and the
most widely used for cell proliferation, mass and energy transfer, and power BioWave, as well as its successor, the
screening experiments, the production input. Among the multitude of BIOSTAT CultiBag RM, which
have become common devices for
process development and scale-up
Animal and plant cell culture-based upstream processing procedures in our cell culture labs.
Their effectiveness is illustrated with
Cell proliferation Screening Experiments Process development and examples from the literature and our
GMP-manufacturing
• Seed inoculum • Cell line
• Glycoproteins own research.
• Human cells for cellular • Medium
therapies • Process parameter • Viruses
• Secondary metabolites
O rbitally Shaken B ioreactors
During the last few decades, orbital
shaking in non-instrumented
disposable containers has become an
Disposable bioreactors accepted technology for the rapid
(10 mL – 2 m3 volume) development of cell culture processes
and seed inoculum production at
Static mL-scale. “Erlenmeyer” f lasks
bioreactors Dynamic bioreactors
(whose engineering principles are
better known today) are regarded as
Mechanically driven
bioreactors Pneumatically Hydraulically Hybrid
reliable devices with which to
driven driven hollow
• Rotating systems
• Shaken systems bioreactors fibre bioreactors
bioreactors perform parallel experiments with
• Stirred systems animal suspension cells in high
• Wave-mixed systems
numbers. Furthermore, the
Figure 1: Main application fields and classes of disposable bioreactors. pioneering work of the Swiss Federal

