Vol.
September
2015
Vol.12,
12, Supplement
Supplement
55
A S p e c i A l S u p p l e m e n t to B i o p r o c e S S i n t e r n At i o n A l
Enabling Cell Therapy Manufacturing
Enabling
Cell Therapy
Manufacturing
in association with
in ASSociAtion with
“ Together we
can advance
cell therapies
worldwide.”
Pall Life Sciences
Pall Life Sciences delivers the expertise and experience
to help our customers realize efficient, effective
development and commercialization of cell therapies.
Working with Pall, you’ll benefit from our industry-leading cell
manufacturing technologies and services – from our unique single-use
bioreactor systems to our innovative SoloHill® microcarriers. You’ll be
fully equipped to overcome the challenges of manufacturing live cells for
therapeutics, making your path to industrialization smoother than ever.
Pall’s business philosophy is about collaboration and helping you achieve
your goals. Together, we can advance the development of cutting-edge
cell therapies... and dramatically improve the lives of patients worldwide.
Pall Life Sciences
Your vision. Our expertise. Their future.
www.pall.com/celltherapy
© 2015 Pall Corporation. Pall,
and SoloHill are trademarks of Pall Corporation.
® indicates a trademark registered in the USA. GN15.9698
September 2015
Expansion and Characterization of Mesenchymal
Stem Cells on Pall SoloHill® Microcarriers . . . . . . . . . . . . . . 22
Heather Woolls, Dave Splan, and Mark Szczypka
Strategies for Microcarrier Culture Optimization . . . . . . . 28
Mark Szczypka and Alain Fairbank
Section 3: Cell Therapy Manufacturing
Enabling Cell Therapy Manufacturing . . . . . . . . . . . . . . . . . . . 2
Alain Fairbank
Pall’s innovative products and capabilities for production of
cellular therapies
Designing the Most Cost-Effective Manufacturing Strategy
for Allogeneic Cell-Based Therapies . . . . . . . . . . . . . . . . . . . . 36
Section 1: Pall in Cell Therapy
Introducing Pall’s commitment to support of cell therapy
manufacturing and commercialization
Positioning for Success: An Interview with Mario Philips . . 4
S. Anne Montgomery and Brian Caine
Meeting Lot-Size Challenges of Manufacturing
Adherent Cells for Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Jon Rowley, Eytan Abraham, Andrew Campbell,
Harvey Brandwein, and Steve Oh
Section 2: Process Development
Tools and technologies from Pall Life Sciences designed to
facilitate process development and scale-up
T-Cell Suspension Culture in a 24-Well Microbioreactor:
High-Throughput Screening of Operating Conditions . . 14
Kenny Choi, Jason N. Carstens, and Shelly Heimfeld
Thierry Bovy, Alain Fairbank, and Suzanne Farid
Production of Viral Vectors Using the iCELLis®
Fixed-Bed Bioreactor System: Beyond Mesenchymal Stem
Cells — Gene-Modified Cell Therapy, Gene Therapy, and
Exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Matt Kremer
Single-Use Bioreactors and Microcarriers:
Scalable Technology for Cell-Based Therapies . . . . . . . . . . 48
Mark Szczypka, David Splan, Heather Woolls,
and Harvey Brandwein
Section 4: Perspectives
Experts at Pall Life Sciences discuss cell therapy
industrialization solutions and future directions
Ask the Experts: Core Technologies Expand Opportunities
for Cell Therapy Manufacturing . . . . . . . . . . . . . . . . . . . . . . . . 54
Scaling Up Stem Cells: Moving from Laboratory
to Commercial Productions with a Single-Use
Multiplate Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
S. Anne Montgomery
Matthieu Egloff and Jose Castillo, with Thierry Bovy
S. Anne Montgomery and Brian Caine
Positioning Tools, Technologies, and Talents for Cell
Therapies: An Interview with Harvey Brandwein . . . . . . . 60
Featured Products and Services . . . . . . . . . . . . 21, 43, 47
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1
BioProcess International
13(8)sp
S eptember 2015
Sponsored Supplement
Introduction
Enabling Cell Therapy Manufacturing
by Alain Fairbank
A
s this special Pall supplement
of BioProcess International
issue goes to press, progress
continues in the field of cell
therapy research. The revival of cell
(and gene) therapy has been driven by
some positive achievements that have
occurred over the past decade.
Cell therapy products differ in many
ways from traditional small-molecular
and biologic products. The main
difference is that, contrary to traditional
biopharmaceutical applications in which
cells secrete the product of interest, in
cell therapy applications cells are the
product. That brings a multitude of
challenges as manufacturers navigate
the industrialization pathway to
determine an optimal manufacturing
strategy for their therapy.
Despite those challenges — or
perhaps because of them — the
demand for manufacturing tools to
support this industry continues to
grow. Some key drivers include the
following:
Scalability — adherent cells such as
mesenchymal stem cells (MSCs)
present a manufacturing challenge as
lot sizes increase from billions to
trillions of cells (1)
Cost — achieving lot sizes of
several hundred billion to trillions of
cells efficiently and cost effectively
will be imperative for commercial
success (1).
Flexibility — predicting market
demand is never easy, particularly for
emerging technologies that lack
historic precedent, so manufacturing
flexibility (both in scheduling and
capacity for rapid production scale-up)
is vital to the commercial success of
the cell therapy industry (2).
Closed systems — one of the most
challenging aspects in cell therapy
manufacturing is conversion of research
2
BioProcess International
13(8)sp
processes to manufacturing campaigns,
which is most readily achieved by
converting to closed systems (3).
The general purpose of this
supplement is to discuss many
industrialization challenges facing
developers of cellular therapies today,
while highlighting how Pall’s platform
of products and services has been
developed to address those challenges
and consequently enable its customers
on their pathway to cell therapy
commercialization.
Pall in Cell Therapy
This special issue commences with an
interview with Mario Philips,
president of Pall’s single-use
technologies division. He discusses
Pall Life Sciences’ commitment to the
cell therapy industry and how Pall has
positioned itself with a platform of
unique and innovative cell expansion
technologies and services.
Pall Life Sciences recognizes that
the advancement of cell therapies —
and, ultimately, that the commercial
success of such products — can be
achieved through the combined
strengths of our customers and the
Pall team. Long recognized for its
expertise in processing and filtration
equipment for the biopharmaceutical
industry, Pall Life Sciences has
S eptember 2015
broadened its offering in upstream
manufacturing in recent years. It has
done so by expanding its core
capabilities in the single-use,
bioreactor, and microcarrier arenas,
with unique and innovative
technologies for cultivation of cells for
therapeutic applications.
“Meeting Lot-Size Challenges of
Manufacturing Adherent Cells for
Therapy” is an abridged article
originally published in BioProcess
International in March, 2012. Readers
of this article will learn from industry
experts, including Pall’s own Harvey
Brandwein, about some hurdles,
challenges, and bottlenecks associated
with bringing a cell-based therapy to
market.
Process Development and
Cell Therapy Manufacturing
Developing the right industrialization
strategy is critical to supporting a
sustainable cell therapy development
and commercialization program.
“Designing the Most Cost-Effective
Manufacturing Strategy for Allogeneic
Cell-Based Therapies” was generated
from a webinar presented in March of
2015. In their presentations, Thierry
Bovy of Pall Life Sciences and Professor
Suzanne Farid from University College
London (UCL) discussed advantages
Sponsored Supplement
and limitations of current technologies
available for the commercialization of
large-scale allogeneic therapies. Farid
also provided some key insights from an
advanced bioprocess economics model
designed by her team. Also discussed in
this article are how the economic
aspects of cell therapy products need to
be addressed from the early phases of
development to enable a viable life cycle.
Some of Pall’s key technologies for
the production of cell-based therapies
— the Micro-24 Microreactor,
Xpansion® Multiplate Bioreactor, and
Pall SoloHill® Microcarriers — are
highlighted in the process development
section of this supplement.
In the article, “T-Cell Suspension
Culture in a 24-Well Microbioreactor”
(originally published in BioProcess
International in April of 2013), Choi et
al. present results from a study using
the Micro-24 MicroReactor from Pall
Life Sciences, a 24-well
microbioreactor with the capability for
agitated suspension culture and
continuous monitoring and control of
pH, temperature, and dissolved oxygen
(DO) in each individual well. The
team valuated its usefulness as a
process development tool to develop
and optimize suspension cell culture
for manufacturing therapeutic T cells,
and they demonstrated the effectiveness
of the Micro-24 MicroReactor as an
accurate, high-throughput experimental
system that properly represents
production-scale systems.
Designed for shear-sensitive,
adherent-cell applications such as stem
cell cultivation, the Xpansion multiplate
bioreactor is part of the Pall Life
Sciences’ single-use bioreactor family.
The article, “Scaling Up Stem Cells”
was first published in May of 2012. In
it, the authors discuss the development
of this unique bioreactor system for use
in scale-up and expansion of stem cells
for therapeutic applications. The authors
highlight three key advantages of
implementing this technology: speed,
ease of adaptation, and reduced
footprint.
Several clinical trials have been
initiated using stem cells in cell therapy
treatments. Fast emerging as a premier
technology for the (very) large-scale
expansion of adherent cells for therapies
Sponsored Supplement
is the microcarrier production platform
used with stirred-tank bioreactors.
“Expansion and Characterization of
Mesenchymal Stem Cells on Pall
SoloHill Microcarriers” was originally
published as an application note. In it,
the authors discuss some additional
benefits of using microcarriers to
expand mesenchymal stem cells and
demonstrate that such cells can in fact
be expanded on a number of
microcarrier types while maintaining
their ability (post expansion) to
differentiate into adipocytes and
osteocytes.
As Oh, et al. discuss in “Meeting
Lot Size Challenges” the ability to
scale up to very large volumes of cells
has been a challenge that the cell
therapy industry has been working to
overcome. Professor Farid and her
team’s work has identified
microcarriers as the most costeffective platform to produce the high
billions to trillions of cells required for
some cellular therapeutics.
In the “Strategies for Microcarrier
Culture Optimization” article, Mark
Szczypka and I outline key
considerations that need to be taken
into account when developing an
optimized microcarrier platform. We
also highlight the strategies necessary
to enable a successful process
development and commercialization
using a microcarrier platform.
In recent years, gene therapies have
rapidly progressed as products move
from academic research laboratories
into commercial development. Many
such therapies in development require
large amounts of viral vectors for use in
modification of cells to be used as
therapeutics. As Matt Kremer reports
in his article, “Production of Viral
Vectors Using the iCELLis® Fixed-Bed
Bioreactor System — Beyond
Mesenchymal Stem Cells: GeneModified Cell Therapy, Gene Therapy,
and Exosomes,” Pall’s iCELLis fixedbed bioreactor has been successfully
applied to this challenge in a number
of academic and industrial institutions.
They have demonstrated scalable
production of viral vectors including
retrovirus, adenovirus, recombinant
adenoassociated virus, and lentivirus in
the iCELLis system. Kremer presents
findings from several experiments
demonstrating the effectiveness of that
system for such applications. He also
discusses its utility in providing a longterm, continuous process environment
for cultivation of stem cells and
harvesting of exosomes — an up and
coming area of research.
Perspectives
Pall Life Sciences has been long
recognized as a leader in processing
and filtration systems for the
biopharmaceutical industry. In recent
years, the company has expanded its
product offerings by moving upstream
with core technology development and
acquisitions in single-use bioreactors
and microcarrier technologies. For this
special supplement, we’ve invited three
of our subject-matter experts —
Fabien Moncaubeig, Thierry Bovy,
and Mark Szczypka — to discuss
some of these technologies. They also
provide an overview of the process
development capabilities now offered
at four of Pall’s sites.
This special issue concludes with
an interview in which Harvey
Brandwein (vice president of business
development at Pall Life Sciences)
provides his perspectives on the cell
therapy industry today, where he sees
it going, and how he sees Pall’s role in
helping to advance cell-based therapies
through the industrialization pathway
to clinical success and the ultimate
reward: commercialization.
We hope that you will find these
articles both constructive and
informative as you navigate your
development journey in this very
exciting therapeutic area.
References
1 Rowley J, et al. Meeting Lot-Size
Challenges of Manufacturing Adherent Cells
for Therapy. BioProcess Int. 10(3) 2012: S16–S22.
2 Davie NL, et al. Streamlining Cell
Therapy Manufacture. BioProcess Int. 10(3)
2012: S24–S28, S49.
3 Sargent B. Best Practices in Cell
Therapy Manufacturing. The Cell Culture Dish
6 February 2013. •
Alain Fairbank is director of cell therapy
marketing for Pall Life Sciences,
alain_fairbank@pall.com.
S eptember 2015
13(8)sp
BioProcess International 3
S e c t i o n O n e PALL IN CELL THERAPY
Positioning for Success
An Interview with Mario Philips
O
n 12 March 2015, BPI met
with Mario Philips,
president, single-use
technologies, at Pall’s Port
Washington, NY, facility, to learn
about Pall’s reasons for entering the
cell therapy market. Also participating
in the discussion was Alain Fairbank,
director of marketing for cell therapies
at Pall Life Sciences.
The discussion began on a personal
note, asking Philips what it was that
launched his interest in single-use
technologies and cell therapies.
Philips: My background is as a
chemical engineer. I had worked for a
couple of years in the
biopharmaceutical industry, more in
the analytical space, and then started
my own business. We targeted the
chemical market for inline and online
process analysis.
We were picked up by the
semiconductor industry and then were
acquired by ATMI in 2002. At that
time ATMI had a materials division
that dealt with chemicals and
packaging of liquids. What not a lot
of people know is that ATMI already
had a relatively large business in
single-use liquid packaging. The
semiconductor industry was facing the
challenge of molecular and particle
contamination when cleaning the
stainless steel containers used for
transportation; and they had a lot of
yield and cleaning issues. So we
developed a bag-in-a-bottle and a bagin-a-container system.
It’s a very different business model
from what you find in life sciences. It
was very standardized. We were
shipping dangerous chemicals, and
there was not a lot of customization.
The film materials were, in 70% of
the cases, also very different — more
20154
BioProcess International
13(8)sp
like working with PTFA because of
the very aggressive chemicals.
ATMI by then had a $40–$50
million business. Meanwhile, I was
staying in touch with my friends in
the life sciences industry, where people
were beginning to use bags. A friend
asked me, “Isn’t that what you do in
microelectronics?” So we started to
look at that. By then, HyClone and
Stedim were already becoming
established in the life sciences.
We knew that we needed to enter
that market with adequate credibility.
So we asked customers what was not
yet addressed by current technology.
Two big answers came out of that:
mixing and bioreactors. At that time
in our corporation we didn’t have a lot
of cell culture competencies. We said:
“Let’s try to do one thing very well
and become the leader in SUT (singleuse technology) mixing.” Then later,
the company evolved its bioreactors
business. So that is how I ended up in
the single-use technology space.
My personal interest in cell therapy
began with my exposure to the
biotechnology industry in Belgium.
That country is quite innovative in
biotech for its size. The big player
there was GSK Biologics, with more
than 10,000 people on site, and a
number of those people were
launching start-ups. A number of
those scientists went into cell therapy.
Not many equipment manufacturers
saw investment in cell therapy
companies as a promising business
model. But we did.
At first we got into cell therapies
more to accelerate our learning curve
than as an investment. But investing
gave us access to CEOs, business
plans, and information about cost of
ownerships. The big lesson was that
S eptember
Brenner Photo Productions (www.brennerphoto.com)
by S. Anne Montgomery and Brian Caine
Mario Philips, President, Single-Use
Technologies at Pall Life Sciences
they were all using static devices —
CellSTACK® culture chambers,
typically. Using multiple containers
for each patient works well when your
target is to make clinical material for
20 patients. But if you want to treat
2,000 patients, that isn’t going to get
you there. That was a problem that we
recognized early on and that we are
still very focused on today, at Pall.
The cell therapy industry is still
largely at laboratory and R&D scales.
But we want to help companies get
more out of their R&D-related
technologies and help them either
scale out (for autologous therapies) or
scale up (for allogeneic therapies).
That was a fundamental problem to
address. Some of the CEOs
acknowledged that if they had to scale
up using static devices, they would
need a square kilometer of clean room
space before they could even think
about going to the FDA. For example,
the first customer we met needed 20
CellSTACK CF10 units for one
patient. His therapy required 725
manual interventions. We were able to
simplify the process and streamline
Sponsored Supplement
that company’s capital investment to
make its industrial plan realistic.
Cell therapy is exciting in that
scientists are doing almost unbelievably
innovative work that can have a
significant impact on us as patients.
We can all be exposed to diseases
requiring such treatments. Bringing
the initial idea to market is a high-risk
project with many challenges. The role
we like to play at Pall is to provide
innovative, cost-effective technology.
That’s a big challenge in all of
biopharma, of course, but definitely in
cell therapy.
And then Pall also brings 60 years
of experience in bioprocessing to these
smaller start-ups — because that’s
what these cell therapy companies
typically are. They are, very often,
very scientific, but they do not have
much expertise in bioprocess
engineering.
The Need for Innovative
Technologies
Caine: How much existing proteinbased technology can be used in the
cell therapy market? What changes or
innovations need to be made for largescale cell therapy manufacturing to
become affordable?
