D i s p o s a b l e s SUPPLIERS RESPOND
Scaling Up Stem Cells
Moving from Laboratory to Commercial Production
with a Single-Use Multiplate Bioreactor
by Matthieu Egloff and Jose Castillo
C
ell-based products are becoming
increasingly important as
potential biotherapies. Cell
therapy is predicted to have a
huge impact on the healthcare sector
over the coming decades. Stem cells, in
particular, are investigated as potential
treatments for a diverse range of
applications (such as heart disease and
metabolic and inflammatory disorders)
in which they might be used to restore
lost biological functions.
The cell therapy industry is starting
to mature. Several emerging
companies are now supporting latestage clinical trials, and stem cellbased products should soon appear on
the market. However, potential
commercial success for such products
is linked to the ability of sponsor
organizations to industrialize
manufacturing processes for ensuring
cell supply and managing costs.
Successful transition from laboratory
scale, which is suitable for producing
just a few batches per year, to an
efficient and robust good
manufacturing practice (GMP)
process will be essential. If the
economic model is to be viable, and if
health authorities and insurers are to
reimburse these products, then prices
must be controlled. That translates to
scaling out for autologous therapies
and scaling up for allogeneics.
The research and development
(R&D) process is based on currently
available laboratory-scale technology,
in which multiple-tray stacks are used
to culture cells. That method is not
practical for large-scale production,
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however, because it requires multiple
manual aseptic operations. Those can
present problems for consistency,
reproducibility, and safety as well as
quality assurance and control (QA and
QC). The multitray option is also
expensive — perhaps prohibitively so.
Although the cost of purchasing a
stack is not always prohibitive, the
cost of running it can be expensive,
costly both financially and in labor.
That was the problem facing
Cardio3 Biosciences, a Belgian
biotech company working on a cellbased therapy to protect patients’
hearts during myocardial injuries,
reducing scar volume and
reconstructing damaged heart tissue.
The company’s developmental
autologous therapy uses cardiopoietic
cells made from a patient’s bone
marrow stem cells, then injected into
the same patient’s heart. Following
successful phase 2 trials, the company
now plans to advance into phase 3
studies. That will require an effective,
efficient, and practical method for
growing batches of autologous stem
cells for more patients.
In 2008, Cardio3 approached
ATMI LifeSciences to develop a
single-container solution that might
solve its scale-up problem. The
company previously used a multitray
stack to grow cells in a laboratory. For
each patient, 20 such devices were
required, with operations during the
cell-production process carried out
under laminar flow. That made
scaling up/out the process very
difficult because Cardio3 was facing
an initial objective of about 3,000
patients per year in the early stages of
commercialization. Using the
laboratory process, that would have
required some 200 aseptic operations
per batch (2,000 aseptic operations per
day). Achieving a current GMP
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(CGMP) process also would have
been difficult. Coupled with the sheer
space and number of operators
required, that made it nearly
impossible to create a viable and
efficient business model. Simulations
demonstrated that this company
would have needed a 5,000-m 2 facility
equipped with 500 incubators and 160
biocabinets, employing 300 operators
in numerous culture rooms.
What Cardio3 needed was a costefficient process that cultured highquality cells to GMP standards on the
necessary scale. For safety reasons, a
closed system was required to
guarantee sterility. The process also
needed to be easily controlled to
ensure reproducibility. Cost efficiency
could be achieved by simplifying and
reducing the number of operations
(and operators) involved.
A Lack of Suitable Solutions
Single-use bioreactors are well
established in biopharmaceutical
production, for which they offer many
benefits. Not only do they simplify QA
and QC functions, but they also reduce
overall costs and provide a flexible
solution for manufacturing. However,
most existing disposable bioreactors are
designed for viral or protein
production. Fragile adherent cells (such
as stem cells) are highly sensitive to the
physical parameters of their
microenvironment. Factors such as the
surface material, pH, dissolved oxygen,
and shear stress all affect the way these
cells grow and differentiate. And
harvesting the cells is not trivial.
It might be possible to use a
standard bioreactor in combination
with microcarriers to provide a surface
area on which such cells need to grow.
But those have not yet been optimized
for stem cell culture. Moreover, shear
stress might affect stem cell cultures
and require intensive process
development. A three-dimensional
(3D) scaffold might be a potential
alternative, but its configuration could
affect stem cell behavior (and
differentiation) by modifying the
niche microenvironment. There is no
guarantee that cells grown in any 3D
bioreactor would be the same as those
grown on a laboratory plate.
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Developing a Specific Solution
All those problems could be prevented
with a new multiplate design
approach. This would mimic the cellgrowth environment of a multitray
stack and minimize risks encountered
during process development. Cells
would still grow on two-dimensional
(2D) plates with similar physical
characteristics to those used in
Cardio3’s laboratory.
ATMI designed the Integrity
Xpansion bioreactor with plates made
from the same plastic (polystyrene) as
is used in multitray stacks. After a
common plasma treatment used to
hydrophilize the plastic, studies show
that the surface (Figure 1) is very
similar to that of a comparator.
It was also important to reduce the
size of the device. A compact design
was created by removing the gas-phase
layer that allows gas exchange into
media in which cells are growing. Gas
exchange is essential, but in the
Xpansion bioreactor it takes place
within a central column in which
medium circulates (rather than
between the plates). That reduces the
gap between plates from 15 mm to
just 1.6 mm, enabling ATMI to fit
≤180 plates in a single device about
60 cm tall (Figure 2 and 3).
