peer-riewed:single-use bioreactors single-use bioreactors …€¦ · · 2015-11-23the article...
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22 BioPharm International www.biopharminternational.com October 2014
In the current competitive bio-
pharmaceutical production land-
scape, the substantially higher
proce s s deve lopment cos t s
mean that new candidate molecules
are increasingly under pressure to
advance to clinical proof-of-concept
stage in the shortest possible time with
a minimum commitment of capital
resources. Single-use bioreactors have
the potential to alleviate both of these
constraints because they confer spe-
cific advantages over conventional
stainless-steel bioreactors. Acquisition
and implementation costs of dispos-
able bioreactors tend to be much lower
compared to traditional stainless-steel
bioreactor systems. Some of these
advantages can be attributed to their
modular nature, which enables the
use of existing manufacturing facili-
ties with minimal modifications to the
current infrastructure, allowing execu-
tion of a straightforward “roll in-roll
out” concept. Furthermore, because
the product contact surface is changed
with each experiment/campaign, carry
over between fermentations is non-
existent, thus removing the need to
perform cleaning verification/valida-
tion and enabling a more rapid turn-
around between products. As a result,
single-use systems from various manu-
facturers are now available on the mar-
ket, and pharmaceutical development
groups have increased the frequency
with which these systems are used to
produce biopharmaceuticals (1, 2).
Although the benefits concerning
this technology are fairly clear, one
of the primary unresolved concerns
focuses on performance comparabil-
ity between single-use and conven-
tional stainless-steel systems. In this
study, the authors present data from
two different fed-batch processes pro-
ducing monoclonal antibodies (mAbs)
using both system types. To achieve
good correlations between bench-scale
development reactors and the single-
ABSTRACT
The article describes successful incorporation of single-use bioreactors as part of
a fed-batch platform technology for the production of clinical biopharmaceuticals.
By matching general reactor characteristics, the authors were able to scale-up
fed-batch processes from traditional bench-scale bioreactors to the large-scale
single-use systems without any significant operational differences or changes
in critical product quality attributes.
single-Use Bioreactors for the rapid Production of Preclinical and clinical
BiopharmaceuticalsRüdiger Heidemann, Christopher R. Cruz, Paul Wu, Mikal Sherman, Jessica Martin, and Christel Fenge
Rüdiger Heidemann, PhD, is senior staff
development scientist, Christopher R. Cruz
is senior associate development scientist,
and Paul Wu, PhD, is director, Upstream
development, all three at cell culture
development, Global Biological development,
Bayer healthcare llc, Berkeley, ca 94701;
Mikal Sherman is application specialist,
fermentation technologies, and Jessica Martin is field marketing manager, single-Use
Bioreactors, both at sartorius stedim north
america, Bohemia, nY 11716, Usa; and Christel Fenge, PhD, is vice-president of marketing for
fermentation technologies at sartorius stedim
Biotech, 37079 Göttingen, Germany.
PEER-REVIEWED
article submitted: april 14, 2014.
article accepted: august 29, 2014.
Ima
ge C
ou
rte
sy o
f S
art
ori
us S
ted
im B
iote
ch
Peer-reviewed: single-Use Bioreactors
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October 2014 www.biopharminternational.com BioPharm International 23
use bioreactors, a continuous stirred tank
reactors (CSTR) design was used through-
out the study as described by De Wilde et
al. (3). One major outcome of this work was
the development of an entirely single-use
upstream platform for the manufacture of
biopharmaceuticals from vial thaw to har-
vest clarification.
Materials and MethodsRecombinant Chinese hamster ovary (CHO)
cell lines were used to produce two different
fully human mAbs in fed-batch processes.
Process A was used to produce an IgG2 and
Process B to produce an IgG1. Both processes
used a combination of commercially avail-
able “off the shelf” and proprietary basal
media and feed solutions.