Philips: There are two types of
therapies. Scaling up autologous therapies
is quite different from what
biotechnology typically does. In the
autologous model of personalized
medicine, a company deals with small
volumes. But if it wants to treat
thousands of patients, it needs to find
a way to scale that process out.
Innovation has to happen. At Pall we
are doing some of that — developing
replacements for traditional static
devices, such as by using expansion
bioreactors. The goal is to close the
process and develop industrial-scale
automation around it.
In the case of allogeneic therapies, I
think the biotechnology and vaccine
models are much more leverageable.
We can give a customer two options.
If a customer is tackling a treatment
for an orphan disease for which there
may be fewer than 10,000 patients,
that company probably can use some
type of expansion reactor. Again, the
goals are to close up the process,
reduce the risk, and bring in some
Sponsored Supplement
controls. For allogeneic therapies,
much of that technology is already
available.
We use microcarriers and single-use
bioreactors in vaccine development. But
the biggest difference between
biotechnology and cell therapy is that
the cells themselves are the product in
cell therapy. That introduces some
challenges. One area where innovation
is still needed is in segregating the cells
from the carriers safely - plastic
material must not end up in a patient.
I believe also that the volumereduction step requires more attention.
We are trying to approach it with
centrifuge technology and tangential
flow filtration (TFF). But that is still
early stage work for us.
Allogeneic development uses basic
cell culture technologies — bioreactors,
microcarriers, and so on. The main
difference is how to work with these
very fragile cells. Again, the
fundamental difference is that you need
the cells not to segregate as in the other
biotechnology applications.
All in all, however, some dedicated
discrete products are available. Our
vision at Pall is that we want to turn
them into an industrial manufacturing
platform so that people can produce
cells safely in a closed environment.
The demand in the market is still
low, with maybe four, five, or six
companies that are really ready for
prime time and have to flesh out their
manufacturing. But if you look at the
clinical trials, the pipeline is huge.
Some won’t make it, but many will.
Our focus is on leveraging existing
technologies from the biotech space
and then trying to close gaps where
needed. The goal is to create an
industrial solution.
When to Enter the Market
Caine: There are other suppliers to the
cell therapy market. Some of them
want to play in this area, but many (if
not most) of them are waiting. They
are waiting for the market, they are
waiting for a return, and they are
waiting for a bit more clarity in the
market. But Pall doesn’t seem to be one
of those companies. Pall is making a
significant investment and a significant
push into cell therapy now. Why is this
the right time?
Cell therapy companies
are more and more
thinking about what
their CORE
competencies are —
where they excel versus
where they need
partners.
Philips: That’s a very good
question. If you look back on the cell
therapy industry, five or seven years
ago a lot of people thought it was
breakthrough time, right? You could
see that at the Cell Therapy Congress
among many vendors and customers.
And then we had a bit of a dip when
the financial crisis prevented some cell
therapy start-ups from obtaining
funding.
Why we feel that now the chances
of a breakthrough are much better are
1), that there is much better funding
and 2), that we have a couple of
commercial therapies on the market.
But it is still fairly early. I think our
industry needs a couple of success
stories now. Otherwise you could look
at it very pessimistically. The two or
three approvals of 2014 didn’t really
take off. All of them are having
problems. But that also helps others
learn how to do it better. One of the
players did think too late about how to
manufacture huge quantities. That
sounds like a positive problem: We will
deal with it when it’s there. But your
investors don’t care. If you are not
delivering on your demand, you are
destroying value, and they will pull
out.
At Pall we believe it is still a risk,
but we believe it is a more calculated
risk, and we want to be first-to-market
with this platform. So we are investing
ahead of the potential. We want to be
associated with the top four, five, or six
that come through now, and we hope
that will give us the long-term benefit.
The Importance of Managing
Long-Term Expectations
Caine: Your prior example was of a
scientific success that was a business
failure. Was it a necessary failure in
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the evolution of cell therapy
companies to understand what they
need to do to be successful?
Philips: Probably. Yes. If you dig
into other industries, they all have
such early examples.
Montgomery: So it’s still an
investment in the future at this point.
Do you feel that Pall’s experience in
the general biopharmaceutical industry
prepares you to wait out the cell
therapy industry — to follow it for a
couple of years before you see more
commercial successes?
Philips: Yes. Of course, we are very
focused on the short-term returns as
well, but we know we also have to
place some bets on the long-term. If
we look at our strategic plans for our
core business in filtration with the
vaccines and the biotech market, three
years is a good strategic plan to look
at. But in cell therapy, we are really
looking more at five to seven years.
Caine: Do you see a change in
behavior and understanding among
cell therapy companies toward earlier
creation of a commercial business
plan? How has that changed in the
past few years?
Philips: I think cell therapy
companies have one big challenge in
finding the right sort of partnerships to
allow them to advance. In the past,
many believed that because the cell is
the product, they had to manufacture
completely in-house. So although some
companies may still set up their
businesses in that way, other are
starting to look at partners. If you are
an investor, you hope that the cell
therapy company will use your money
for reaching important clinical results.
Then you will attract more investors
and be able to fund your scale-up.
So cell therapy companies are more
and more thinking about what their
core competencies are — where they
excel versus where they need partners.
For example, I know that some of
them start to do some in-house work
and make some clinical materials, but
also immediately involve CMOs. They
recognize that they need to drive
down the needed capital and not put a
lot of money into clean rooms and
manufacturing infrastructure. That
makes any investor quite nervous. The
lesson is that some of the work has to
be done in-house, but more and more
there are specialized CMOs who are
helping with the clinical stages.
The Role of Contract
Manufacturing Organizations
Montgomery: Are you seeing more
dedicated CMOs coming into this
space?
Philips: Yes, and pretty much
everywhere. In the United States we
have Progenitor Cell Therapy and of
course Lonza. In Europe, in Belgium,
a company there is MaSTherCell. I
have met a company in Asia that wants
to dedicate itself only to cell therapy.
It’s a little bit of a different animal. A
lot of the CMOs today have large
stainless steel reactors, so they do not
necessarily have the right infrastructure
to serve a cell therapy customer. They
also understand some of the time issues
involved, too, especially with
autologous therapies, and that the
timing differs from any classic model.
Caine: Your target customer is the
end-user and the CMO. From a
technology development standpoint, is
one a better partner than the other? If
you want to understand the allogeneic
need to produce billions, trillions of
cells, who do you find to be a more
involved or leading partner?
Philips: Well, it is definitely always
both. But the immediate focus is
definitely on the end-user customers
because I think in most cases they will
prescribe to the CMO what to use.
Caine: But whose responsibility is it
to develop new technologies? We
S eptember
talked earlier about gathering
information and doing trend analysis
regarding what’s needed and where.
Would a CMO be more of a partner
with you because it is actually
manufacturing a cell therapy and can
tell you what is lacking, what is
needed? Do you have that interaction
with PCT and some of these other
CMO partners?
Philips: Sure. But they bring the
same messages to us as the end-users.
They are facing the same problems.
Fairbank: The challenges exist
whether the end-user or CMO is
scaling up. There are gaps and
challenges that need to be overcome.
The CMO is somewhat, I would say,
the voice of their customer.
Technology Transfer
Expectations
Montgomery: Are there unique
technology transfer issues that people
are grappling with?
Philips: I think not unique, but it’s
a very good question. It’s quite
challenging from being at a couple of
milliliters, then going to large types of
reactors. If you look at the clinical
market today, many people today are
in phase 1 and in CellSTACKs. So
going from a CellSTACK to a
microcarrier type of process is quite
disruptive. It will be challenging.
These cells will be more sensitive than
the ones we used to deal with and,
honestly, the scale-up and the transfer
later on is highly complicated.
That is one of the major reasons
why we introduced our Xpansion®
bioreactor technology first rather than
using microcarriers with a bioreactor,
because in the Xpansion system, we
are really not changing the
environment for the cell. So although
it’s a big change, with all the benefits
of scalability and closed systems, at
the end the cells feel completely the
same. We have been very successful in
tech transfer from static devices into
what I could call a huge redesigned
CellSTACK. That’s a big benefit
because otherwise the technology
transfer gets very complicated.
Now customers that want to serve
markets of ten thousand or hundreds of
thousands of patients will have to go to
Sponsored Supplement
To Manufacture or Be Acquired?
Caine: Do you see a trend overall in
the strategy of cell therapy companies
right now toward being acquired
rather than planning to manufacture?
Philips: I think we are going to see
a lot more therapy companies that
only want to bring their product to
phase 2 and then try to sell off, which
will largely be to big pharma.
One of the trends you also see in
that respect is that big pharma is
starting to get into these start-up
companies through its venture capital
groups. That’s with only one
intention: to have a preferred position
to one day acquire the company. So I
think the tendency will be that more
people will just create value at the
point of that investment.
Also, bringing a therapy to market
requires understanding the logistics
and the dynamics, and for that, of
course, big pharma has all the
experience. I think we’ll see a lot of
companies change ownership after
initial therapeutic proof that a therapy
works. So although some companies
will want to go to the end, I think
we’ll see more of them being acquired
and merged into large pharma.
Caine: Do you also see cell
therapy–cell therapy mergers and
acquisitions?
Philips: There are one or two that
have happened, but it is still quite
exceptional.
Sponsored Supplement
Caine: Pharma does bring greater
financial resources and manufacturing
capabilities to the table, however.
Montgomery: And established
distribution networks.
Fairbank: And related logistics.
Philips: That’s huge for a standalone cell therapy company to
undertake logistics. The one other
tendency that I see is that big pharma
may be waiting a little bit longer to
acquire an early stage cell therapy
company. They used to buy companies
in phase 1. It was cheaper, despite the
risk. Now the trend is to say, “Let it
be more expensive, but more proven.”
So that’s another thing that we’ll see
in cell therapy. Investors don’t want to
necessarily get in too early. It’s still
cheaper, but there is far more
uncertainty.
The Role of Quality by Design
Brenner Photo Productions (www.brennerphoto.com)
something like a reactor, and they will
have to go through that change.
But it’s always a challenge in our
industry. People get into phase 2 and
start thinking about how to
manufacture product for a hundred
thousand patients — and everybody
gets nervous about changing the
process and explaining it to the FDA.
You’re probably going to have a
setback and have to go back a phase
and re-do a lot of work. Clearly for
cell therapy companies it is very
important at the very early (stages) to
figure out what their end game is. I
think some of them are facing issues
today because their tech transfer will
be highly complicated and explaining
the technology to the regulators will
be almost impossible.
Brian Caine, Publisher of BPI
Pall’s Technologies
— Present and Future
Montgomery: To what extent will
current risk-based approaches apply to
cell therapies? Does QbD have a
place?
Philips: For manufacturing
allogeneic therapies, it’s quite the
same sort of process, and cell-therapy
CEOs realize that the need to bring
in bioprocess expertise. More and
more of these company leaders come
from large biotech. They will start
using the same risk-based approaches
because they bring their expertise
into this field. I have seen many,
many examples of this already. People
who have been with Amgen, Biogen
or from vaccine companies are
implementing the same type of
approaches to risk. I think bringing
that expertise into the cell therapy
space will definitely give us better
chances that this industry will
prosper because these people have
lived it.
Fairbank: This was really validated
just yesterday, when I attended a
presentation by the FDA. They were
discussing the importance of
identifying critical process parameters.
Applying quality by design and the
whole risk assessment approach for
cell therapy is just as important. Down
the road, we know that there were be
more regulations to come.
Caine: Can you tell us about Pall’s
technologies, their current application
to this market segment, and your
unique position? Where do you see
yourself as a leader in the cell therapy
market, and what products does Pall
have to support that?
Philips: Our focus is currently on
industrial manufacturing, from smallto large-scale devices. I think we have
a strong position in what I call the
expansion step of the cells. We have
more product development to do in
what I call the volume reduction step.
Then we want to leverage expertise
from our current business — one such
area is process development.
We have four process development
hubs across the world: one in Brussels,
one in Portsmouth in the United
Kingdom, one in Ann Arbor,
Michigan, and in Westborough,
Massachusetts. A lot of our customers
are cell therapy people. They ask us,
for example, to start the expansion
process in a small-scale Xpansion
reactor, scale it up, and then either
transfer it back to them or transfer it
to a CMO.
The second area that we want to
leverage is our internal biopharm
automation and process group. We
build large chromatography skids. We
set up complete single-use suites with
the bioreactor in the middle and the
mixers around it. We are building on
that expertise to connect these great
products into a real platform solution.
So I would say our focus is
currently on industrial manufacturing;
helping customers get out of a relative
International
S eptember 2015
13(8)sp
BioProcess
7
crunch that they are in. In looking at
future strategies, one desire for us is to
be able to offer cell culture media. We
are also dedicated to bringing
technology solutions to the
downstream process for volume
reduction. We have a centrifuge
technology in development. We have
single-use TFF. These are strengths in
our vaccine and the biotechnology
businesses, so we hope to close the
gaps related to cell therapies. We can
do that by combining existing
expertise looking into mergers and
acquisitions. Because our mission is to
really have a total manufacturing
solution for future cell therapy
companies.
We will be less involved in
analytical aspects, cell isolation and all
that. So even in cell therapy, we will
try to focus on our core capabilities.
Caine: Not too long ago when
protein titers went from one to 10+
grams, there was concern about the
downstream bottleneck and not being
able to clarify or purify at those
volumes. Is the challenge for cell
therapies going to be dealing with the
very large volumes needed for trillions
of cells, or is it about the gentleness
with which you handle the cells?
What do you see as being perhaps a
solution to the current challenges on
the downstream side?
Fairbank: We know that the cells
for cell therapy are the product and
they need to be treated more gently,
and so I agree that that’s an issue.
We’ve got some things in development
as Mario said, but scaling up to deal
with processing lot sizes of several
hundred billion or up to a trillion cells
is still a gap that needs to be
addressed.
Philips: Probably the biggest
challenge of the customers is more in
the downstream volume reduction
step. You see presentations in which
people say that have expanded cells,
and somebody asks them: “Okay, now
what?”
Caine: You can’t have a process if
you have half a process.
Fairbank: The good news is nobody
is really at that “now what?” stage
where it’s so critical, it’s going to stop
the works. Everybody has been talking
about it enough that people who are
20158
BioProcess International
13(8)sp
Bringing
QUALITY BY
DESIGN
EXPERTISE
into the cell therapy
space will definitely
give us better chances
for this industry to
prosper.
indeed listening. Issues of scale are
clearly identified as an unmet need.
There is still a window of opportunity
to develop the solution before it’s
necessary or, worse, becomes an
impediment
Montgomery: Thinking about
these steps so early in the game is still
kind of a hard sell for some people, it
seems.
Fairbank: You know, it is. I was at a
conference in Brussels in December
and the question was put out to the
people who were attending the
conference: “How many are in 2D?”
Just about everybody in the room
raised their hands. “How many are in
3D?” and a few raised their hands.
Then the question was posed: “How
many people are thinking about the
transition from 2D to 3D when you
scale?” Only one responded to that
question. I found it quite surprising to
find that so few people were thinking
about it yet.
Driving that point home, especially
if you know you are developing a
treatment for a large population, you
need to concurrently — in phase 1,
before your process is locked in —
develop side-by-side what your
commercial process is going to be.
Montgomery: Including looking at
eventual reimbursement issues?
Fairbank: Yes, reimbursement
issues — which will help to drive the
entire manufacturing strategy.
Montgomery: Is this message
getting across?
Fairbank: It’s starting to more and
more. We certainly have been making
some noise about it or the past year
since I joined Pall. The noise level
has certainly been elevated, and the
message is getting across.
S eptember
Bridging the Commercial Gaps
— Who Are the Leaders?
Caine: I’ll come at it from a
publication standpoint. This will be
our fifth year with cell therapy and
trying to bridge that gap between
research and commercialization and
the technologies. Three or four years
ago, even two years ago, you heard
very little about phase 3 commercial
scale — understanding the financial
business dynamics. You just heard:
“Check this out. We can do this. Look
how cool this is.” The whole tone has
changed in the marketplace. I take
it this is also one reason why you’re
committing to this market — that
you see a transition toward a maturing
market. What geographic area do you
see as being a leader in development,
either from a regulatory standpoint or
a scientific standpoint?
Philips: I would say the United
Stated is definitely leading; that’s
where most activity is. There is
activity in Europe, but from a leading
perspective, it’s the United States, and
then Europe, and now Asia. The
Japanese industry is receiving quite a
bit of support from its government.
That’s another element leading toward
a breakthrough in the industry: Many
governments are starting to
incentivize and give subsidies to cell
therapy developers, as well as offering
big manufacturing initiatives.
One example is the Catapult
Initiative in the United Kingdom. Its
vision is to build a manufacturing
hotel where a small cell therapy
company can come in to produce its
cells. Initially maybe it needs the
building for three or four months to
do its clinical work, and then it can go
out and say, “For my next phase, now
I have all the clinical results, and I’m
going to find an investor.” That’s
another area where you can see some
governments becoming more
supportive in their regulatory climates,
as in Japan recently.