18 BioProcess International
Bio-process Supplement
Institute of Technology Lausanne
and ExcellGene SA supported the
introduction of frustoconical shake
tubes as suitable cultivation
containers for such purposes, which
culminated in the development of
the TubeSpin cultivation vessel (trade
name CultiFlask 50 disposable
bioreactor) (3). This bioreactor,
which resembles the classic 50 mL
centrifuge design, is equipped with a
vented screw cap. Integrated holes
and a PTFE membrane guarantee
optimal gas exchange, sterility and
minimized liquid loss by evaporation.
Presupposing moderate agitation
speeds and working volumes, the
high cell densities (>1 x 107 cells/mL)
that have been reached in CultiFlask
50 disposable bioreactors with a
CHO DG44 suspension cell line are
comparable with those cell densities
that are normally achieved in
optimized stirred bioreactors.
Thus, it was assumed that the
CultiFlask 50 disposable bioreactor
ensures a non-limiting oxygen
transfer for the growth and
production of animal suspension
cells. This hypothesis was
corroborated by the reported
volumetric oxygen mass transfer
coefficients (k L a values), which
range between 5 and 30 per hour at
culture volumes of 10–20 mL, and
have agitation speeds between 180
and 220 rpm (4).
In our investigations, aimed at
analysing growth courses and
metabolite profiles of insect
suspension cells (Sf 21), similar
behaviour was detected in CultiFlask
50 disposable bioreactors, 125 mL
shake f lasks (Corning) and 2 L
wave-mixed CultiBags. Under batch
conditions and appropriate
cultivation parameters, we
determined cell densities to be
between 1.8 and 2.2 x 107 cells/mL,
Supplement
Figure 2: The BIOSTAT CultiBag RM (laboratory version).
growth rates to be between 0.031
and 0.033 per hour, doubling times
to be between 21–22.1 hours, glucose
uptake rates to be 0.48–0.61 x 10 -17
mol/cell/s, and glutamine uptake
rates to be between 0.11 and 0.2 x
10 -17 mol/cell/s (5). Moreover,
comparative expression studies with
the aim of producing the catalytic
domain of B-Raf kinase showed
similar results (personal
communication, C. Ries) and
revealed TubeSpin technology’s
potential applicability in scaling-up
the culture volume, even if different
cultivation systems are used. Thus,
these findings suggest the high
suitability of CultiFlask 50
disposable bioreactors as scale-down
systems for serum-free cultivated
insect suspension cells, which are
frequently employed to generate tool
proteins and new vaccines in
conjunction with baculovirus
expression vector systems.
S mall -S cale Stirred
D isposable B ioreactors
SuperSpinner D 1000 case study: In
contrast to previously described
shaking bioreactors, which have no
internal elements, stirred bioreactors
are equipped with a rotating or
tumbling impeller that ensures mass
and temperature homogeneity, as
well as gas dispersion. Whereas in
magnetically driven non-
instrumented stirred bioreactors —
so-called spinner f lasks — surface
aeration is typically accomplished,
aeration is effected by a tumbling
hollow fibre membrane in the
disposable SuperSpinner D 1000.
The underlying idea of bubble-free
aeration by a hollow fibre membrane
wound around a tumbling impeller
was elucidated in the early 1990s by
Lehmann et al. and resulted in the
development of SuperSpinners made
of glass (6). Owing to the
opportunity for membrane aeration,
k L a values about 3.5 times higher
than in standard spinners have been
estimated (7). These values
correspond with volumetric oxygen
mass transfer coefficients determined
for membrane aerated stirred cell
culture bioreactors at laboratory
scale. Consequently, SuperSpinners
should provide higher cell densities
than surface aerated standard
spinners, which are principally used
for the expansion of animal
suspension cells.
Cell densities that were two to
three times higher than in standard
spinners were guaranteed by the
disposable SuperSpinner D 1000 for
two CHO suspension cell lines
(CHO DG44 ST1-C6, CHO XM
111-10) that have been grown,
serum-free or chemically defined, in
BioProcess International 19
 B i o p r o c e s s Disposable Bioreactors
batch mode at culture volumes of 0.8
and 1 L (8,9). From a comparison of
standard spinners and SuperSpinner
D 1000 results, the same trend was
observed for murine hybridoma cells
cultivated in feeding mode at culture
volumes up to 800 mL (8). Finally,
serum-free batch cultivation of Sf 9
suspension cells (1 L culture volume)
1 = non-infected, 2 = recombinant, 3 = culture volume.
Table I: Overview of animal and plant cell cultures successfully grown in wave-mixed CultiBags.
20 BioProcess International Supplement
in the SuperSpinner D 1000 resulted
in high cell densities after only 3
days of cultivation. The maximum
cell density achieved, 1.66 x 107
cells/mL is, again, about three times
higher than those observed in
standard spinners, and is similar to
typical cell densities obtained in
wave-mixed bioreactors (9,10). These
results suggest that the SuperSpinner
D 1000 can even be used for the
production of protein samples for
clinical trials.
Wave -M ixed B ioreactors
In the case of wave-mixed bag
bioreactors, the rocking movement of
the bioreactor’s platform containing
the f lexible culture bag facilitates
mixing. In this way, the surface of
the culture medium is continuously
renewed and a bubble-free surface
aeration takes place. Mass and
energy transfer in wave-mixed
bioreactors, and therefore their
growth and product formation
efficiency, are highly dependent on
wave development and propagation.
Both features are mainly inf luenced
by the rocking rate, rocking angle,
filling level, bag geometry and
culture broth viscosity. BioWave,
BIOSTAT CultiBag RM (see Figure 2),
Wave Bioreactor, AppliFlex, Tsunami
Bioreactor, CELL-tainer and Wave
and Undertow Bioreactor belong to
the group of wave-mixed bag
bioreactors. Despite their identical
working principle, major differences
affect their control unit, platform
movement and culture bag design
(material, scale, dimension,
instrumentation with sensor probes
and filters).
Like Wave Bioreactor, BioWave
and BIOSTAT CultiBag RM
originated from the first prototype of
a wave-mixed bag bioreactor and is
characterized by a one-dimensional
rocking movement. Modified
Reynolds numbers (characterizing
the f luid f low), mixing times,
residence time distribution data,
volumetric oxygen mass transfer
coefficients and values for specific
power input have demonstrated the
comparability (and sometimes
superiority) of the BioWave and
BIOSTAT CultiBag RM with
surface or bubble-free aerated cell
culture bioreactors made of glass or
stainless steel (10). Hence, their
prevalence in small- and middle-
volume-scale processes based on
animal and plant cells with low
oxygen demands is easy to explain.
The cultivation overview presented
in Table 1 clearly indicates the
popularity of wave-mixed CultiBags
operating in batch, feeding or
perfusion modes and allowing the
online control of pH and dissolved
oxygen via optical sensors.
Independent of the cell culture
type, encouraging cultivation results
have been described for wave-mixed
CultiBags in which biologically
active cells, recombinant
glycoproteins, virus vaccines and
secondary metabolites were
produced in cultures of up to 100 L
(2,10,11–20). Even plant cell
cultures, which tend to aggregate
and become increasingly viscous
during cultivation, have been grown
up to high biomass concentrations
(2,18,20). To achieve successful
scale-up, a guarantee of similar
f luid f low conditions and an
optimal oxygen supply —
independent of scale — is
recommended for wave-mixed
culture bags in general (10). In the
future, it is expected that CFD
models will identify local velocity
and concentration prof iles in
extant wave-mixed culture bags to
support their use in scale-up and
will make comparisons with
stirred and shaking cultivation
containers possible.
Conclusions and Prospects
In summary, the CultiFlask 50
disposable bioreactor, SuperSpinner
D 1000, BioWave and BIOSTAT
CultiBag RM have been very
successful in growing cell cultures
and expressing their products.
Supplement
Cryopreserved vial
CultiFlask 50 disposable
bioreactor Disposable shake flasks
(20 mL)* (20 mL)*
SuperSpinner D 1000 Disposable shake flasks t-flasks
(1 L)* (200 mL)* (30 mL)*
BIOSTAT® CulitBag RM BIOSTAT® CulitBag RM CELLine CL 1000
(10 L)* (1 L)* (20 mL)*,**
BIOSTAT® CulitBag RM * culture volume
(50 L)* ** high cell density system
BIOSTAT® CulitBag RM BIOSTAT® CulitBag STR
(300 L)* (250 L)*
Figure 3: Completely disposable upstream processing procedures in which static, shaken,
stirred and wave-mixed bioreactors are used. Additional time and cost savings can be
achieved by reducing the intermediate cultivation steps.
Although they differ in scale, disposable bioreactor. In doing so,
design, working principle and the special features of the respective
instrumentation, they all provide bioreactor type have to be taken into
excellent cell growth and account (1,10,21). Because of the
comparable or even higher cell demands of the market, it is even
numbers and product yields than expected that the increase in use of
their counterparts made of glass or disposable bioreactors for cell
steel. In addition, their availability culture-based processes will continue
enables the design of completely in the future. Moreover, in
disposable upstream processes, as personalized medicine, where ex vivo
shown in Figure 3 and as realized in generated functional human cells
our cell culture labs. (including stem cells) in clinically
We would like to mention that, in relevant numbers are required,
addition to the BIOSTAT CultiBag disposable bioreactors with a culture
RM, the corresponding BioWave or volume between 1 L and 1000 L will
Wave Bioreactor and the AppliFlex become a necessity. Lastly, there is
can also be used. It is also possible to evidence of an expansion of the
replace the recently introduced already existing small number of
BIOSTAT CultiBag STR with microorganism-based applications
ThermoFisher’s well-established (such as microbial immunomodulator
SUB, Xcellerx’s XDR-Disposable secernation and the production of
Stirred Tank Bioreactor or Artelis- chiral building blocks and biological
ATMI Life Sciences’ Pad-Drive insecticides by the in vitro cultivation
BioProcess International 21
 B i o p r o c e s s Disposable Bioreactors