Initial process development was per-
formed in traditional bioreactor designs at
the 2-L, 5-L, and/or 10-L scale (Applikon
Biotechnology, Netherlands). Preclinical and
clinical material was generated in the single-
use Biostat STR (Sartorius Biotech, Germany)
at the 200-L and 1000-L scales. A standard
bag design consisting of two marine-type
impellers, a macro-sparger and optical sen-
sors (PreSens, Germany) for pH and dissolved
oxygen was used. Stirrer geometry, tip speed,
and overall power input were matched as
closely as possible among all bioreactors,
similar to the approach taken by De Wilde
and Adams (4) and Noack et al. (5). Figure 1
illustrates tip speed and power input values
among the different bioreactors. Although
the power input of the 10-L system is slightly
higher than the rest, the overall tip speed
remains comparable to that of the disposable
units.
Standard shaker flasks were used during
the seed train expansion process. Cells were
cultivated in a CO2 incubator maintained at
37.0 oC and 5–7% CO2. Scale-up passaging
occurred every two to three days until suf-
ficient cell mass had accumulated to directly
inoculate the bioreactors regardless of scale,
with the exception of the 1000 L. The 200-L
bioreactor was inoculated at an initial vol-
ume of 50–80 L operation range due to varia-
tion in the accumulated cell mass of the seed
train. Basal medium was added to the reactor
to “passage” the cells by dilution and consid-
ered in “scale-up” mode until a predefined
total cell number was reached; this time
point was defined as fed-batch day zero. If
required, the 200-L unit was used as part of
the seed train expansion process and culture
was directly transferred to the 1000-L reactor
to achieve a target starting cell density on
inoculation day.
Fermenter process parameters (e.g., pH,
temperature, pO2) were maintained by bio-
controllers manufactured by either Sartorius
(DCU 2/3/Biostat STR, Sartorius Biotech,
Germany) or Applikon (iControl, Applikon
Biotechnology, Netherlands). All bioreactors
were configured with a macro-sparger to
deliver mixed gas consisting of air, N2, CO2,
AL
L F
IGU
RE
S A
RE
CO
UR
TE
SY
OF
TH
E A
UT
HO
RS
Peer-reviewed: single-Use Bioreactors
Figure 1: Tip speed and power input values for different bioreactors. Two different types of
impellers were used in the 2 L reactors.
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
25
20
15
10
5
0
Tip speed = Nidπ
Power input =nNpρ Ni
3d5
V
Ni - agitation rate
Np - impeller power number
n - number of impellers
ρ - fuid density
d - impeller diameter
V - reactor volume
2L a 2L b 5L 10L
Tip
sp
eed
(m/ s
)
Po
wer
inp
ut
(W/ m
3)
200L
SUB
1000L
SUB
Tip speed and power input
Contin. on page 26
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26 BioPharm International www.biopharminternational.com October 2014
and O2. Dissolved oxygen was controlled at
40% air saturation, pH at 6.7–7.3, and the
initial process temperature was set to 36.5 oC.
For Process B, a temperature shift to 33.0 oC
was performed once the cell density reached
8 x 106 cells/mL.
The bioreactors were sampled daily to
determine viable cell density and viabil-
ity (Cedex cell counter, Roche Diagnostics,
Germany) as well as glucose and lactate val-
ues (YSI, Yellow Springs Instruments, OH,
USA). Dissolved oxygen, pH, and pCO2 lev-
els were measured offline using a blood gas
analyzer (Siemens Diagnostics, NJ, USA) and
used to verify the pH and dissolved oxy-
gen probes of the bioreactors. mAb titers
were measured using a protein A high-per-
formance liquid chromatography (HPLC)
method. The fed-batch process was termi-
nated after reaching predefined harvest cri-
teria (viability and/or time based), and the
crude harvest was clarified by dead-end fil-
tration.
The 200-L Biostat, in addition to being
the seed source for the 1000-L system, was
also used to verify the bench-scale fed-batch
process and to produce preclinical material.