If you were to ask me what one big
risk remains for this industry, I think
the most uncertainty rests in the
reimbursement strategies of local
governments. Personal medicine is
always going to be quite expensive as a
therapy. That’s a reality. Allogeneic
Sponsored Supplement
Sponsored Supplement
Philips: As I mentioned earlier, our
industry desperately needs a couple of
success stories that will attract more
investors, more big pharma. They will
be seen as providing a proven business
model. In the short term it’s all about
that. Five years from now, if we have
these success stories, I am confident
that cell therapy will be a wellaccepted industry already largely
dominated by and enhanced by
biopharma. It will be almost a
condition for cell therapy to succeed
that the biotechnology industry will
invest into this space and bring in
expertise in logistics and
infrastructure — as well as experience
managing the regulatory piece. So big
pharma stepping in will probably
increase the chances of success for the
industry.
Some Final Words
Caine: Do you have an opinion on
where you think Pall’s role will be in
this market if you are, again, to look
forward five years?
Philips: I hope to be recognized as
a leader in industrial manufacturing
for cell therapy. Everybody goes to
work and does that typically for
money. But one thing I’ve learned
with my employees is that cell therapy
is a very motivating area. Our people
like to be successful with the company
because everybody can see the impact
of our work on the lives of patients.
The Pall retrospective in five years
will be to say that we have helped
world-class scientific people live their
dream by offering them cost-effective,
innovative technologies. That would
be awesome. I think helping them get
to an economical, viable
manufacturing strategy has to be our
role. That’s where we can make an
impact. Drive cost out of it, drive risk
out of it. We will benefit, of course,
from that. But this is the key: Besides
reimbursement, making
manufacturing economical will also
drive cost down the cost of current
therapies.
Caine: It’s a challenging but
exciting place to be. How often you
get to be at the early stages of a
developing industry?
Fairbank: It’s a good reason to get
up and go into the office in the
morning. It is a very exciting industry,
and it’s motivating to be part of it.
Philips: When people ask me, “So,
Mario. What are you doing?” I very
often talk about cell therapy because
of the passion that these people bring
to their work. They are very
entrepreneurial and very scientific,
and know what a significant impact
their success can have on the lives of
other human beings. c
S. Anne Montgomery is cofounder and
editor in chief, and Brian Caine is cofounder
and publisher of BioProcess International,
amontgomery@bioprocessintl.com;
bcaine@bioprocessintl.com.
Brenner Photo Productions (www.brennerphoto.com)
therapies might be more affordable,
but there are investors that want a
return. Only therapies that really
make significant impacts on the lives
of patients will get reimbursed.
It will depend, that is, on how we
treat the disease today and what the
cost is — and the cost of ownership. If
you have a patient with heart disease
and poor quality of life and who has
to go into the hospital a lot, he might
cost the healthcare system $100,000
per year. If you then come in with a
fundamental fix that has a one-time
cost of $100,000, that’s an easy
reimbursement discussion. But there
are also going to be therapies where
the impact is perhaps quite good, but
there is not much benefit to the
healthcare system. Will those
therapies be reimbursed? That is an
ethical question, and there is still
uncertainty in our space. But, again,
really disruptive therapies will get
reimbursed.
Montgomery: Do you see the startup companies doing a pretty good job
examining their competitive position
with regard to existing therapies? Is it
something they have to get better at?
Philips: I think they are getting
better at it. But I think that in the
past, our very scientific industry was
not paying much attention to market
potential. The pipeline definitely has
companies targeting the same
diseases. But I think newer companies
do scan the market to validate the
areas where they can play. It’s a more
mature industry. Each cell therapy
needs easily $60 million to get
through the clinical phases. You’re
going to have to raise that capital.
You’re going to have to show investors
what your target markets are, where
the competitors are — even among
alternative competitive treatments
today. The companies that we have
invested in have very solid business
plans including that type of analysis.
Caine: If you had a crystal ball and
wanted to flash forward two, three, or
five years, where do you think the cell
therapy market will be then? What
lessons do you think you’ll look back
on and say: “These were important
lessons or important steps that we had
to go through.”
(Left to Right) Brian Caine (Publisher, BPI); Mario Philips (President, Single-Use
Technologies); Alain Fairbank (Director of Marketing, Cell Therapies); Harvey Brandwein
(Chief Technology Officer, Senior Vice President of Research); and
Anne Montgomery (Editor in Chief, BPI)
International
S eptember 2015
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BioProcess
9
S e c t i o n O n e PALL IN CELL THERAPY
Meeting Lot-Size Challenges
of Manufacturing Adherent
Cells for Therapy
by Jon Rowley, Eytan Abraham, Andrew Campbell, Harvey Brandwein, and Steve Oh
A
dherent cells such as adult
primary cell lines and human
multipotent (MSCs) and
pluripotent stem cells (hPSCs)
present a manufacturing challenge as
lot sizes increase from 109 (billions) to
1012 (trillions) cells (1). Typically,
manufacturing platforms are good for
one log of expansion. So new methods
will be required to achieve
commercially relevant lot sizes.
Traditional two-dimensional culture
methods have been used to grow
anchorage-dependent cell types.
Although such methods are reliable
and well defined, they are very labor
intensive and limited in scale-up
production potential by the available
growth surface area (Table 1).
Allogeneic “off-the-shelf ” therapies
based on adherent-cell platforms may
require manufactured lot sizes from
100 billion to a trillion cells depending
on a given indication’s market size (2).
Here, we examine the three
platforms available for producing
adherent cells — planar technologies,
packed-bed systems, and suspension
platforms such as microcarriers and
aggregate cultures — for their
potential of meeting lot requirements
at different scales. As new production
methods are introduced, we propose
addressing downstream processing
bottlenecks before they occur and
introduce some large-volume
downstream process technologies.
Scaling of Planar Flask Cultures
Adherent therapeutic cells (e.g., dermal
fibroblasts, chondrocytes, and MSCs)
10 BioProcess International
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Figure 1: Closed system manufacturing of therapeutic adherent cells in 10-layer vessels using
bagged media
are typically produced using planar
technologies (flasks). Ten-layer vessels
(Figure 1) have been used to progress
several allogeneic cell therapy products
into mid- to late-stage clinical
development. By some estimations,
planar technologies will reach lot-size
limitations of 3–5 million cm2 per lot
(Table 1) — capping lot sizes in the
100–400 billion cell range for most
adult primary cells.
Scaling up traditional flask-based
culture processes from laboratory scale
usually involves commercially available
stacked-plate systems such as Nunc
Cell Factories and Corning
CellStacks. These multilayer vessels
have been used for over 30 years for
large-scale cell culture (3). They were
first published for therapeutic dendritic
cells (DCs) (4) and large-scale culture
of MSCs (5) in the early 2000s. More
S eptember 2015
recently, Millipore and Becton
Dickinson (BD) have brought smallscale parallel-plate vessels to market.
Traditional 10-layer vessels have been
adopted for closed-system processing
and are being used as a platform in the
good manufacturing practice (GMP)
production of allogeneic therapeutic
adherent cells (6).
The main strategy for
maximizing lot size in planar vessels
is to scale up the total surface area
manipulated per unit operation and
then scale out multiple units. Scaleup can be achieved by increasing the
size and number of layers per vessel
and then scaling out that unit
operation to a manageable size for
manufacturing. Traditional 10-layer
vessels with ~6,300 cm 2 of surface
area have successfully been scaled
out to lot sizes of 50–70 vessels for a
Sponsored Supplement
Table 1: Projected lot sizes by production platform in number or unit operations, total surface area per harvest, and total cell numbers, with robotic
manipulation of four large vessels in a single unit operation; cell density at harvest used in the calculations represent harvest densities at confluence for
multipotent stem cells, fibroblasts, and pluripotent stem cells.
Types of Planar Methods
Vessels/Unit Operations
Total Surface Areas (cm2)
Harvest Density
Cell Types
(cells/cm2)
MSCs
25,000
HDFs
80,000
hPSCs
160,000
Manual 10/12 Layers
Low 60/60 High 100/100
372,000
620,000
9
30
60
16
50
99
total of ~400,000 cm 2 (6). These
vessels were designed to be scaled-up
to 40 layers (3).
Robotic instrumentation has been
used to manipulate four 40-layer vessels
in a single manipulation — or 16-fold
greater surface area per unit operation
than a single 10-layer vessel. Recent
innovations in Hyper technology
(Corning) has tripled the surface area
per unit volume of traditional
multilayer vessels (7). Applying robotics
to that technology could allow the
manipulation of four 120-layers to
achieve >240,000 cm2 per unit
operation, with the potential to scale
out to several million square
centimeters per harvest (Table 1). Table
1 shows approximate surface area per
harvest that is achievable for various
flask-based vessels, with total harvest
yields for typical cell types at estimated
harvest densities. Harvest densities of
GMP processes can vary greatly
depending on media composition, cell
type, and confluency at harvest.
The two main variables dictating
harvest size in planar culture methods
are total surface area harvested per lot
and cell density at harvest. Increases in
lot size require maximizing both
variables. Because different cell types
can achieve different cell densities at
harvest, we estimated lot sizes in
billions of cells per harvest over various
culture systems (Table 1). For adult
primary cells such as MSCs, lot sizes
>100 billion cells are not readily
achievable with planar technologies
outside of massive automation and
parallel processing. Suspension
technologies are required to achieve
scales of 1 trillion cells per lot. Human
dermal fibroblasts (HDFs) can achieve
much higher densities at harvest, and
lot sizes may approach 500 billion
Sponsored Supplement
Manual 36/40 Layers
Low 40/40 High 60/60
1 million
1.5 million
Robotic 36/40 Layers
Low 64/16 High 80/20
1.6 million
2 million
Estimated Billions of Cells Produced
25
38
40
50
80
120
128
160
160
240
256
320
using planar technologies. Human
pluripotent stem cells (hPSCs) are very
small and grow as tight clusters. So if
more robust cell lines are developed,
they may achieve 1 trillion cells per lot
more readily than adult primary cells.
Researchers have made several
attempts to apply bioreactor control to
planar culture systems, including the
RepliCell (Aastrom) system (8),
CellCube (Corning) cell culture unit
(9), and Xpansion (Pall Life Sciences)
bioreactor (10). Those systems
automate many operations and
monitor and control many traditional
culture variables such as dO2, dCO2 ,
and pH.
Suspension Microcarrier
and Aggregate Cultures
Achieving lot sizes of several hundred
billion to trillions cells efficiently and
cost effectively will be imperative for
commercial success. Suspension
culture of therapeutic cells in existing
single-use bioreactor manufacturing
platforms is likely to be the only way
to accomplish that.
Nonetheless, other strategies for
adherent therapeutic cell scale-up have
been investigated. One such method
uses microcarriers in a bioreactor-based
system. A potential benefit of using
microcarriers for large-scale production
is that the surface-area-to-volume ratio
is greatly increased over traditional
static culture processes. So cell density
may be increased and the required
footprint reduced.
Many different types of
microcarriers are commercially
available. Microcarriers can be made
from polystyrene such as the Hillex
brand (Pall Life Sciences) or made
from other materials such as collagen
or dextran. Although most
Robotic 120 Layers
Low 64/16
High 80/20
3.84 million
4.8 million
120
384
768
150
480
960
microcarriers are spherical and smooth,
others have macroporous surfaces and
alternatives such as rod-shaped carriers
(10). Additional technological advances
include infusion of magnetic particles
that may help in cell separation from
beads (GEM particles from Global
Cell Solutions) and chip-based
microcarriers such as the µHex product
(Nunc) that provide a traditional flat
surface for cell growth while
maintaining the high SA:V ratio of
traditional microcarriers. Selecting a
microcarrier for cell expansion is not a
trivial task. Different properties of
microcarriers may significantly affect
expansion rates and cell multi- or
pluripotency (11). Some surface
chemistry modifications can
improve cell adhesion. Such methods
include applying positive or negative
charges and coating with extracellular
matrix proteins such as laminin or
vitronectin (12).
One advantage of using
microcarriers is increased control of a
culture’s environment in a bioreactorbased system. Technology borrowed
from the development of single-use
bioreactors for biopharmaceutical
processes can be applied to growing
therapeutic adherent cells (13). MSCs
typically achieve <1 × 106 cells/mL,
whereas ESCs can achieve 1 × 106 to
3 × 106 cells/mL. Using similar
bioreactor-based systems of >1,000-L
volume for scaling up stem cells may
result in lot sizes approaching or
surpassing 1 trillion cells (Table 2).
Bioreactor technology offers the
ability to precisely control process
parameters such as gas exchange,
nutrient feeding, and pH. In smallerscale systems, however, factors such as
shear stress must be controlled closely
because stem cells are susceptible to
S eptember 2015
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BioProcess International
11
Table 2: Achievable cell densities projected to commercial-scale lot sizes (total volumes and cell
harvests) for bioreactors
Methods for Producing
One Lot of Stem Cells
Suspension
(microcarriers/aggregates)
Low
High
Cell Density
(cells/mL)
0.5 million
5 million
Volumes (L)
1,000
1,000
Total Cell
Harvest
500 billion
5 trillion
systems can achieve homogenous
cultures of high densities, but that will
require fine control to maintain stem
cell function as the volumes increase.
Downstream Processing
Table 3: Estimated harvest volumes and number of product doses per lot produced by planar and
bioreactor technologies
Number of Doses per Lot
Harvested
Volume (L) 50 million/dose 250 million/dose
30
200
40
Bioreactor Types
10-layer trays
Scale
60 vessels
40 layers per rack
20 racks, 80 vessels
200
1,000
200
120 layers per rack
20 racks, 80 vessels
600
3,000
600
Bioreactor*
250 L
250
5,000
1,000
Bioreactor*
1,000 L
1,000
20,000
4,000
*
Assuming a cell density of 1 million cells/mL
spontaneous differentiation in an
unoptimized system (14, 15). An
alternative to a traditional impellerdriven bioreactor system is the XRS20 (Pall Life Sciences) that uses a
single-use biocontainer and a rocking
motion to agitate cell suspension. As a
result, shear stress is potentially
reduced. The system chosen,
microcarrier, and cell culture medium
will influence cell proliferation.
Maintaining cell phenotype and
differentiation potential is critical.
Harvesting: For the biotherapeutic
and vaccine markets, in which a
supernatant contains the product,
there is no need to separate cells from
a microcarrier. In cell therapy
processes, cells are the product and
must be harvested. Cell harvesting
and yield of microcarrier-based
methods depends on efficiency of cell
dissociation and separation from
beads.
Enzymatic treatment using
commercially available recombinant
animal-origin–free proteases is
commonly used to remove cells from
microcarriers. Using new surface
chemistries that allow nonenzymatic
removal of cells may increase a
system’s effective yield. Tangential
flow filtration (TFF) and sequential
differential centrifugation techniques
are options for cell harvesting but
require extensive optimization and
validation for processing large lot sizes
(>1,000 L) to ensure that all
microcarriers or particulates are
removed from a cell suspension.
12 BioProcess International
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Because many cell therapies will
be administered intravenously,
carryover of particulates or intact
microcarriers into final products
poses a serious safety risk. Using
magnetic-particle infused beads can
facilitate cell separation, and
incorporating biodegradable and
thermosensitive materials may help
reduce that concern (16).
An alternative approach is culturing
stem cell as aggregates. The advantage
is that it does not need a carrier or
extracellular matrix. The key to
ensuring success of this technique is
using single-cell seeding and
maintaining high viability (e.g., using a
ROCK inhibitor) (17). Aggregate size,
however, is more difficult to control,
and large aggregates will suffer
transport limitations. To overcome that
problem, aggregates will have to be
broken up or passaged every few days.
But high cell-density limitations will
pose a greater challenge.
Studies have demonstrated
suspension aggregate cultures for both
pluripotent hESCs and iPSCs. Other
studies have shown the same for MSCs
— albeit at relatively low cell densities
— and sometimes slower doubling
times result (18–20). Similar to
microcarrier cultures, agitation can
cause cell differentiation and unstable
cell growth in aggregate cultures (17).
Regardless of the method used for
harvest and cell concentration, robust
quality control (QC) assays will be
necessary to demonstrate product
consistency and efficacy. So suspension
S eptember 2015
As therapeutic-cell lot size is scaled
from several billions to hundreds of
billions (Table 1), manufacturing
bottlenecks will shift to the
downstream processing (DSP) areas.
The cell therapy industry will need to
proactively address DSP requirements
so that technology is in place to accept
larger lot sizes as new culture
technology is implemented.
Process bottlenecks will shift to
two DSP process steps: volume
reduction and wash, and final product
filling. Volume reduction and washing
process requirements will be driven by
harvest volume, which is dictated by
the culture platform and volumes used
during harvest (Table 3). Volumes
>5–10 L cannot be easily reduced
using laboratory centrifugation or
blood processing equipment, and
scale-out is cumbersome. Scalable
single-use technologies have been
adapted from bioprocessing to enable
presterilized, closed systems. Other
technologies include process
automation such as therapeutic cell
TFF (21) and continuous
centrifugation (e.g., from kSep
Systems). Both TFF and continuous
centrifugation processes are scalable
from tens of liters to hundreds of
liters. The kSep technology has the
potential to scale up to 1,000 L
processing volume.