of fungal species) with disposable

bioreactors (10). Selecting the most
suitable disposable bioreactor type
and, thus, the decision to shake, stir
or rock the disposable cultivation
container is dependent on the
cultivation task, the production
organism and its characteristics, the
scale, the legal regulations and,
finally, the infrastructure and know-
how of the user.
Interestingly, for biomanufacturing
processes with culture volumes
greater than 500 L, only stirred
versions of disposable bag bioreactors
(1000 L SUB [Figure 4] and 1000
L/2000 L XDR-Disposable-Stirred
Tank Bioreactor) are available to
date. They are basically equipped
with microspargers or sparger rings
and rotating axial f low impellers.
Whereas bottom-driven XDR-
Disposable Stirred Tank
Bioreactors operate with a
magnetically coupled impeller,
SUBs are top-driven and have
mechanical seals. The cylindrical
culture bags are always shaped and
fixed in customized steel support
containers, allowing the sterile
insertion of standard sensors for
IPC. However, in addition to
further stirred bag bioreactors, 1800
L bag bioreactors with Vibromixers
(Zeta) and 2000 L bag bioreactors
that apply a buoyant force of inlet
gas to pneumatically power mixing
using air-wheels (PBS Biotech) have
also been announced.

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Figure 4: A 1000 L SUB. A Fast Track Approach for Process

22 BioProcess International Supplement

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Facility Management, Institute of
Biotechnology, August 2008;
unpublished.
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Wave-Induced Motion: Characteristics
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School of Life Sciences and Facility
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September 2008; unpublished.
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Production in Disposable Bioreactors.
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Inf luenza A Virus Production in
Microcarrier Systems? Poster
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Groot T. Vaccine Production Utilizing
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Verlag, Berlin, Germany; in press.
For more information
Prof. Regine Eibl (regine.eibl@zhaw.ch) lectures
in biotechnology and cell cultivation techniques,
and Prof. Dieter Eibl (dieter.eibl@zhaw.ch)
lectures in biochemical engineering and the
planning of biotechnological production facilities
at the Institute of Biotechnology of the School of
Life Sciences and Facility Management at the
Zurich University of Applied Sciences
(Switzerland). Their working group (biochemical
engineering and cell cultivation techniques) has
been using disposable bioreactors since the
middle of the 1990s (Campus Grüental, PO Box,
CH-8820 Wädenswil, Switzerland).
Abbreviations
ADV Aujeszkys disease virus
BEV Baculovirus expression vector
BHK Baby hamster kidney
BY-2 C ultivar Bright Yellow-2 from
the tobacco plant
CFD Computational f luid dynamics
CHO Chinese hamster ovary
E-FL E mbryogenic feline lung
fibroblast
EIV Equine inf luenza virus
FDA Food and Drug Administration
HEK Human embryogenic kidney
his Histidine
hERG Human ether-a-gogo related gene
IPC In Process Control
k L a G as-liquid mass transfer
coefficient
MDCK Madin-Darby canine kidney
MEV Mink enteritis virus
PBS Pneumatic Bioreactor System
PTFE Polytetraf luorethylene
RM Rocking motion
SEAP Secreted alkaline phosphatase
Sf Spodoptera frugiperda
STR Stirred reactor
SUB Single-Use Bioreactor
Vero cells K idney epithelial cells from African
green monkey
BioProcess International 23