The 1000-L reactor was used to produce GMP
material for clinical trials. The entire clinical
upstream process consisting of the 200-L and
1000-L bioreactors as depicted in Figure 2,
uses disposable materials throughout, includ-
ing bags for basal media, feeds, and clarified
harvest.
resUlts and discUssionTo compare the performance of the single-
use bioreactors to traditional fermenters, crit-
ical process parameters for each vessel type
are plotted together. Data from multi-use
reactors are plotted as grey dots to define a
point cloud of expected values and single-
use disposable runs are shown as continuous
lines. Figure 3 and Figure 4 summarize the
data obtained in Process A and B, respec-
tively. As shown for Process A, cell growth,
viability, glucose, and lactate concentra-
tions obtained with the single-use systems
follow the general trends defined by the
bench-scale reusable bioreactors. Lactate val-
ues were slightly higher in the bench-scale
reactor cultures. These cultures were used
for the initial process development, specifi-
cally to optimize the feeding process. The
slightly higher viability can be attributed to
slight differences in the Cedex cell counter
used during these cultures. Offline pH and
pO2 measurements are well aligned, dem-
Peer-reviewed: single-Use Bioreactors
Figure 2: Schematic overview of the upstream fed-batch process using disposable bioreactors.
Duration ~ 14 days
• 1mL cryo-vial• shaker �asks (10L required for inoculation)
Seed-Train
Expansion
200L
BIOSTAT
STR
1000L
BIOSTAT
STR
Cell Sep:
Dead-End
Filtration
Clari3ed
Harvest
Clari3ed
Harvest
• Single-Use / Disposable 3lters already implemented for downstream processes
• 50L min working volume in 200L• 250L min working volume in 1000L
Duration ~ 25 days Duration ~ 1 day
Contin. from page 23
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October 2014 www.biopharminternational.com BioPharm International 27
onstrating that single-use bioreactor con-
trol is comparable and was not affected by
vessel type or the measurement technology
(i.e., conventional glass electrodes and Clark
oxygen probes vs. single-use fluorescence
patches). As a result, most Biostat STR data
Peer-reviewed: single-Use Bioreactors
Figure 3: Process A parameters. Grey squares: bench-scale data (5 and 10 L), blue lines: 200 L
Biostat STR, green lines: 1000 L Biostat STR.
30
25
20
15
10
5
0
8
7
6
5
4
3
2
1
0
7.5
7.4
7.3
7.2
7.1
7.0
6.9
6.8
6.7
6.6
6.5
100
90
80
70
60
50
40
30
20
10
0
140
120
100
80
60
40
20
0
2.5
2.0
1.5
1.0
0.5
0.0
110
100
90
80
70
60
0 2 4 6 8 10 12 14 16
VC
D (
10
6 c
ell
s / mL)
Glu
cose
(g
/L)
pH
pC
O2(m
mH
g)
pO
2(m
mH
g)
Tit
er
(re
lati
ve
un
its)
Via
bil
ity
(%
)
Time (days)
0 2 4 6 8 10 12 14 16
Time (days)
0 2 4 6 8 10 12 14 16
Time (days)
0 2 4 6 8 10 12 14 16
Time (days)0 2 4 6 8 10 12 14 16
Time (days)
offine pO2
pCO2
Titer
offine pH
0 2 4 6 8 10 12 14 16
Time (days)
0 2 4 6 8 10 12 14 16
Time (days)
0 2 4 6 8 10 12 14 16
Time (days)
Viable cell density
Glucose
Cell viability
LactateLa
cta
te (
g/ L
)
Contin. on page 30
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30 BioPharm International www.biopharminternational.com October 2014
were highly comparable to the data obtained
at small scale, and certain parameters shifted
towards more desirable profiles (i.e., reduced
lactate concentrations and increased titer
per unit time). Process B in Figure 4 shows
similar behavior, with tighter clustering of
Peer-reviewed: single-Use Bioreactors
Figure 4: Process B parameters. Grey squares: bench-scale data (5 and 10 L), blue lines: 200 L
Biostat STR, green lines: 1000 L Biostat STR.