Larger lot sizes may shift
bottlenecks to the final-product dosefilling step, which is driven by lot size
and the number of cells per dose. Lot
sizes will range from several hundred
to several thousand doses per lot,
requiring a shift from traditional
blood bags to pharmaceutical vials and
compatible filling automation. New
plastic vials from West (22) and
Aseptic Technologies (23) coupled
with traditional pharmaceutical fill
line automation will enable lot sizes in
the several hundred to several
thousand doses per lot.
Holding times for cells in dimethyl
sulfoxide will dictate process timing,
Sponsored Supplement
so the ability to fill several thousand
vials per hour should enable lot sizes
of at least three to five thousand vials
per lot. The DSP manufacturing
bottleneck is likely to shift depending
on manufacturing platform, harvest
process volumes, and product dose
size. Final fill will be the
manufacturing bottleneck of greatest
concern at the highest culture volumes
and lowest product doses where fill
time may become unmanageable.
Challenges Ahead
As lot sizes increase from tens of
billions to trillions of cells, alternatives
to planar technologies will have to be
considered. The two main variables
dictating harvest size in planar culture
methods are total surface area
harvested per lot and the density of
the cells at harvest. Increases in lot
size will require maximizing both.
Manipulation of multiple units,
however, will limit the scale of this
technology to one trillion cells.
Packed-bed reactors can achieve
very high densities (108/mL), up to
200× more than planar surface
densities. The challenge is to increase
volumes beyond 40 L with good
process control to achieve a trillion
cells. Suspension technologies such as
microcarriers and aggregate cultures
can already achieve densities >106/mL
and potentially can scale to thousands
of liters. So they are the most
promising approaches for meeting lot
sizes of trillions of cells. As those
commercial-scale lot sizes are reached
in the upstream portion of a process,
bottlenecks downstream should be
addressed proactively so that
technology is in place to accommodate
large product doses.
References
1 Brandenberger R, et al. Cell Therapy
Bioprocessing: Integrating Process and Product
Development for the Next Generation of
Biotherapeutics. BioProcess Intl. 9(3) 2011: S30–
S37.
2 Kirouac D, Zandstra P. The Systematic
Production of Cells for Cell Therapies. Cell
Stem Cell 9 October 2008: 369–381.
3 Davis J. Medicines from Animal Cell
Culture. Glyn Stacey and John Davis, Ed. John
Wiley & Sons: New York, NY, 2007; 145–172.
4 Tuyaerts S, et al. Generation of Large
Numbers of Dendritic Cells in a Closed System
Sponsored Supplement
using Cell Factories. J. Immuno. Methods 264,
2002: 135– 151.
5 Colter DC, et al. Rapid Expansion of
Recycling Stem Cells in Cultures of PlasticAdherent Cells from Human Bone Marrow.
PNAS 97(7) 2000: 3213–3218.
6 Rowley JA. Developing Cell Therapy
Biomanufacturing Processes. Chem. Eng. Prog.
November 2010, S50–S55.
7 Titus K, et al. Closed System Cell
Culture Protocol Using HYPERStack Vessels
with Gas Permeable Material Technology. J. Vis.
Exp. (45) 2010: e2499.
8 Goltry KL, et al. Adult Stem Cell
Therapies for Tissue Regeneration: Ex Vivo
Expansion in an Automated System. Stem Cell
Research and Therapeutics. Yanhong Shi and
Dennis O. Clegg, Eds. Springer Science: New
York, NY, 2008; 251–274.
9 Aunins JG, et al. Fluid Mechanics,
Cell Distribution and Environment in
CellCube Bioreactors. Biotechnol. Prog. 19,
2003: 2–8.
10 Oh SK, et al. Long-Term Microcarrier
Suspension Cultures of Human Embryonic
Stem Cells. Stem Cell Res. 2(3) 2009: 219–230.
11 Chen A, et al. Critical Microcarrier
Properties Affecting the Expansion of
Undifferentiated Human Embryonic Stem
Cells. Stem Cell Res. 7(2) 2011: 97–111.
12 Heng BC, et al. Translating Human
Embryonic Stem Cells from 2D to 3D
Cultures in a Defined Medium on Lamininand Vitronectin-Coated Surfaces. Stem Cell
Dev. 23 December 2011 (epublication).
13 Kehoe D, et al. Scalable StirredSuspension Bioreactor Culture of Human
Pluripotent Stem Cells. Tissue Eng., Part A 16
(2) 2010: 405–421.
14 Santos FD, et al. Toward a ClinicalGrade Expansion of Mesenchymal Stem Cells
from Human Sources: A Microcarrier-Based
Culture System under Xeno-Free Conditions.
Tissue Eng. Part C 17(12) 2011: 1201–1210.
15 Leung HW, et al. Agitation Can Induce
Differentiation of Pluripotent Stem Cells in
Microcarrier Cultures. Tissue Eng. Part C 17(2)
2011: 165–172.
16 Yang HS, et al. Suspension Culture of
Mammalian Cells Using Thermosensitive
Microcarrier That Allows Cell Detachment
without Proteolytic Enzyme Treatment. Cell
Transplant 19(9) 2010: 1123–1132.
17 Zweigerdt R, et al. Scalable Expansion of
Human Pluripotent Stem Cells in Suspension
Culture. Nat. Protoc. 6(5) 2011: 689–700.
18 Larijani MR, et al. Long-Term
Maintenance of Undifferentiated Human
Embryonic and Induced Pluripotent Stem Cells
in Suspension. Stem Cells Dev. 20(11) 2011:
1911–1923.
19 Amit M, et al. Dynamic Suspension
Culture for Scalable Expansion of
Undifferentiated Human Pluripotent Stem
Cells. Nat Protoc. 6(5) 2011: 572–579.
20 Bartosh TJ, et al. Aggregation of
Human Mesenchymal Stromal Cells (MSCs)
into 3D Spheroids Enhances Their
Antiinflammatory Properties. Proc. Natl. Acad.
Sci. 107(31) 2010: 13724–1379.
21 Pattasseril J, Rowley JA. High Shear Rates
Negatively Affect Cell Viability and Final Product
Quality in TFF Processing (poster). Society for
Biological Engineering Biannual Meeting on
Stem Cell Engineering, Boston, MA, 2010.
22 Woods EJ, et al. Container System for
Enabling Commercial Production of
Cryopreserved Cell Therapy Products. Regen.
Med. 5(4) 2010: 659–667.
23 Thilly J, Conrad D, Vandecasserie C.
Aseptic Filling of Closed, Ready to Fill
Containers. Pharm. Eng. 26(2) 2006: 1–6. c
At the time of this article’s original
publication, Jon Rowley, PhD, was
innovation director, cell processing
technologies, at Lonza Walkersville. Eytan
Abraham, PhD was 3D cell culture research
and development manager at Pluristem
Therapeutics. Andrew Campbell was
senior manager of PD direct services at Life
Technologies. Harvey Brandwein was vice
president at Pall Life Sciences.
Corresponding author Steve Oh is associate
director and principal scientist at
Bioprocessing Technology Institute; steve_
oh@bti.a-star.edu.sg.
This article was first published in BioProcess
International 10(3)s 2012: 16–22.
S eptember 2015
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S e c t i o n T w o PROCESS DEVELOPMENT
T-Cell Suspension Culture in
a 24-Well Microbioreactor
High-Throughput Screening of Operating Conditions
by Kenny Choi, Jason N. Carstens, and Shelly Heimfeld
C
ell therapy promises
revolutionary new therapeutic
treatments for cancer and other
serious diseases and injuries.
For example, T-cell therapy response
rates of >50% and durable complete
response rates of 20% have been
reported in patients with metastatic
melanoma who had failed other
therapies (1). In another example,
sustained remissions of up to a year
were achieved among a small group of
advanced chronic lymphocytic
leukemia patients upon treatment with
autologous T-cells expressing an
anti-CD19 chimeric antigen receptor
(2). Numerous other examples use cell
therapy for cardiac repair, bone or
cartilage regeneration, organ repair
(pancreas or liver), neurological repair
(spinal cord or brain injury), correcting
genetic defects, and treating infectious
diseases such as human
immunodeficiency virus (HIV) (3).
Such therapies involve the ex vivo
expansion and manipulation of
different cell types including stem cells
and T-cells. Because many are still in
early clinical research phases, often
their manufacturing processes are
based on research-scale methods using
static flasks and bags, with very
limited process monitoring and
control. Although such methods have
been manageable for enabling phase 1
and 2 clinical trials treat a small
number of patients per month, the
need to produce tens of billions of cells
per subject significantly diminishes the
practicality of static processes. This is
especially true when you take into
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Figure 1: The Micro-24 microbioreactor is a 24-well agitated reactor system; each well has
individual pH, DO, and temperature control.
Controller
Microbioreactor
Sterile
membrane
pH sensing
material
Temp.
Thermal sensor
conductor
Optical pH
sensor
consideration the future prospects of a
manufacturing facility that will
provide treatments for hundreds of
patients at any given time. Static cellculture processes often require a vast
array of small-volume vessels, which
are both labor intensive and present
risks in aseptic fluid handling.
Furthermore, static systems are prone
S eptember 2015
DO sensing
material
Heater
Dilute CO2
lowers pH
Thermal
conductor
Purge Gas
O2/Air to raise DO
Optical DO
sensor
to mass-transfer limitations due to
their heterogeneity. They do not offer
the capacity to easily monitor and
control physical system parameters
such as dissolved oxygen (DO), pH,
and temperature — variables that
almost certainly influence cell culture
expansion and the critical quality
attributes of a cellular product.
Sponsored Supplement
Figure 2: Comparing theoretical DO concentration delivered to the
microbioreactor with the average steady state on-line value; the off-line
values were determined by running samples on a blood-gas analyzer.
Figure 3: Comparing off-line and on-line pH measurements
Off-line pH (GEM 3000)
8.0
Measured DO
(% air saturation)
100
80
60
40
Average on-line Micro-24 DO
20
0
Off-line DO
Theoretical DO
0
20
40
60
The current state of cell therapy
manufacturing is similar to that of
therapeutic protein manufacturing of
30 years ago. That industry also
commonly relied on static culture
systems and lacked bioprocess knowhow, well-defined raw materials,
sensitive analytical methods, and
sophisticated equipment that would
permit precise control over the process
and therapeutic product. Over
decades, the therapeutic protein
manufacturing industry evolved,
particularly with advances eliminating
the need for static culture and instead
permitting manufacture of product in
suspension culture using fed-batch or
perfusion stirred-tank bioreactors. The
body of knowledge around bioreactor
scaling issues, media composition, and
feed strategy has grown considerably
over the past few decades, allowing for
development of more productive and
efficient cell culture manufacturing
processes. In addition, improved
monitoring and process control
capabilities of bioreactors have
continually helped to progress the
development of increasingly robust
and reproducible processes. Finally,
the introduction of disposable
bioreactors and associated components
has reduced costs without
compromising aseptic processing and
final-product quality.
The cell therapy industry faces
unique challenges unlike those in
protein manufacturing. Nonetheless,
many tools originally developed to
support therapeutic protein
development and manufacturing can
be adapted to cell therapy
applications. One such technology is
the Micro-24 microbioreactor from
Sponsored Supplement
80
Theoretical DO (% air saturation)
y = 0.9811x + 0.1853
R2 = 0.8834
7.8
7.6
7.4
7.2
7.0
6.8
6.8
100
7.0
Pall Life Sciences, a 24-well
bioreactor with the capability for
agitated suspension culture and
continuous monitoring and control of
pH, temperature, and DO in each
individual well (Figure 1). The small
operating volume (3–7 mL) is well
suited to cell therapy applications, in
which patient-specific starting cells
are in short supply and culture media
may contain very expensive growth
factors. Finally, the system already
has a successful history as a scaledown model for culturing
mammalian cells in stirred-tank
bioreactors.
Because this microbioreactor
system had never before been used in
a cell therapy application, our
laboratory set out to evaluate its
usefulness as a process development
tool supporting efforts to develop and
optimize suspension cell culture for
manufacturing therapeutic T-cells.
As a first step, we qualified the
equipment for use by demonstrating
both its ability to accurately measure
and control process parameters and
its reproducibility in cell growth.
Then we evaluated the system for its
ability to serve as a scale-down model
compared with a 3-L stirred-tank
bioreactor.
7.2
7.4
On-line pH (Micro-24)
7.6
7.8
Materials and Methods
To validate the precision of on-line
DO readings, a range of known
oxygen concentrations (from 0 to
100%) were delivered by purging to
each bioreactor well with a rotometergoverned blend of oxygen, carbon
dioxide, and nitrogen. Using the
onboard Micro-24 controls, we set the
purge flow rate at 5.0 sccm, the
platform agitation at 800 rpm, and the
temperature at 37.0 °C. Each well
received 5.0 mL of a cell culture
medium based on the familiar Roswell
Park Memorial Institute (RPMI)
medium. For comparative analysis, we
took off-line DO readings with a
GEM 3000 blood-gas analyzer from
Instrumentation Laboratory. That
involved removing samples from the
microbioreactor using a syringe and
needle to puncture the cap, then
introducing them to the gas analyzer
as rapidly as possible in hopes of
minimizing changes in the dissolvedgas concentration.
For qualification of on-line pH
readings, we performed a retrospective
analysis by taking samples during the
course of several T-cell culture
experiments. We measured the offline pH using the GEM 3000 bloodgas analyzer and compared the
Table 1: An example of the instantaneous on-line dissolved oxygen (DO) reading reported in each
microbioreactor well; theoretical oxygen concentration is 11.2%.
Online DO Readings of Cassette (%)
A
1
2
3
4
5
6
16
15
15
15
14
14
B
14
14
16
15
15
13
C
16
16
16
16
16
15
D
13
13
14
13
15
15
Average DO reading of cassette 14.75%, standard deviation 1.07%
S eptember 2015
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BioProcess International
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Figure 4: Viable cell density (VCD) over time
under the same growth conditions in each well
VCD (106/mL)
10
9
8
7
6
5
4
3
2
1
0
8
11
13
Day
It is sometimes
desirable to maintain
certain cell types under
hypoxic conditions or
otherwise ensure that
conditions remain
CONSTANT while
the microbioreactor is
manipulated.
resulting values with the Micro-24
on-line readings. We’d set the
temperature, agitation, and purge rates
at the same values as in our dissolved
oxygen qualification experiments.
We qualified the cell culture
growth performance capabilities of the
Micro-24 system using a polyclonal
antigen-specific CD8 T-cell line. The
T-cells were stimulated under static
conditions using irradiated PBMC
feeder cells for four days before we
transferred them to suspension culture
in the microbioreactor. In experiments
to assess well-to-well reproducibility,
we inoculated all 24 wells identically
with a pH set point of 7.0, a DO
setting of 25% air saturation, a
temperature of 37 °C, and an agitation
rate of 500 rpm. When comparing the
Micro-24 system with a 3-L stirredtank reactor, we inoculated both from
the same starting culture and operated
them under similar conditions.
Results and Conclusions
DO Qualification: From preliminary
experiments using a nitrogen purge,
we observed that with a constant
purge-gas flow rate of 1.0–2.0 sccm it
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Table 2: Means and standard deviations from Figure 4
Day
8
11
13
Number
24
24
24
Mean
1.91625
4.83750
6.74792
Standard Deviation
0.34810
0.71843
1.22061
was impossible to drive the DO
reading to 0% in any of the wells. In
fact, the lowest DO levels we achieved
were 10–15%. Presumably that was
due to oxygen leaking into the system
through the bottom seal or elsewhere
in the gas-delivery system. However,
as we increased the purge-gas flow
rate to >3.0 sccm, the system reached a
0% DO reading in all wells. So we
chose a standard operating condition
of 5.0 sccm for purge gas.
Next we wanted to confirm that
the microbioreactor would be capable
of maintaining integrity under closed
conditions. First, to demonstrate how
the system would respond when
purposefully breached, we showed that
under low-oxygen conditions it would
rapidly reabsorb oxygen when exposed
to open atmosphere. For example,
after 800-rpm agitation under a pure
nitrogen purge (with all wells reading
0% oxygen), when agitation was
stopped and a well cap was removed
for 30 seconds (and then replaced), the
DO reading increased by 15–20%
almost immediately. When we
conducted the same experiment at a
DO setting of about 50%, the reading
increased by about 5% — not a
surprising result when you consider
the reduced driving force for oxygen
mass transfer.
We then demonstrated that the
closed system could maintain its
integrity when left sealed and closed.
Under the same starting conditions —
800 rpm and pure nitrogen purge
with all wells uniformly reading 0%
DO — the capped plate was
unclamped from its platform and
placed on the laboratory bench for
120 seconds. After the plate was
reclamped and returned to normal
agitated operating conditions, all
wells had increased their dissolved
oxygen levels by only 2–3%,
indicating that this microbioreactor
could generally maintain a closed,
leak-tight system. This feature is
important because it is sometimes
S eptember 2015
Lower 95%
1.7693
4.5341
6.2325
Upper 95%
2.0632
5.1409
7.2633
desirable to maintain certain cell
types under hypoxic conditions or
otherwise ensure that conditions
remain constant while the
microbioreactor is manipulated.