The surface of each Xpansion plate
is ~614 cm 2, roughly equivalent to
each plate of a multitray stack. So one
180-plate bioreactor has the same cellgrowing capacity as 18 stacks (11 m 2)
in Cardio3’s laboratory with a much
smaller footprint than they would
require.
These circular plates contain 16
radial channels to circulate media.
Liquid moves up through the first
channel, flows horizontally over a
plate, then rises to reach a second
plate, and so on through all plates
until it reaches the top of the reactor.
It is then recirculated and returned to
the bottom of the stack — a design
optimized to minimize shear stress on
cells. The culture operates similarly to
multitray stack cultures without their
need for complex manual
manipulation. And there is only one
device to operate, not many.
Other technological improvements
involve regulation and control of cell
Figure 1: An Xpansion plate with 614 cm2
available for cell growth
Figure 2: (left) multitray stack 10; (right) 10
Xpansion plates
Figure 3: (left) 18-multitray stack 10; (right)
180-plate Xpansion unit
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Figure 4: Bone-marrow mesenchymal stem
cells cultured on an Xpansion plate (shown by
an Ovizio microscope)
culture parameters. An Xpansion
bioreactor can be operated as a fully
closed system, guaranteeing safety and
sterility during a cell culture process.
In addition to temperature
monitoring, ATMI added pH and
dissolved oxygen patches to each plate.
Combined with sensors on a unit’s
head plate, they enable monitoring
and control of gas exchange within the
bioreactor and maintenance of correct
pH and dissolved oxygen (DO).
Throughout a process, the precise
environment in which cells are
growing can be regulated and
controlled, which is particularly
important for fragile stem cells that
are very sensitive to their environment.
This will greatly increase process
reproducibility. For autologous cells, it
is important to remember that each
batch will be different. Stem cells
harvested from one patient will react
differently from those harvested of
another, so environmental control is a
necessity.
Another important feature of the
Xpansion system allows operators to
observe cells as they grow within the
bioreactor. Cell density can be
calculated automatically, and cell
morphology can be checked.
Specialized light microscopy
developed by Ovizio enables cells to
be observed on multiple layers of the
bioreactor with very high image
quality. This is also important for
autologous cells. Those from one
patient might take four days to reach
confluence, whereas cells from another
could take a week — and that is
unpredictable. Proper monitoring of
their growth will ensure that cells are
harvested at the optimum time
(Figure 4).
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Scale-Up Advantages
The biggest advantage of this new
single-use bioreactor is that switching
to it from multilayer stacks does not
affect the quality or nature of the
cells. That makes it possible to speed
up the scaling process while
decreasing risks. It eliminates the need
to start from scratch and develop a
new 3D manufacturing process in a
traditional cell culture bioreactor,
which would not be guaranteed to give
the correct cell morphology. No
aseptic operations are required
(compared with 2,000 such procedures
each day using previous technologies),
and both space and operator
requirements are cut by >60%. Only a
class C cleanroom is required for
transfer of cells into the bioreactor
because no open handling takes place.
And that is much less expensive to
install than the class B room needed
for multilayer stacks.
Reducing bioreactor and facility
footprint enables economically feasible
commercial-scale production, as
Cardio3 found. Scale-up from the
multitray stack process to one
supporting several thousand patients
would have been impractical in space
and operator requirements. The
Xpansion system makes that possible
with a significant reduction in both
those parameters. A quick calculation
of the number of batches and patients
indicates that 300 operators would
have been required for a multitray
stack scale-up; a validated cost
simulation performed with two
different customers showed that the
number could be halved with an
Xpansion system. The potential cost
savings are dramatic. We calculate
that for an autologous cell therapy to
treat 3,000 patients per year, the
annual operational expenses would be
reduced by 40%.
There are also benefits in capital
investment. The number of operations
that must to be carried out under
laminar flow is significantly reduced.
With a smaller footprint, fewer
incubators are required. These cost
savings are important because cellbased therapies are predominantly the
domain of small biotech companies
with limited access to capital. They
can’t risk investing several million
dollars in constructing new
manufacturing facilities. And
commercialization becomes a more
realistic prospect when capital
expenditure requirements are reduced
by 50%.
Using a close, compact, single-use,
multiplate bioreactor provides a
realistic solution to the problem of
scaling up a fragile, adherent-cell
manufacturing process —
guaranteeing that stem cells retain the
quality and morphology of those
grown in an R&D laboratory. This
would not otherwise be possible at a
commercial scale without prohibitive
investment and running costs.
Further Reading
Placzek MR, et al. Stem Cell
Bioprocessing: Fundamentals and Principles.
J. R. Soc. Interface 6, 2009: 209–232.
Rowley JA. Developing Cell Therapy
Biomanufacturing Processes. Stem Cell Eng.
106(11) 2010: S50–S55; www.aiche.org/
uploadedFiles/SBE/Restricted/
SBEOnlyNew/111050.pdf.
Rowley JA, et al. Meeting Lot-Size
Challenges of Manufacturing Adherent Cells
for Therapy. BioProcess Int. 10(3) 2012: S16–S22.
Integrity Xpansion. ATMI: Bloomington,
MN, 2012: www.atmi.com/lifesciences/
products/bioreactors/xpansion.html. •
Matthieu Egloff is product manager, and
Jose Castillo is director of cell culture at
ATMI LifeSciences; 32-2264-1868; megloff@
atmi.com; www.atmi.com. ATMI, Integrity,
and Xpansion are trademarks or registered
trademarks in the United States, other
countries, or both. Other names are
trademarks of their respective companies.
To order reprints of this article, contact
Rhonda Brown ([email protected])
1-800-382-0808. Download a low-resolution
PDF online at www.bioprocessintl.com.
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