14
12
10
8
6
4
2
0
0 2 4 6 8 10 12 14
VC
D (
10
6 c
ell
s / mL)
Lact
ate
(g/ L
)
Time (days)
0 2 4 6 8 10 12 14
Time (days)
0 2 4 6 8 10 12 14
Time (days)
0 2 4 6 8 10 12 14
Time (days)
0 2 4 6 8 10 12 14
Time (days)
0 2 4 6 8 10 12 14
Time (days)
0 2 4 6 8 10 12 14
Time (days)
0 2 4 6 8 10 12 14
Time (days)
Viable cell density110
100
90
80
70
60
Via
bilit
y (
%)
Cell viability
8
7
6
5
4
3
2
1
0
Glu
cose
(g
/L)
Glucose 3.0
2.5
2.0
1.5
1.0
0.5
0.0
Lactate
7.5
7.4
7.3
7.2
7.1
7.0
6.9
6.8
6.7
6.6
6.5
pH
offine pH 140
120
100
80
60
40
20
0
pC
O2(m
mH
g)
pCO2
100
90
80
70
60
50
40
30
20
10
0
pO
2(m
mH
g)
offine pO2
Tit
er
(rela
tive u
nit
s)
Titer
Contin. from page 27
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October 2014 www.biopharminternational.com BioPharm International 31
data among the reactor types and scales.
For some of the bench-scale reactors as well
as the 1000-L disposable unit the tempera-
ture shift was programed into the control
unit at a rate of 0.5 oC per 30 minutes. The
temperature decline was linear over the 3.5
h time span with less than 0.5 oC under
shoot. The 200-L disposable unit used a heat-
ing blanket, therefore, active cooling like in
the 1000-L unit was not possible. The tem-
perature shift here took approximately 11 h
with a slightly larger under shoot (data not
shown). Nevertheless, this second process
also demonstrates the feasibility of imple-
menting temperature shift control schemes
with single-use bioreactors. Overall, the tem-
perature shift for Process B was necessary to
obtain the correct product quality attributes.
In addition, it increased the space-time pro-
ductivity of the antibody.
In addition to similar in-process culture
performance, critical product quality attri-
butes of the harvest like protein aggregation
rates, charge heterogeneity, and glycosyl-
ation profiles remained consistent across
all bioreactor systems (data not shown).
Overall, the data show good comparability
between the bench-scale cultures and the
large-scale fermentations, demonstrating
both the scalability of each processes and
the successful integration of single-use bio-
reactor technology into the authors’ clinical
manufacturing platform for mAb fed-batch
processes.
One of the primary concerns surround-
ing the adoption of single-use reactor tech-
nology for full commercial manufacturing
is bag robustness. Due to the nature of the
materials employed, defects can poten-
tially be introduced at any point during the
life of the bag; at manufacture, shipping,
unpacking/setup, or operation. During
the preliminary implementation at Bayer
HealthCare, a 200-L culture was terminated
one day after inoculation because a 24 h
medium sterility-hold at 50 L did not reveal
a pinhole defect. That incident triggered
an in-house integrity test of all single-use
reactors prior to use which consisted of
pressurizing a mounted bag to 0.25–0.35
psi (17–24 mbar) and monitoring the pres-
sure for 10 min (200 L) or 20 min (1000
L), shown in Figure 5. An intact bag will
only lose ~0.02 psi (1.4 mbar) during the
test period whereas a defective bag will rap-
idly depressurize during the test procedure.
Unfortunately, this practice does not guar-
antee success, as the metal wall of the bag
Peer-reviewed: single-Use Bioreactors
Figure 5: Pressure drop profles for all STR bags. A pressure profle from a defective bag is
shown for comparison.
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0 5 10 15
200 L bag
1000 L bag
defective bag
20
Pre
ssu
re d
rop
(p
si)
Time (min)
Contin. on page 34
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34 BioPharm International www.biopharminternational.com October 2014
Peer-reviewed: single-Use Bioreactors
holder can mask potential defects during
the pressure hold and despite a passing test.