Next, we evaluated the precision
and accuracy of oxygen readings using
a rotometer manifold that delivered a
gas mixture of known oxygen
composition (0–100%) by purging. We
allowed the system to achieve a
steady-state operation condition over a
period of at least 30 minutes. In an
effort to identify performance changes
that might occur over time, we
performed this evaluation over two
weeks. At each setting, we recorded
the on-line DO in each well and
calculated the average and standard
deviation. In all cases, the well-to-well
deviation was <3%. Table 1 provides
example data collected from each well
at a DO setting of 11.2%.
Figure 2 compares the known DO
concentration delivered to the
microbioreactor with the average
on-line DO reading in each well. In
general, there was excellent agreement
between the on-line DO reading and
the theoretical value of the known
DO. In addition, we also analyzed offline samples with the blood-gas
analyzer. Despite our efforts to place
samples on the analyzer as rapidly as
possible and minimize their
atmospheric exposure, reabsorption of
ambient oxygen into the samples
produced artificially high DO
measurements (a more prominent effect
at lower DO conditions). These results
demonstrated acceptable accuracy and
precision of the DO readings. In fact,
the on-line instrument readings should
be viewed with greater confidence than
off-line samples tested with the bloodgas analyzer.
pH Qualification: During a threemonth period, we performed several
cell culture experiments using T-cells
in an RPMI-based medium. At
intervals, we pulled samples for offline measurement, including for pH
Sponsored Supplement
Sponsored Supplement
pH 7.1, DO 40%
pH 7.0, DO 10%
5
4
3
2
1
0
pH 6.8, DO 40%
30
Fold Expansion (×100)
Cell Density (106/mL)
6
pH 7.0, DO 40%
pH 6.8, DO 10%
0
5
Run Days
10
25
20
15
10
5
0
15
0
5
Run Days
10
15
Figure 6: Lactate and glucose concentration profiles
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
2.5
pH 6.8
3-L bioreactor
pH 7.1
pH 7.0
pH 6.8
2.0
pH 7.1
pH 7.0
3-L bioreactor
0
5
Run Days
10
15
be aimed at optimizing the media
composition and feed strategy. More
important to the purposes of equipment
qualification, the information provides
confidence in the scalability of results
from the microbioreactor to the stirredtank bioreactor.
A Process Development Tool
Glucose (g/L)
using the GEM analyzer. Whenever a
sample was pulled, we also recorded
the instantaneous on-line pH
measurement for that particular well.
From a retrospective analysis, Figure 3
compares on-line and off-line pH
measurements. The 0.98 slope shows
excellent agreement between those
measurements at the given pH values.
Cell Culture Qualification: We
initiated a CD8 T-cell culture in the
microbioreactor by inoculating and
operating each well under identical
conditions. As Figure 4 shows, cells
successfully expanded over a 13-day
period with a reasonably comparable
cell density in each well (day 13 mean
of 6.75 million cells/mL, standard
deviation of 1.2 million cells/mL).
The total cell number expanded an
average of 1,850-fold and exhibited
exponential growth through day 11,
with an average doubling time of 24.2
hours. Those results were consistent
with what we observed in larger-scale
bioreactors.
Figure 5 illustrates a second cell
culture experiment, in which we
inoculated a 3-L stirred-tank bioreactor
and the Micro-24 system using the
same seed culture. Here we operated
the microbioreactor in a matrix with
three pH set points (6.8, 7.0, and 7.1)
and two DO set points (10% and 40%).
Observed cell densities and foldexpansion values were reasonably
similar in all cases, with data suggesting
that perhaps the pH 7.0 condition
might be best. Occasionally we sampled
the cultures for their glucose and lacticacid concentrations (Figure 6), with
data demonstrating that glucose was
becoming depleted. Those results
suggest that future experiments should
3-L bioreactor
pH 7.1, DO 10%
Lactate (g/L)
More important to the
purposes of equipment
qualification, the
information provides
CONFIDENCE in
the scalability of results
from the
microbioreactor to the
stirred-tank bioreactor.
Figure 5: Comparing cell growth in a 3-L stirred-tank bioreactor and in the microbioreactor at
three different pH set points and two DO set points
Our instrument qualification results
show that the Micro-24
microbioreactor can be used as an
accurate, high-throughput
experimental system that properly
represents production-scale systems.
The system offers a sufficient degree
of accuracy and precision in its on-line
DO and pH measurements and
demonstrates a reasonable degree of
well-to-well reproducibility.
Furthermore, cell growth rates and
metabolic profiles are similar to what
we observed with a 3-L stirred-tank
bioreactor. Our results suggest that
the Micro-24 system is suitable as a
scale-down model for high-throughput
evaluation of operating parameters for
therapeutic cell culture.
1.5
1.0
0.5
0.0
0
5
10
Run Days
15
donation from the Bezos Family Foundation
and NIH grants P30 DK56465, P01 CA18029,
and P30 CA15704.
References
1 Rosenberg SA. Durable Complete
Response in Heavily Pretreated Patients with
Metastatic Melanoma Using T-Cell Transfer
Immunotherapy. Clin. Cancer Res. 17(13) 2011:
4550–4557.
2 June CH. Chimeric Antigen ReceptorModified T Cells in Chronic Lymphoid
Leukemia. NEJM 365, 2011: 725–733.
3 Trounson A. Clinical Trials for Stem
Cell Therapies. BMC Medicine 9, 2011: 52. c
Kenny Choi is a research technician;
corresponding author Jason N. Carstens,
PhD, is director of process development;
and Shelly Heimfeld, PhD, is scientific
director of the cellular therapy and cell
processing facilities — all in
the clinical research division at Fred
Hutchinson Cancer Research Center, 1100
Fairview Avenue North, MS D5-100, Seattle,
WA 98109-1024; 1-206-667-6131; jcarsten@
fhcrc.org.
This article first appeared in BPI’s April 2013
issue.
Acknowledgments
This work was supported by a generous
S eptember 2015
13(8)sp
BioProcess International
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S e c t i o n T w o PROCESS DEVELOPMENT
Scaling Up Stem Cells
Moving from Laboratory to Commercial Production
with a Single-Use Multiplate Bioreactor
by Matthieu Egloff and Jose Castillo, updated by Thierry Bovy
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. This method is not
practical for large-scale production,
18 BioProcess International
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however, because it requires multiple
manual aseptic operations, which 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 a
biotech company working on a cellbased therapy to protect patients’
hearts during myocardial injuries. The
developmental autologous therapy uses
cardiopoietic cells made from a
patient’s bone marrow stem cells, then
injected into the patient’s heart.
Following successful phase 2 trials,
the company then planned to advance
into phase 3 studies. This required an
effective, efficient, and practical
method for growing batches of
autologous stem cells for more
patients.
ATMI, later acquired by Pall
Corporation, was approached to
develop a single-container solution
S eptember 2015
Figure 1: An Xpansion plate with 614 cm2
available for cell growth
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
Sponsored Supplement
difficult because the company’s
objective was to treat approximately
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
(cGMP) process also would have been
difficult. Coupled with the sheer space
and number of operators required, this
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 our customer needed was a
cost-efficient process that cultured
high-quality 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.
Figure 2: (left) multitray stack 10; (right) 10
Xpansion plates
Figure 3: (left) 20-multitray stack 10; (right)
200-plate Xpansion unit
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
DO), 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
Sponsored Supplement
(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.
Developing a Specific Solution
All of these problems could be
prevented with a new multiplate
design approach. This would mimic
the cell-growth 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 traditional multiplate
systems.
The Xpansion® bioreactor was
designed 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) was 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 the
Xpansion bioreactor allows it to take
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 ≤200 plates to
fit in a single device about 80 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
200-plate bioreactor has the same cellgrowing capacity as 20 stacks
(122,400 cm 2) 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
involved regulation and control of cell
S eptember 2015
13(8)sp
BioProcess International
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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, pH and dissolved oxygen
patches were added 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 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 from
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).
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
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Figure 4: Bone-marrow mesenchymal stem
cells cultured on an Xpansion plate (shown by
an Ovizio microscope)
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. 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
S eptember 2015
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.
At the time of this writing, Matthieu Egloff
was product manager, and Jose Castillo
was director of cell culture at Pall Life
Sciences. Thierry Bovy is global product
manager, Xpansion multiplate bioreactor
systems, Pall Life Sciences, Brussels,
Belgium.
Xpansion is a trademark or registered
trademark in the United States, other
countries, or both. Ovizio is a trademark of
Ovizio Imaging Systems SA. Other names
are trademarks of their respective
companies.
An earlier version of this article was
published in BPI’s May 2012 Cell Therapy
supplement.
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Even experienced scientists can feel
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Features: SoloHill microcarrier training
courses are offered at Pall’s sites in
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Applications: Flexible for suspensionadapted and adherent cell cultures
Features: The PadReactor family of
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cells and adherent cells on microcarriers
(e.g., Pall SoloHill® microcarriers).
• Adapting flatware and roller bottle
processes to microcarriers
• Handling microcarriers and
optimizing attachment conditions
• Small-scale microcarrier processes
(spinner flasks)
A PadReactor unit incorporates a cubeshaped biocontainer design with a
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dynamic sparging supports high cell
densities. Paddle and sparger are
enclosed in a sleeve made from the
same medical-grade ultralow-density
polyethylene (ULDPE) material as the
biocontainer itself. The flexible drive unit
is compatible with different biocontainer
sizes and a mobile retaining tank
supports the biocontainer and provides
mobility. A user-friendly design allows
short set-up times. The fully transparent
front cover ensures easy cell culture
observation during cultivation and
harvest. An available controller provides
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Contact Pall Life Sciences
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Pall’s training team works with you
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Contact Pall Life Sciences
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S eptember 2015
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BioProcess International 21
S e c t i o n T w o PROCESS DEVELOPMENT
Expansion and Characterization of
Mesenchymal Stem Cells on
Pall SoloHill® Microcarriers
by Heather Woolls, Dave Splan, and Mark Szczypka
M
esenchymal stem cells
(MSCs) are self-renewing
cells that differentiate into
several terminally
differentiated cell types. These cells
have been isolated from multiple
sources such as bone marrow, adipose
tissue, peripheral blood, and other
adult tissues(1-6). The interest in these
cells is that they hold the potential to
cure disease and are being pursued in
clinical trials. Three emerging fields
of interest for stem cells are cell
therapy, regenerative medicine and
screening of candidate drugs. In many
cases, poor correlation between
efficacy of candidate drugs in animal
models and humans is observed. This
leads to high attrition rates of
candidate drugs from the
developmental pipeline and also
contributes to large losses in revenue
spent on animal model testing.
The ability to isolate, expand, and
differentiate human stem cells in vitro
will streamline drug testing by
allowing candidate drug testing on
human cells at early stages thereby
better predicting how human
populations may react to new and
developing drugs. It is hoped that the
ability to reproducibly isolate and
expand these cell types will facilitate
22 BioProcess International
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the identification of candidate drugs
earlier in the development process. In
addition, the ability to differentiate
stem cells into various cell lines should
allow for more relevant toxicity
testing. These achievements should
ultimately lead to overall cost savings
and decreased health risks in the
future.
In addition to drug product testing,
several clinical trials have been
initiated using stem cells in cell
therapy treatments. Research has
shown stem cell characteristics such as
differentiation potential, angiogenic
potential, immunosuppression, or
immune-privilege may be effective in
the treatment of many diseases.
Clinical trials using stem cells for the
treatment of osteoarthritis, spinal cord
injuries, Parkinson’s disease, ischemia
due to stroke, cardiac arrests, or
diabetes, are seeing promising results.
However, for toxicology screening and
cell therapy applications, large
numbers of cells are needed.
Expansion of adult stem cells is
difficult since they have a finite life
span and pluripotency can be lost.
Two-dimensional (2D) culture systems
such as T-flasks, cell cubes/factories,
and roller bottles are common
production platforms for vaccine and
S eptember 2015
Mesenchymal stem cells were seeded at low
density and expanded for eight days on Pall
SoloHill’s Plastic microcarrier. Cells were
stained with DAPI (blue) and FITC-labeled
phalloidin (green) for visualization.
biologics manufacturing as well as cell
therapy. These systems are typically
used for expansion of cells to seed
large bioreactors. Although wellestablished, these formats occupy a
large footprint, are labor intensive,
and are susceptible to contamination
problems due to numerous open
handling steps. Microcarriers offer a
large surface area for growth of
anchorage-dependent cell types, and
could thereby facilitate use of
bioreactors for stem cell expansion in
fewer passages.
Herein we characterized MSC
expansion on flatware and five Pall
SoloHill microcarriers (Collagen,
C102-1521; Plastic, P102-1521; Plastic
Sponsored Supplement
Plus, PP102-1521; Pronectin F,
PF102-1521; and Hillex II®,
H112‑170) in stirred vessels.
Retention of multipotency of the
MSCs expanded in stirred culture was
verified by immunostaining with stem
cell specific antibodies and by
assessing their ability to differentiate
into osteocytes and adipocytes.
Materials and Methods
Culturing of MSCs: Human bone
marrow-derived MSCs (Passage 1)
were purchased from EMD Millipore
(SCR108) and expanded in spinners or
on flatware in DMEM (GIBCO
11054) supplemented with 10% fetal
bovine serum (FBS) (HyClone
SH30071.03), 2 mM l-glutamine
(HyClone SH30034.02), penicillin/
streptomycin (ATCC 30-2300), and
basic Fibroblast Growth Factor
(bFGF) (EMD Millipore GF003).
Unless otherwise noted, medium refers
to this complete formulation.
For growth experiments on
flatware, MSCs were cultured on
Corning T-flasks (430825, 430639, and
430641). To subculture cells, medium
was decanted and cells were rinsed
once with Dulbecco’s phosphate
buffered saline (DPBS; HyClone
SH30028.03). The DPBS was
immediately decanted and 1–3 mL of
TrypLE Select enzyme (Life
Technologies 12563) was added
(depending on T-flask size). Flasks
were incubated at 37 °C until cells
detached (5–8 minutes). The cells were
resuspended with medium and then
centrifuged at 300g for five minutes to
pellet the cells. Medium and trypsin
were decanted. Cells were resuspended
in 2–5 mL plus 10% FBS but without
bFGF (volume depends on T-flask size
and number) and counted using trypan
blue stain and a Nexcelom Cellometer
with associated software. Fresh
T-flasks were seeded at 3 × 103 cells/
cm2. Fresh bFGF was added (8 ng/mL)
to seeded T-flasks, which were then
incubated at 37 °C ± 0.5 with 5% CO2
in complete medium. 100% media
exchanges (with fresh bFGF) were
performed every other day beginning
on the second day of culture.
Incubation: For initial microcarrier
attachment studies 200,000 cells were
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MICROCARRIERS
offer a large surface
area for growth of
anchorage-dependent
cell types, and could
thereby facilitate use of
bioreactors for stem
cell expansion in fewer
passages
seeded onto the equivalent of
seven cm 2 of each microcarrier type.
Although this seeding density of ~3 ×
104 cells/cm 2 was higher than
anything used subsequently, this
density was used to provide enough
cells for counting and visualization on
the microcarriers. Cells were
incubated with microcarriers in 1 mL
of medium (either ± FBS) in 1.5 mL
Eppendorf tubes at 37 °C ± 0.5 with
5% CO2. At various time points, tubes
were removed from the incubator and
microcarriers were allowed to settle.
Cell counting: 20-µL samples of the
supernatant were taken for counting
on the Nexcelom counter. Time
courses for percent cells attached
versus unattached were determined for
each condition and plotted (Figure 2).
For growth experiments on the various
Pall SoloHill microcarriers, 0.5 g of
microcarriers were used per 50 mL of
medium in each Corning brand
125 mL spinner vessel (Fisher
Scientific 10-203B). All microcarriers
were prepared according to
manufacturer’s instructions by
autoclaving at 121 °C in deionized
water. Spinner cultures were
essentially performed as described in a
previous microcarrier protocol (7).
Briefly, spinners were seeded in
complete medium low protein
concentration (less than 0.25% FBS
and no bFGF) for 30 minutes for
initial attachment. After 30 minutes
>85% of cells had attached to the
microcarriers. Final protein
concentrations of 10% FBS and 8ng/μL
bFGF were added slowly to prevent any
osmotic shock from the serum. The
spinners were incubated at 37 °C ± 0.5
with 5% CO2. Cell counts were
performed using standard assays to
quantify cell numbers and determine
viability. Spinners containing cells on
Hillex II microcarriers were grown at
60 rpm, whereas all other microcarrier
spinners were kept at 40 rpm. Media
exchanges of 25 mL (50% volume)
were performed every other day
beginning on the second day of
culture. Samples were retrieved daily
for nuclei counts using the citric acid/
crystal violet method. Nuclei were
counted using the Nexcelom counter.
The number of nuclei per cm 2 surface
area was calculated for each sample.
Trypsinization: For trypsinization
of cells from microcarriers, cells/
microcarriers were allowed to settle
and medium was removed. Cells and
microcarriers were washed with DPBS
for five minutes at room temperature
with occasional rocking back and
forth by hand to resuspend
microcarriers. After five minutes, the
microcarriers were allowed to settle,
and the DPBS was removed. Five mL
of TrypLE Select was added. The
spinners were gently pipetted once or
twice to thoroughly mix and then
incubated at 37 °C for 10–15 minutes
(with occasional rocking by hand).
Cells and microcarriers were pipetted
after five minutes and again after ten
minutes to achieve a single-cell
suspension that could be used to
reseed fresh microcarriers.
Visualization: To visualize the
expression of several stem cell markers
on MSCs expanded on Pall SoloHill
microcarriers, samples were
transferred from the spinners into
15-mL tubes. Once the microcarriers
settled, medium was removed and
cells/microcarriers were carefully
washed with DPBS for five minutes at
room temperature. Once cells and
microcarriers settled, DPBS was
removed and cells and microcarriers
were fixed in 4% paraformaldehyde for
ten minutes at room temperature. The
paraformaldehyde was removed and
cells were washed in DPBS and stored
at 4 °C until use. To visualize stem
cell markers, 250 µL of each sample
was transferred to a 1.5 mL tube,
microcarriers settled and DPBS
removed. Nonspecific binding was
blocked by incubation with 5% FBS in
S eptember 2015
13(8)sp
BioProcess International
23
Figure 1: MSC growth curve on flatware; cells seeded at 3 × 103 cells/cm2 were grown for 10 days.
Data are presented here as means ± SEM (n = 3).
4.5
4.0
Determining Differentiation
Potential: Samples were then incubated
Cell Density (×104 cells/cm2)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
0
1
2
3
4
5
6
7
8
Days
Figure 2: MSC attachment studies; removing FBS increased attachment rate to all SoloHill
microcarriers in semistatic conditions.
A
B
100
80
Collagen
60
Plastic
40
Plastic Plus
Pronectin F
20
Hillex II
Percent Attachment
Percent Attachment
100
DPBS for one hour at room
temperature. Samples were washed in
500 μL DPBS three times for five
minutes at room temperature.
1
0 30 60 90 120 150 180 210 240
80
Collagen
60
Plastic
40
Plastic Plus
Pronectin F
20
1
Attachment Time (minutes)
Hillex II
0
5 10 15 20 25 30 35 40
Attachment Time (minutes)
Figure 3: After 30 minutes of attachment at 37 °C, high percentages of cells remained unbound to
microcarriers (left column). Decreasing FBS concentrations in medium increased attachment rate,
and almost no cells were visible in medium after 30 minutes (right column).
ProNectin F
Plastic Plus
Plastic
Collagen
– FBS
+FBS
Hillex II
Figure 4: MSC attachment to microcarriers in spinner cultures
Hillex II
Plastic Plus
ProNectin F
Plastic
Collagen
in 250 µL of the dye/antibody
solutions. All antibodies were used at
1:1000 except for Stro-1 (1:500). Dyes
and antibodies used were DAPI (Life
Technologies, D3571), phalloidinFITC (Life Technologies, A12379),
FITC antihuman CD44 (BioLegend
338803), APC antihuman CD90
(BioLegend 328113), Alexa Fluor 647
antihuman Stro-1 (BioLegend 340103),
FITC antihuman CD18 (BioLegend
302105), FITC antihuman CD19
(BioLegend 302205), Alexa Fluor 647
antihuman CD14 (BioLegend 325611),
and Alexa Fluor 647 antihuman
CD146 (BioLegend 342005).
To determine differentiation
potential of MSCs expanded on Pall
SoloHill microcarriers, spinners were
seeded at 3 × 103 cells/cm2 and grown
for eight days. Spinners were
subsequently passaged into new
spinners or flatware (24 well plate) at
3 × 103 cells/cm2. Spinners at passage 2
on microcarriers were allowed to
expand until near-confluency (2–3 ×
104 cells/cm2). Samples from the
spinners were transferred to a 24-well
plate to determine differentiation
capabilities on microcarriers compared
to cells grown on flatware.
Growth/expansion medium was
removed, and 1 mL of either
osteogenesis induction medium
(EMD Millipore SCR028) or
adipogenesis induction medium
(EMD Millipore SCR020) was
added. Induction and maintenance
media were changed according to
EMD Millipore’s protocol (as
recommended by supplier). Osteocyte
differentiation was determined by
Alazarin Red S staining, and
adipocyte differentiation was
determined by Oil Red O staining
(protocols with EMD Millipore kits).
Results
Characterization on Flatware: To
characterize MSCs on flatware, T-25s
were seeded at 3 × 103 cells/cm2 and
incubated for up to ten days to generate
growth curves (Figure 1). As shown in
24 BioProcess International
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S eptember 2015
Sponsored Supplement
Figure 5: Nuclei counts for MSC spinner cultures; counts show maximal confluent densities
6 and 10 × 104 nuclei/cm2. Data are presented as means ± SEM (n = 3).
14
12
Cell Density (× 104/cells/cm2
Figure 1, MSCs seeded at 3 × 103 cells/
cm2 MSCs reached a maximum
confluent density of ~4 × 104 cells/cm2.
Over the course of the ten day growth
curve, cells had an average doubling
time of about 48 hours.
Attachment Studies: To determine
initial attachment conditions for
MSCs to the Pall SoloHill
microcarriers, attachment studies in
which FBS was removed from the
attachment media were performed as
described earlier. As shown in Figure
2A and 2B, MSCs were 70–80%
bound to all microcarriers after 15
minute incubations at 37 °C. However,
with 10% FBS present in the medium,
attachment ranged from 30% to 80%
after two hours and from 50% to 90%
after four hours. Observations under
light microscopy after 30 minutes of
incubation at 37 °C supported these
cell counts (Figure 3). Since these
experiments were performed under
semistatic conditions, the faster
attachment rates in the conditions
with low FBS were chosen for future
spinner cultures.
To determine the growth
capabilities on Pall SoloHill
microcarriers, spinner cultures were
seeded at 3 × 103 cells/cm 2. The
attachment was done in low serum
concentration conditions for 30
minutes. As shown in Figure 4, after
this 30 minute attachment period in
the spinner flasks, very few cells
remain unbound. The low seeding
density of 3 × 103 cells/cm 2 is
approximately 2–3 cells/bead. Some
microcarriers were observed to have
more than three cells and some had no
cells attached.
10
Collagen
8
Plastic
6
Plastic Plus
ProNectin F
4
Hillex II
2
0
0
2
4
Days
Figure 6: MSC expansion in spinner culture;
MSCs tended to clump once higher densities
were reached in microcarrier spinner cultures
(days 5–0).
6
8
10
Figure 7: Uniform attachment to
microcarriers; using low FBS concentration and
no bFGF medium during attachment
increased efficiency from ~75% to >95%.
Figure 8: (a) Stem cell marker expression of microcarrier-expanded MSCs; MSCs expanded on Pall
SoloHill microcarriers (collagen shown) were incubated with anti-CD44 (green), anti-CD90 (red), and
DAPI (blue). Populations of CD44 expressing cells are indicated by green arrows. Populations of
CD90 expressing cells are indicated by red arrows. Populations of cells expressing both markers are
indicated by yellow arrows. (b) MSCs were incubated with anti-CD44 (green), anti-Stro1 (red), and
DAPI (blue). Populations of CD44 expressing cells are indicated by green arrows. Populations of
Stro‑1 expressing cells are indicated by red arrows. Populations of cells expressing both markers are
indicated by yellow arrows.
MSC Attachment to Microcarriers in
Spinner Cultures: When seeded at low
densities, low serum attachment
conditions led to quick attachment,
although not completely uniform
attachment.
At this low seeding density, uniform
attachment was not possible in medium
with 10% FBS. However, cells attached
to approximately 70% of the
microcarriers allowing good expansion
over the 10-day growth period.
Nuclei Counts: Figure 5 shows the
nuclei counts for spinner samples over
the ten day growth periods. Nuclei
density reached 6–10 × 104 nuclei/cm 2.
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MSC density on microcarriers
appeared to reach a higher maximal
confluent density than what was seen
in T-flask growth.
As shown in Figure 6, cells grown
on all microcarriers tended to stretch
across two or three microcarriers
(with Hillex II as the exception). By
stretching between multiple
microcarriers, cells could effectively
have a larger available threedimensional volume in which to
grow, which is not possible on 2D
surfaces.
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Figure 9: Potentiality of microcarrier-expanded MSCs; (a) MSCs grown on T-flasks and Plastic
microcarriers remain in an undifferentiated state; (b) MSCs grown on T–flasks and plastic
microcarriers underwent adipogenesis and osteogenesis. Cells expanded on microcarriers appeared
to have similar differentiation capabilities compared with cells expanded on T-flasks only.
Plastic
Adipogenesis
Osteogenesis
Undifferentiated
A
T-Flask
Plastic
T-Flask
B
Figure 10: Differentiation; MSCs grown on plastic microcarriers for multiple passages are shown
undifferentiated in the left two panels and differentiated into adipocytes and osteocytes in the two
right panels.
To increase initial attachment,
bFGF was removed from the medium
during attachment (complete medium
with low FBS and without bFGF).
After 30 minutes all cells appeared
attached to greater than 90 percent of
microcarriers. Expansion for seven
days verified uniform attachment and
excellent growth, shown in Figure 7.
To verify the stem cell-like
character of MSCs grown on Pall
SoloHill microcarriers, the expression
of MSC-associated cell surface
markers was determined as well as
26 BioProcess International
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the ability of these cells to
differentiate after being expanded on
microcarriers. To check for the
expression of cell surface markers,
samples were incubated with
f luorophore-conjugated antibodies.
Shown in Figure 8, imaging on a
Nikon (Ti65) determined these cells
to be CD44+, CD90+, Stro1+ and
CD146+ when grown on all five Pall
SoloHill microcarriers. The
hematopoetic cell markers CD14 and
CD19 were not expressed in MSCs
(data not shown).
S eptember 2015
Stem Cell Marker Expression:
(Figure 8).
Expansion on Microcarriers: To
determine the potentiality of MSCs
when expanded on microcarriers, cells
were grown on Plastic microcarriers
for multiple passages. MSCs were
seeded at 3 × 103 cells/cm 2 and
expanded in spinner flasks for eight
days. Cells were trypsinized from
microcarriers and seeded onto flatware
for differentiation into adipocytes and
osteocytes. After 21 days cells grown
on plastic microcarriers were able to
differentiate into adipocytes and
osteocytes at a level comparable to
cells grown on T-flasks alone (Figures
9a and 9b).
Investigating Differentiation: To
determine if several passages on
microcarriers affected the
differentiation ability of MSCs, cells
were passaged multiple times on
plastic microcarriers. After six
passages on microcarriers, cells were
seeded onto flatware for
differentiation. Table 1 shows that
over the six passages on microcarriers,
the harvesting density was consistently
above 3 × 104 cells/cm 2 with a
doubling time of 48–51 hours when
seeded at 3 × 103 cells/cm 2, showing
that multiple passages on microcarriers
did not decrease maximal confluent
density or doubling rates. The ability
of cells grown on microcarriers for six
passages is shown in Figure 10. The
cells grown on plastic microcarriers
appear undifferentiated (top panels).
Additionally, these cells were able to
differentiate into both adipocytes and
osteocytes (bottom panels) similar to
levels seen previously on earlier
passages and on flatware (Figure 9,
Table 1)
Several passages on microcarriers
demonstrate the ability to expand
MSCs continuously on microcarriers
without decreasing maximal confluent
density or doubling time when seeded
at 3 × 103 cells/cm 2.
Conclusions
Current obstacles limiting the use of
stem cells for therapeutic benefits
include a limited number of cell
divisions and the potential loss of
pluripotency. Due to the restricted
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number of population doublings,
achieving maximal possible expansion
in the fewest passages is vital. We
have shown here that MSCs can be
expanded on various types of Pall
SoloHill microcarriers. The benefit of
MSC expansion on microcarriers is
two-fold. First, expansion on
microcarriers allows growth on large
surface areas within single containers,
and second microcarrier expansion
increases the ratio of apparent surface
area to medium volume due to the fact
that MSC growth on microcarriers
outpaces growth on flatware. This is
particularly important with stem cells
grown in medium that contains
expensive supplements. Therefore, the
use of microcarriers allows minimal
passages for expansion of cells while
decreasing the overall cost required to
grow enough cells for a therapeutic
dose in clinical trial treatments. We
have shown that multiple passages on
microcarriers do not affect the ability
of MSCs to differentiate into
adipocytes and osteocytes. The ability
to maintain pluripotency while
expanding MSCs on microcarriers for
five or six passages allows for the
isolation of cells from bone marrow
onto a T-150 flask. Cells expanded in
this fashion can subsequently seed a
small scale spinner culture which
could be used to seed a small
bioreactor. For example, the maximal
confluent cell density in a T-150
results in 2.5–3 × 106 cells and 3.5–4 ×
104 cells/cm 2 on microcarriers. To seed
a 200-mL spinner volume requires 3.1
× 10 6 cells using 5,150 cm 2/L. The
maximal densities on spinner cultures
achieved here would result in enough
cells for a minimum 10-fold expansion
into a 2 L bioreactor, by the third
passage after isolation and the second
passage on microcarriers.
Considering the recoverable cell
numbers presented here, a 6.7 L
bioreactor volume (at 5,150 cm 2/L)
would result in enough cells for one
therapeutic dose (~1 × 109 cells).
Assuming similar growth between
small scale spinners and bioreactors, a
2-L bioreactor could be used to seed a
20-L bioreactor, which would result in
enough cells for three doses from a
single T-150 and multiple passages on
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Table 1: Continuous passage on microcarriers in spinner cultures; several passages on microcarriers
demonstrate the ability to expand MSCs continuously on microcarriers without decreasing maximal
confluent density or doubling time when seeded at 3 × 103 cells/cm2.
Plastic Spinners
Seeding Density1
Harvest Density1
Days of Growth
Number of Doublings
1 P1
0.3
4.6
8
4
P2
2
3.3
5
<1
P3
1
2.7
5
1.3
P4
0.3
3.6
7
3.6
P5
0.3
4.3
8
3.8
P6
0.3
4.5
8
3.9
× 104 cells/cm2
Table 2: Extrapolated liters needed for therapeutic doses for various concentrations of plastic
microcarrier cultures
Microcarrier
Concentration
(plastic)
10 g/L
20 g/L
Surface Area
0.3
Maximum
Confluent Density
(cells/cm2)
2
Total Cells/L
1
L/Therapeutic
Dose
0.3
4.6
3.3
2.7
3.6
microcarriers. Additionally, increasing
the microcarrier concentration beyond
5,150 cm 2/L would decrease
bioreactor volume required for a large
number of recoverable MSCs. Table 2
extrapolates the expected number of
cells if the Plastic microcarrier
concentrations used were 10 g/L and
20 g/L. The resulting liters needed for
therapeutic doses of stems cells would
be 7–8 liters and 3–4 liters, for
concentrations of 10 g/L and 20 g/L,
respectively.
Work will continue to further
characterize stem cells grown on Pall
SoloHill microcarriers, such as
defining various populations within
bone marrow-derived MSCs, any
fluctuations in these populations when
grown on microcarriers for multiple
passages, and additional stem
characteristics such as the ability of
microcarrier-expanded cells to produce
angiogenesis signals such as VEGF.
Future work will also expand to
include other stem cells such as
induced-pluripotent stem cells, mouse
embryonic stem cells, and
mesenchymal stem cells from other
sources such as placental-derived or
adipose-derived. Additionally, work
will continue to define growth and
expansion conditions under animal
component free environments, as well
as direct expansion on microcarriers of
isolated stem cells, thereby bypassing
flatware tissue culture and increasing
the number of available passages
before senescence.
References
1 Friedenstein AJ, Gorskaja JF, Kulagina
NN. Fibroblast Precursors in Normal and
Irradiated Mouse Hematopoietic Organs. Exp.
Hematol. 4, 1976: 267–274.
2 Fraser et al. Fat Tissue: An
Underappreciated Source of Stem Cells for
Biotechnology. Trends Biotechnol. 2, 2006: 150–
154.
3 Cao C, Dong Y. Study on Culture and
In Vitro Osteogenesis of Blood-Derived
Human Mesenchymal Stem Cells. Zhongguo
Xiu Fu Chong Jian Wai Ke Za Zhi. 19, 2005:
642–647.
4 Griffiths M, Bonnet D, Janes SM. Stem
Cells of the Aveolar Epithelium. Lancet. 366,
2005: 249–260.
5 Beltrami et al. Adult Cardiac Stem
Cells Are Multipotent and Support Myocardial
Regeneration. Cell. 114, 2003: 763–776.
6 Pittenger et al. Multilineage Potential
of Adult Human Mesenchymal Stem Cells.
Science. 284, 1999: 143–147. c
Corresponding author Mark Szczypka,
PhD, is senior director of applications and
new product development,
mark_szczypka@pall.com. David Splan is
senior research scientist; and Heather
Woolls, PhD, is a research scientist, all at
Pall Life Sciences. SoloHill and Hillex are
registered trademarks of Pall Corporation.