Bag failure can result in contamination and
lost cultures.
To mitigate this risk, Sartorius Stedim
Biotech developed and qualified a pressure
test comparable to the one described by the
authors but utilizing a patented fleece that
inserts a mesh gap between the bag and
holder surfaces to prevent these potential
masking effects. The approach allows post
bag installation and pre-use testing of the
entire single-use bioreactor assembly (6). The
fleece is designed to be easily removed prior
to the start of a run, to ensure normal bio-
reactor temperature control. This method
has been qualified for reliable defect detec-
tion for various bag sizes (i.e., a correlation
between reliably detectable defect size and
pressure decay has been established) (7).
This method is, therefore, a more reliable
detection method and fully encompasses
any typical defects that might be incurred
during transportation, storage, unpacking,
and installation that cannot be identified
by simple visual inspection. In essence, this
approach is similar to conventional stainless-
steel bioreactor practices, where the steriliza-
tion method is qualified during installation
qualification/operation qualification (IQ/
OQ) and a pressure hold test is often per-
formed for risk mitigation purposes to
detect any miss-assemblies during regular
operation. The sterilization IQ/OQ can be
compared to the vendor assembly and ster-
ilization qualification of single-use bags and
the pressure decay testing serves in a compa-
rable way as a risk mitigation tool.
conclUsionAs more and more single-use bioreactors and
bags are used in late-phase and commercial
production, especially with the availabil-
ity of 2000-L single-use stirred tank biore-
actors, the need for consistent bag quality,
robustness, improved assurance of supply,
change management, and business continu-
ity planning become crucial. To satisfy these
requirements, Sartorius Stedim Biotech
developed a new polyethylene film in close
collaboration with resin and film suppli-
ers to meet future industry needs and to
further improve bag consistency, robust-
ness, and performance for single-use biopro-
cessing applications. During development,
attention was paid to working with ven-
dors to ensure a stable supply chain, clearly
defining and controlling the resin and addi-
tive packages, establishing acceptable film
extrusion ranges with design space studies,
and optimizing of the bag welding process.
The result was a system with significantly
improved strength and flexibility charac-
teristics, alleviating many of the potential
avenues to introduce defects during the
manufacture and handling of these single-
use bioreactors (8). Furthermore, cell culture
and leachable studies were performed during
all stages of the new film development to
ensure the new formulation is not toxic and
does not impede cell growth (9).
All these aspects help to pave the way
towards wide implementation of commercial
scale single-use biomanufacturing, benefit-
ting from the initially mentioned advantages
of reduced upfront investment, flexibility,
quick change-over, and minimal validation
effort.
references 1. C. Heath and R. Kiss, Biotechnol. Prog. 23,
46–51 (2007).
2. M. George et al., Biotechnol. Bioeng. 106, 609–
917 (2010).
3. D. De Wilde et al., BioProcess Int. 7 (Suppl 4)
36–41 (2009).
4. D. De Wilde and T. Adams, Eur. J. Parent. Pharm.
Sci. 15, 41–46 (2010).
5. U. Noack et al., “Single-Use Stirred Tank
Reactor BIOSTAT CultiBag STR: Characterization
and Applications,” in Single-Use Technology in
Biopharmaceutical Manufacture (Eds. R. Eibl and
D. Eibl), John Wiley & Sons, Inc. (Hoboken, NJ,
USA, 2010).
6. M. Stering et al., Genetic Engineering &
Biotechnology News, 33 (11) (2013).
7. M. Stering et al., BioProcess Int. 12 (Suppl 5)
58–61 (2014).
8. E. Vachette et al., BioProcess Int. 12 (Suppl 5)
38–42 (2014).
9. E. Jurkiewicz et al., Verification of a new
biocompatible single-use film formulation
with optimized additive content for multiple
bioprocess applications. Biotechn. Prog., Epub
ahead of print, accepted: 21 May 2014, DOI:
10.1002/btpr.1934. ◆
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