S eptember 2015
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BioProcess International
27
S e c t i o n T w o PROCESS DEVELOPMENT
Strategies for Microcarrier
Culture Optimization
by Mark Szczypka and Alain Fairbank
T
he process of delivering an
allogeneic stem-cell therapy to
patients requires isolation and
expansion of rare tissue-specific
stem cells, which are subsequently
delivered to individual patients for
treatment. One type of cell used for
such therapies is commonly known as
human mesenchymal stem cells
(hMSCs). They have been isolated
from a number of tissues: e.g., bone
marrow, heart, brain, placenta, and
umbilical cord. And they have been
shown to be immune-privileged in that
hMSCs elicit no graft-versus-host
(GvH) response such as those observed
with allogeneic organ transplants.
Thought to provide therapeutic
benefits for a range of diseases,
hMSCs have hypothesized
mechanism of actions of promoting
intrinsic organ repair and performing
immunomodulatory activities (1).
Those functions are postulated to be
temporally and site specific. In vitro,
hMSCs secrete growth factors and
other molecules that may regulate
intrinsic repair mechanisms in vivo.
Those molecules may represent the
therapeutic effectors when they reach
sites of damage. It is important to note
that hMSCs do not integrate into
tissues and “transdifferentiate” into
adult-cell types at appreciable
frequencies, and their residence time
within a patient’s body is relatively
short (1–3).
That provides a challenge for
achieving an effective therapy, making
it necessary for therapeutic cells to
rapidly reach their site of action to
perform their necessary functions
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Figure 1a: Monitoring kinetics of cell attachment to microcarriers; samples retrieved from dynamic
culture are fixed and stained with DAPI — nuclei can be visualized and counted using a microscope
equipped for fluorescence
Suboptimal
(few cells attached)
Optimal
(10–12
cells/bead)
before they are cleared. Early results
from clinical trials indicate that
relatively large numbers of cells — in
the range of tens of millions to
billions — are needed to produce a
therapeutic effect. Furthermore, some
indications may require multiple
treatments to guide and promote
curative processes (4).
If those observations hold true,
then allogeneic hMSC therapies will
require implementation of large-scale
manufacturing processes similar those
performed for many years in
mammalian-cell–based manufacturing
of biologics and vaccines (5). That
requirement is complicated by the fact
that hMSCs expanded in vitro must
retain their therapeutic potential
throughout the entire manufacturing
process so that healthy cells can be
efficiently harvested for delivery to
patients.
However, when cells are products,
special challenges arise in developing a
manufacturing platform that are not
found in more traditional biologics
S eptember 2015
manufacturing. The theoretical basis
and practical demonstrations of the
concepts for using adult stem cells to
treat disease have come from academic
institutions, research institutes, and
medical schools. Methods used by their
investigators to generate cells for
seminal studies were similar to
traditional cell culture techniques used
in research laboratories: static twodimensional (2D) platforms consisting
of cell culture dishes and T-flasks. In
many cases, such platforms have been
sufficient for small-scale animal studies
and some phase 1 clinical trials
requiring limited numbers of cells.
However, as cell therapies transition to
larger clinical trials for multiple
indications in humans, developers need
to identify more user-friendly
manufacturing platforms for cell
expansion. The methods are too
onerous for handling large numbers of
dishes or T-flasks needed to generate
enough cells for such purposes.
Fortunately, procedures for
isolation, in vitro expansion,
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formulation of hMSCs into a final
product, and ultimate delivery to
patients have undergone continual
process improvements over the past
10–15 years (6–8). Multiplate units are
commonly used to expand hMSCs;
however, this platform provides only a
short-term solution for the expansive
surface area (SA) requirements needed
for hMSC expansion. Different
iterations of multiplate technologies
provide sufficient SA to generate tens
of billions cells. But even the most
sophisticated devices require an
unmanageable number of units to
generate the trillions of cells required
to treat many predicted patient
populations, with the required multiple
manual manipulations increasing the
risk of culture contamination (9).
Hollow-fiber units also provide a
solution for generating millions to
billions of cells. However, when cell
number requirements even modestly
increase, an unmanageable number of
units become required. These
limitations have spurred efforts to
identify simple, cost-effective, and
efficient solutions for generating much
larger numbers of cells. Implementing
a microcarrier-based production
platform has become an obvious
option for filling this need (10).
Microcarriers for
Expansion of hMSCs
Microcarriers provide a large SA-tovolume ratio for propagation of
adherent cells, making expansion to
trillions of cells possible in a single
culture vessel. Implementation of this
technology greatly reduces the number
of production lots needed per year as
well as lowering risk and cost while at
the same time increasing process
control (5, 10, 11). All these
improvements lead to product
consistency and quality.
Microcarriers used for propagation
of mammalian cells are micron-sized
particles. Manufactured at relative
densities that promote their suspension
in vessels using gentle stirring, they can
take different shapes and sizes.
Different core materials include
synthetic compounds such as
polystyrene and biologic materials such
as extracellular matrix molecules. The
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Table 1: Properties of Pall SoloHill microcarriers
Microcarrier Core Material
Collagen
Cross-linked
polystyrene
Fact III
Cross-linked
polystyrene
Plastic
Cross-linked
polystyrene
Plastic Plus
Cross-linked
polystyrene
ProNectin F Cross-linked
polystyrene
Hillex II
Cross-linked
modified polystyrene
Relative
Density
1.02–1.04
Animal
Protein Free?
No
1.02–1.04
No
1.02–1.04
Yes
Charged surface
1.02–1.04
Yes
Recombinant protein
1.02–1.04
Yes
1.10
Yes
Surface Treatment
Type I porcine
gelatin coating
Charged surface with type
I porcine gelatin coating
Uncharged
Cationic amine
surfaces of microcarriers also can be
chemically modified or coated with
different molecules to facilitate efficient
cell attachment, spreading, and growth
(Table 1).
Availability of a broad range of
microcarriers provides an opportunity
to select an optimal substrate that
promotes robust and efficient cell
growth while allowing use of simple
methods for harvest and separation of
cells from the microcarriers after
expansion. Unique, innovative
approaches to accomplishing harvest
across the range of manufacturing
scales continue to be developed to
ensure that highly viable, healthy, and
therapeutically active hMSCs can be
efficiently expanded on microcarriers
and isolated from bioreactors for
delivery into patients.
Growth of Adherent Cells
on Microcarriers
For cell culture processes, bioreactor
vessels are assembled, and culture
media and microcarriers are added to
them. Environmental conditions such
as pH and gas concentrations are
allowed to stabilize at desired set
points prior to cell addition, then
adjusted and maintained at preferred
levels. Cells are then added at an
appropriate concentration for
attachment to the microcarriers.
Cells are harvested from a smaller
bioreactor or static platform (e.g., an
Xpansion multiplate bioreactor from
Pall Life Sciences) and introduced into
a large bioreactor containing
microcarriers. Cell densities that
promote occupation of all microcarriers
in the vessel are determined empirically
and used for seeding. Seeding density
influences overall culture efficacy
because cells must come into contact
with a microcarrier in suspension and
subsequently attach to its surface. Once
attached, cells must spread over the
microcarrier through cell division while
remaining attached.
Dynamic microcarrier culture can
be described as a series of discrete,
sequential events. Several important
physical parameters regulate this
process. Kinetics of cell attachment to
microcarriers, distribution of cells
across the microcarrier population
within a vessel, cell spreading, and
maintenance of adherence to
microcarriers during cell division are
therefore all important processes to
characterize. Those events can be
monitored using qualitative and
quantitative methods in small-scale
studies that help culturists identify
conditions that lead to efficient
microcarrier culture. Microcarrier
concentration, cell seeding density,
microcarrier surface characteristics,
medium formulation, mixing
dynamics, and environmental controls
are process parameters that directly or
indirectly influence culture efficiency.
Cell Attachment and Distribution:
The kinetics of cell attachment to
microcarriers is of primary importance
because adherent cells that remain in
suspension for extended periods can
form aggregates and will eventually
perish. To measure the kinetics of
attachment, representative samples
retrieved at predetermined time points
after cell seeding are formaldehyde
fixed, then stained with a fluorescent
dye for visualization of nuclei using
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fluorescence microscopy. The number
of cells attached to microcarriers then
can be quantitated (Figure 1a). The
resulting measure helps culturists assess
the quality of cell attachment and
reveals the distribution of cells on
microcarriers suspended in solution
over time.
Ideally, cells will attach in a
predictable time frame, and all
microcarriers will have the same
number of cells attached to their
surfaces. In practice, however,
attachment times depend on cell type
and medium. A calculation using cell
seeding density (cells/cm2) and the
physical properties of the microcarriers
can predict the number of cells that
will occupy each microcarrier in a
culture (Figure 1b). The observed
distribution of cell-containing
microcarriers in suspension — and the
number of cells attached to each
microcarrier within a sample — can be
compared with the theoretical number
of cells expected to attach to each one.
Performing that comparison helps
culturists determine the attachment
efficiency and microcarrier occupancy
under different environmental
conditions and facilitates identification
of conditions that lead to rapid and
efficient cell attachment.
An efficient culture should have a
roughly equivalent number of cells on
each microcarrier, and every
microcarrier in suspension should have
at least one cell bound to its surface.
Such a scenario increases the efficiency
of culture by facilitating synchronous
cell growth and promoting coverage of
the entire available SA within a vessel.
Environmental conditions such as
protein concentration in the medium,
pH level, cell-seeding density, mixing
dynamics, and microcarrier
concentration can be modified to
promote cell attachment. Optimal set
points for those parameters should be
identified and refined in small-scale
cultures before further optimization
with larger bioreactors.
Cell spreading onto microcarriers is
indicative of a favorable cell-substrate
interaction and thus another critical
parameter to assess. Monitoring this
process can be subjective because there
are no simple and robust methods to
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Figure 1b: Monitoring kinetics of cell
attachment to microcarriers; calculation to
determine theoretical cell numbers attached
to microcarriers using seeding density and
physical characteristics of microcarriers
Cs(SAg)
Bg
Cells/microcarrier =
Cs = seeding density (cells/cm2)
}
SAg = surface area (cm2) per gram of
microcarriers
Bg = number of beads
quantify the phenomenon on
microcarriers. With some guidance and
practice, however, this measure can be
used to effectively gauge microcarrier
performance in early screening efforts.
Cells initially attach to substrates
with a rounded appearance. If the
substrate is favorable, they will rapidly
begin to spread across the microcarrier
surface and adopt a gumdrop-shaped
morphology. As cells continue to
spread, they flatten to their typical in
vitro morphology — which
(depending on cell type) can make
them more difficult to visualize. For
example, hMSCs adopt a flattened
fibroblast-like morphology that makes
visualization more challenging. Use of
fluorescent dyes and microscopy can
help analysts visualize those cells once
they have spread onto the surface of
nontranslucent microcarriers.
Adherence and Growth: Successful
propagation of cells on microcarriers
in dynamic culture is more complex
than on flatware because cells are
constantly exposed to forces they do
not encounter in static culture. Shear
forces make cell division more
difficult. Many cell types have specific
requirements for attachment and
growth, and a number of parameters
control their ability to grow effectively
in dynamic cultures.
As mentioned above for other
cellular processes, identification of the
optimal combination of these variables
for a specific cell type is best
performed in small-scale cultures.
This can be difficult sometimes
because representative scaled-down
models that mimic larger-scale vessels
are not always available. But this
approach can provide initial insight
into important process-control
variables for specific cells in a selected
S eptember 2015
medium type and also identify
starting conditions for a particular
larger (production-sized) vessel.
Cell seeding density can greatly
influence the implementation and
efficiency of microcarrier cultures (12).
Fine tuning this parameter leads to
efficient use of the SA within a vessel.
Total SA in a bioreactor depends on
the mass of microcarriers used, and
that SA is distributed among the
number of beads within each gram of
microcarriers.
Using the physical characteristics of
microcarriers, culturists can calculate
the number of cells expected to occupy
individual microcarriers within a
bioreactor. This establishes a lower
theoretical limit for cell seeding. It is
preferable to seed with enough cells to
have at least one cell attached to each
microcarrier in suspension. Seeding
densities used in static cultures should
be used as a starting point for smallscale studies. The lowest density that
allows for population of all
microcarriers should be tested along
with several other (higher) seeding
densities.
Microcarrier Concentration: Total
available SA is determined by the
concentration of microcarriers added to
a bioreactor. Microcarrier properties
(e.g., size and shape) influence the
maximum SA that can be occupied by
cells. For example, the SA of spherical,
nonporous microcarriers is dictated by
the diameter of those particles.
Individual microcarriers with smaller
radii have a smaller SA than that of
larger microcarriers, but the SA
equivalent of their entire population is
greater because the number of
microcarriers of a given density in a
single gram is much larger. That larger
number of individual spheres will
inverse-exponentially increase the
overall surface so that solid
microcarriers >300 µm have a very low
overall SA per gram.
It is important to note that
increasing microcarrier concentration
is not directly linked to culture
performance. Some investigators use
concentration of cells (cells/mL) to
describe microcarrier culture
efficiency, but that measure does not
accurately assess the actual SA use.
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Figure 2: In hMSC attachment studies, removing fetal bovine serum (FBS) increased attachment rate to all SoloHill microcarriers in semi-static
conditions. After 30 minutes attachment at 37 °C, high percentages of cells remained unbound to microcarriers (bottom panel). Decreasing FBS
concentration in media to 0.5% increased attachment rate after 30 minutes.
Low Serum (0.5%)
100
80
Attachment (%)
Attachment (%)
80
60
40
Collagen
60
Plastic
Plastic Plus
40
Pronectin F
20
20
0
Standard Serum (10.0%)
100
0
5
10
15 20 25
Time (minutes)
Increasing microcarrier concentration
will expand SA within a vessel, but if
the available substrate is not colonized
by cells, then the system is inefficient.
Microcarrier concentration can
influence other culture parameters as
well: e.g., the formation of cell
microcarrier aggregates and possible
increases in shear forces encountered
by cell-laden microcarriers. It
therefore becomes very important to
identify the optimal microcarrier
concentration for a given combination
of cells and media that will support
the highest concentration of cells.
Environmental Control: Standard
environmental-control methods used
in dynamic suspension cultures of
mammalian cells are appropriate for
implementation with adherent-cell
microcarrier cultures. Parameters such
as pH and dissolved oxygen (DO)
levels, nutrient supplementation, and
metabolite removal must be tightly
controlled because of their influence
on culture health (13).
Changes to pH result from cellular
metabolic activities, including release
of carbon dioxide (CO2), lactic acid,
and other metabolites. Those shifts
can affect cell adherence to
microcarriers. Culture pH can be
controlled with the same CO2bicarbonate buffering systems used in
flatware along with infusions of CO2,
oxygen (O2), nitrogen (N2), and air
mixtures. Other buffering systems are
available; however, the bicarbonate
buffering system is most commonly
Sponsored Supplement
30
35
40
0
Hillex II
0
5
10
15 20 25
Time (minutes)
used for many microcarrier-based
processes.
Gases can be infused into a vessel
using either a gas overlay or sparging.
Overlay entails infusing a gas mixture
into the vessel as a constant stream
over the surface of the liquid–air
interface to promote gas exchange.
Sparging a vessel with gas occurs
through an apparatus that contains
differently sized holes, and is
immersed into the culture medium.
Although the latter increases gas
exchange, the former helps to prevent
some of its shortfalls (e.g., bubble
formation and foaming, both which
can lead to shear stress and loss of
microcarriers from solution). Base
addition can also be used to maintain
pH; however, care should be taken to
not expose cells directly to highly
concentrated alkaline solutions, which
can cause membrane damage and cell
death.
Batch feeding, perfusion, and
nutrient supplementation all can be
implemented in microcarrier cultures.
Bioreactors should be outfitted with
spin filters or dip tubes that can be
covered with mesh screens that
facilitate removal of spent medium
without removing microcarriers from
the vessel. Medium can be replenished
through additional vessel ports. The
amount of medium removed and
replaced is regulated through the use
of level probes. Feed strategies for
large cultures (including nutrient or
growth factor supplementation) along
30
35
40
with metabolite monitoring should be
optimized first in small-scale cultures
to save time and cost.
Microcarrier Surface Characteristics:
Commercially available microcarrier
types are composed of a range of
materials and possess unique surfaces.
Unique combinations of core materials
and surface modifications control
physical characteristics of microcarriers,
including relative density, rigidity, size,
surface porosity, and charge density.
Some microcarriers feature a coating
with recombinant or animal-sourced
extracellular matrix proteins and/or
synthetic peptides. Such molecules
promote attachment and adherence of
cells. The types of molecules used and
the manner in which they are attached
to the microcarrier influences how
effective the substrate is at promoting
cell attachment and growth (14–17).
Plasma membranes of specific cell
types have unique assortments of
attachment molecules and distinct
molecular properties. The membrane
composition influences how cells attach
to a surface and also governs whether
one substrate is preferred over another.
It is therefore important to identify an
ideal surface chemistry for specific cells
to achieve optimal culture efficiency.
Using charged moieties to modify
microcarrier surface chemistry also has
proven to be useful and can sometimes
mitigate the need for protein coatings.
Polymeric microcarriers can be
chemically charged by modulating
monomeric composition using well-
S eptember 2015
13(8)sp
BioProcess International
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known chemistries or charged-molecule
coatings. Charges can also be imparted
onto the surface of microcarriers using
plasma-emission coating, a technology
commonly used to generate tissueculture plastic. It’s not surprising that
some microcarriers combine charges
and proteins and have proven to be
useful in a number of mammalian cell
culture processes. Commercially
available Pall SoloHill microcarriers
present a number of available surface
modifications. Microcarriers containing
positive charges, animal-derived
attachment proteins, recombinant
proteins, and combinations of all those
substrates are offered.
Many other types of surface
modifications have been applied to
microcarriers. Application of
temperature-responsive molecules to
microcarriers has been performed in
the past, but new technologies and
thermal-responsive molecules thought
to improve utility are being explored.
The potential benefits of such
technology are that cells can be
removed from microcarrier surfaces
without enzymatic treatment.
Media Formulations: It is well known
that culture media formulations
significantly influence cell growth.
Therefore, it follows that the
composition of a medium (including
pH, osmolality, and protein
concentrations) also has a major
influence on cell attachment, adherence,
and growth in microcarrier cultures.
Media components such as serum,
recombinant proteins, and synthetic
peptides can passively absorb onto
microcarrier surfaces during
acclimation in reactors, or they can
attach to cells and directly or indirectly
influence their attachment to
microcarriers. Attachment kinetics and
cell distribution have been shown to
change according to media components
and concentrations (18–20).
For example, the commonly used
culture supplement fetal bovine serum
(FBS) contains many proteins and
other molecules such as bovine serum
albumin (BSA), fibronectin,
vitronectin, and galectin. It is wellknown that those and other molecules
can affect cell attachment, spreading,
and adhesion (21). Some such
32 BioProcess International
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molecules can facilitate cell
attachment when they are affixed to
surfaces, but modulation of protein or
serum concentration in culture media
also can influence cell attachment and
distribution on microcarriers. We have
shown that decreasing concentrations
of those media components can
improve cell attachment and facilitate
even cell attachment to microcarriers
(Figure 2).
Optimization Roadmap
and Time Considerations
It is paramount that a simple and
straightforward experimental strategy
be adopted when developing and
implementing microcarrier cultures,
requiring a high degree of attention to
detail. Careful planning and timely
execution of experimental plans and
implementing multiparameter
experiments (where applicable) will
expedite developmental timelines and
save money.
The first step in the process is to
fully characterize the selected cell type
in flatware cultures. Parameters that
should be characterized and optimized
include doubling time, population
doubling levels, expression of cellspecific markers, cell behavior in
functional/potency assays, attachment
factor requirements, and conditions for
cell harvesting. Those cell
characteristics and behaviors in static
culture will be used to guide selection
of initial conditions used for
microcarrier culture. They also can
help culturists assess potential changes
in cell function that could be
attributable to expansion in a dynamic
microcarrier culture.
Cells must retain their critical
phenotypic characteristics when grown
in a suspension microcarrier culture.
For example, adult hMSCs should
retain their differentiation capacity,
expression of characteristic markers,
and functionality. However, minor
differences in some parameters
sometimes can be seen. Doubling times
might be slightly longer for some cell
types, and expression levels of some
“house-keeping” proteins can be altered
by exposure to dynamic conditions.
Attachment kinetics and efficiency
are the first parameters to optimize.
S eptember 2015
Examination of cell attachment to
different microcarrier types and growth
in static culture can be used as a
method to identify microcarriers that
are compatible with a given cell type
and medium combination. Using this
method, cells and microcarriers
combined in plates that have not been
treated for tissue culture are gently
mixed at defined intervals over the first
few hours of culture. Attachment and
growth are monitored over two to three
days. This is a rapid and economic way
to obtain an indication of productive
cell–microcarrier interaction. Although
useful, this technique does not expose
cells to dynamic conditions, so it is not
always fully predictive of their behavior
on specific microcarriers in stirred-tank
vessels.
Characterization of attachment and
binding in dynamic culture should
closely follow. These studies can be
performed using small-scale formats
such as multiwell bioreactors or smallscale spinners of 50–200 mL. Such
formats are convenient for multifactorial
studies and can be used to identify and
optimize growth conditions in dynamic
culture. Parameters such as cell seeding,
microcarrier concentration, and
medium formulation optimization
(including feeding and/or
supplementation) can be optimized
effectively, expeditiously, and costeffectively using this type of format.
This is especially useful for
optimization when expensive medium
formulations are required.
Cell harvest techniques should be
optimized at every stage of
development, beginning with
optimization of conditions for
enzymatic treatment (e.g., trypsin,
chymotrypsin) in flatware (22–24).
Investigators must establish temperature
limits and determine minimum and
maximum exposure times that do not
affect cell health. Harvesting of cells
from microcarriers is a multistep
process that includes enzymatic
treatment to initiate cell detachment,
dislodging cells from microcarriers
using some type of physical disruption,
and then separating the cells from the
microcarriers. Durable, rigid
polystyrene microcarriers made of
materials that are similar to standard
Sponsored Supplement
culture flatware are ideally suited for
harvesting cells. Additionally, the
properties of such microcarriers allow
for implementation of a range of
techniques to dislodge cells from their
surfaces with simple processes for
capturing cells from the resulting slurry.
Although small-scale formats are
informative, further optimization in
larger formats is required. Transitioning
to small (2-L to 5-L) bioreactors will
allow for further optimization of
conditions required to increase cell
numbers and further optimize feed
strategy. The intrinsic environmental
and process-control options available in
such platforms provide an opportunity
to further optimize conditions that
support superior cell growth. Physical
conditions present in these platforms
are closer to those observed in larger
systems. But for most such platforms,
each increase in scale presents specific
challenges that must be addressed for
optimization of environmental
conditions. Fortunately, a large amount
of historical information and expertise
have accumulated from suspensionculture manufacturing. Such literature
can provide starting points for
optimization of microcarrier celltherapy cultures.
Conclusion
Until recently, a common paradigm in
the biopharmaceutical industry has
been that development of a
microcarrier-based production
platform is a lengthy and onerous
process when compared with the effort
needed to scale out static platforms.
But new microcarriers have emerged
to present a reliable and accessible
technology for generating large
numbers of cells for allogeneic cell
therapies. Although slight differences
may arise with transition from static
2D systems to dynamic microcarrierbased technology, the cost benefits
and process efficiencies achieved with
microcarrier culture far outweigh the
time and effort required for bioreactor
process development.
To facilitate successful production
at commercial scale, process
development efforts should be
initiated as early as possible in product
development, which also helps
Sponsored Supplement
products transition to clinical trials at
the earliest practical time point.
Implementing the strategies of careful
planning, understanding the critical
process parameters, and timely
execution of experimental designs will
expedite developmental timelines; save
money; and facilitate development of
robust, reproducible, and cost-effective
manufacturing platforms for cellbased therapies (Table 1).
References
1 Phinney DG, Prockop DJ. Concise
Review: Mesenchymal Stem/Multipotent
Stromal Cells: The State of Transdifferentiation
and Modes of Tissue Repair — Current Views.
Stem Cells 25(11) 2007: 2896–2902.
2 Kean TJ, et al. MSCs: Delivery Routes
and Engraftment, Cell-Targeting Strategies,
and Immune Modulation. Stem Cells Int. 13
August 2013.
3 Chamberlain G, et al. Concise Review:
Mesenchymal Stem Cells: Their Phenotype,
Differentiation Capacity, Immunological
Features, and Potential for Homing. Stem Cells
25, 2007: 2739–2749.
4 Van't Hof W, et al. Direct Delivery of
Syngeneic and Allogeneic Large-Scale
Expanded Multipotent Adult Progenitor Cells
Improves Cardiac Function After Myocardial
Infarct. Cytother. 9(5) 2007: 477–487.
5 Rowley J, et al. Meeting Lot-Size
Challenges of Manufacturing Adherent Cells
for Therapy. BioProcess Int. 10(3) 2012: 16–22.
6 Want AJ, et al. Large-Scale Expansion
and Exploitation of Pluripotent Stem Cells for
Regenerative Medicine Purposes: Beyond the
T Flask. Regen. Med. 7(1) 2012: 71–84.
7 Kehoe D, et al. Scalable StirredSuspension Bioreactor Culture of Human
Pluripotent Stem Cells. Tiss. Eng. Part A 16,
2010: 405–421.
8 Santos F, et al. Toward a ClinicalGrade Expansion of Mesenchymal Stem Cells
from Human Sources: A Microcarrier-Based
Culture System Under Xeno-Free Conditions.
Tiss. Eng. Part C Meth. 17, 2011: 1201–1210.
9 Davie NL, et al. Streamlining Cell
Therapy Manufacture: From Clinical to
Commercial Scale. BioProcess Int. 10(3) 2012:
S24–S27.
10 Simaria AS, et al. Allogeneic Cell
Therapy Bioprocess Economics and Optimization:
Single-Use Cell Expansion Technologies.
Biotechnol. Bioeng. 111(1) 2014: 69–83.
11 Szczypka MS, et al. Single-Use
Bioreactors and Microcarriers: Scalable
Technology for Cell-Based Therapies.
BioProcess Int. 12(3) 2014: 54.
12 Hu WS, Meier J, Wang DIC. A
Mechanistic Analysis of the Inoculum
Requirement for the Cultivation of
Mammalian Cells on Microcarriers. Biotechnol.
Bioeng. 27, 1985: 585–595.
13 Wlaschin KF, Hu WS. Fed-Batch
Culture and Dynamic Nutrient Feeding. Adv.
Biochem. Eng. Biotechnol. 101, 2006: 43–74.
14 Varani J, et al. Growth of Three
Established Cell Lines on Glass Microcarriers.
Biotechnol. Bioeng. 25, 1983: 1359–1372.
15 Giard DJ, et al. Virus Production with a
Newly Developed Microcarrier System. Appl.
Environ. Microbiol. 34, 1977: 668–672.
16 Varani J, et al. Substrate-Dependent
Differences in Growth and Biological
Properties of Fibroblasts and Epithelial Cells
Grown in Microcarrier Culture. J. Biol. Stand.
13, 1985: 67–76.
17 Varani J, et al. Cell Growth on
Microcarriers: Comparison of Proliferation on
and Recovery from Various Substrates. J. Biol.
Stand. 14, 1986: 331–336.
18 Blüml G. Microcarrier Cell Culture
Technology. Animal Cell Biotechnology. Pörtner
R, Ed. Humana Press: New York, NY, 2007:
149–78.
19 Butler M, et al. High Yields from
Microcarrier Cultures By Medium Perfusion. J.
Cell Science 61, 1983: 351–363.
20 Serra M, et al. Improving Expansion of
Pluripotent Human Embryonic Stem Cells in
Perfused Bioreactors Through Oxygen Control.
J. Biotechnol. 148(4) 2010: 208–215.
21 Grinnell F. Cellular Adhesiveness and
Extracellular Substrata. Int. Reve. Cytol. 1978:
67–129.
22 Schriebl K, et al. Stem Cell Separation:
A Bottleneck in Stem Cell Therapy Biotechnol.
J. 5, 2010: 50–61.
23 Heathman TR, et al. Expansion,
Harvest and Cryopreservation of Human
Mesenchymal Stem Cells in a Serum-Free
Microcarrier Process. Biotechnol Bioeng. 112(8)
2015: 1696–1707.
24 Weber C, et al. Expansion and
Harvesting of hMSC-TERT. Biomed. Eng. J.
7(1) 2007: 38–46. •
Corresponding author Mark Szczypka is
senior director of applications and new
product development at Pall Life Sciences in
Ann Arbor, MI 48108; 1-734-973-2956;
mark_szczypka@pall.com. Alain Fairbank
is director of cell therapy marketing at Pall
Life Sciences in Port Washington, NY;
alain_fairbank@pall.com.
S eptember 2015
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“ We’ve been finding
solutions to complex
challenges for over 60 years.
Just imagine what we
can achieve together.”
Pall Life Sciences
Your vision. Our expertise. Their future.
www.pall.com/celltherapy
© 2015 Pall Corporation. Pall,
, Xpansion, iCELLis and PadReactor are trademarks of Pall Corporation.
® indicates a trademark registered in the USA.
S e c t i o n T h r e e CELL THERAPY MANUFACTURING
Designing the Most Cost-Effective
Manufacturing Strategy for
Allogeneic Cell-Based Therapies
by Thierry Bovy, Alain Fairbank, and Suzanne S. Farid
R
apid progress is occurring in
the field of stem cell therapy
research, and increasing
numbers of products will begin
reaching the market in the near
future. But new cell therapy
treatments must fit into a competitive
and highly regulated healthcare
environment. Succeeding in that
environment requires alignment
between a company’s business model
and its manufacturing strategy.
Cell therapy products are different
in many respects from traditional
small-molecule and even biologic
drugs. So developers may need to
reconsider preconceptions about the
manufacturing process to
accommodate the unique
characteristics of these therapeutics.
Economic aspects should be addressed
from early phases of development to
enable a viable product life cycle.
Identifying cost drivers early on will
help companies develop the right
manufacturing strategies and
determine necessary batch sizes.
Commercial strategy helps to identify
available resources — and risks — for
bioprocess development and full-scale
manufacturing.
Some key drivers of the rapidly
expanding demand for manufacturing
tools include cost, scalability, and
flexibility. Achieving lot sizes of several
hundred billion to trillions of cells both
efficiently and cost effectively will be
imperative for commercial success.
Scaling the culture of adherent cells,
such as mesenchymal stem cells, has
presented manufacturing challenges as
36 BioProcess International
13(8)sp
Implementing Technology Options
in Cell Therapy Development
lot sizes increase from billions to
trillions of cells. Predicting market
demand is never easy, particularly for
emerging technologies that lack
historic precedent. Manufacturing
flexibility, in both scheduling and
capacity for rapid production scale-up,
is therefore vital to commercial success
of a cell therapy.
On 25 March 2015, Pall Life
Sciences presented a webcast on
designing cost-effective
manufacturing strategy for allogeneic
cell-based therapies. The speakers
were Thierry Bovy (Pall Life
Sciences’ global product manager of
Xpansion multiplayer bioreactor
systems) and Suzanne Farid
(professor in bioprocess systems
engineering at University College
London’s department of biochemical
engineering).
Below, Bovy discusses
industrialization challenges and the
advantages and limitations of
implementing different technology
options across the productdevelopment pathway.
S eptember 2015
Industrialization of a cell therapy
process can be challenging. The
product is, of course, our center of
attention. But some aspects of
increasing importance will have to be
considered when moving through
drug development.
First, the scale of a manufacturing
process must match market needs for
increasing quantities of cellular doses.
Raw materials and other supplies need
to be carefully managed, as well. No
one wants to see a therapy’s progress
halted by a shortage in raw materials
or consumables. And cost of the final
product must allow the sponsor
company to make proper benefit while
marketing product that the patient/
payer community can afford to buy.
Finally, facility design and size will
need to match the production process
and manufacturing volumes. The
quality system will have to be robust
enough to cope with increasing
workloads associated with
intensification of manufacturing and
associated validation efforts.
Scale of adherent cell culture is
associated with the extent of surface
available for cells to colonize. The
vertical axis of Figure 1 represents the
surface area per unit, and the
horizontal axis plots the different
currently available technologies for
adherent cell cultures. Most people are
familiar with open, multilayered
stacks. But Pall proposes the Xpansion
bioreactor as a compact and closed
alternative for lateral-scale
Sponsored Supplement
Figure 2: Upstream options — adherent cells for therapeutic applications
>10
6
122,400
25,000
Ho
Bi llow
or ea Fi
ct be
o r
M rs
ul
til
St ay
ac er
k
M
Bi u s
or lti
ea p l
c a
M torste
i
St cro
irr ca
ed rr
Ta iers
nk
18,000
manufacturing with up to
>122,000 cm 2 per single unit. And
microcarrier storage tanks are currently
under investigation at most advanced
cell therapy companies working on
allogeneic treatments. But the
Xpansion bioreactor technology is
currently the only one able to reach
millions of square centimeters per unit.
Developers must select the best
manufacturing strategy with their end
target in mind, taking into account
the amount of cells that will need to
be produced in the commercial phase.
Figure 2 shows different technical
options for adherent-cell culture,
considering the number of patients to
treat each year and the amount of cells
per patient.
The dose size for autologous
treatment is generally smaller than
that of allogeneic therapies. Open
T-flasks or multilayer stacks are
typically used for manufacturing lowdose products. Increasing the
production capacity involves scaling
out rather than up. Use of bioreactors
and/or automation will contribute to
reducing cost of goods (CoG).
Allogeneic treatment doses can go
up to a billion cells per patient (or per
injection). When multiplied by the
number of patients treated per year,
that requires a huge amount of cells to
be produced. Existing planar
technologies are unable to achieve more
than 5 × 1011 cells per lot. Pall’s
Xpansion system is currently the only
single-use bioreactor providing
>100,000 cm2/ unit. Threedimensional (3D) systems such as
microcarriers in stirred-tank bioreactors
could generate up to 1013 cells per lot.
The Xpansion multiplate
bioreactor was developed for largeSponsored Supplement
Allogeneic (scale-up)
Millions of
cells/patient
Autologous
(scale-out)
Surface Area (cm2/unit)
Figure 1: Available upstream technologies
3D
Stirred-tank
bioreactors with
microcarriers
500
Xpansion (multilayer),
automated multilayer
2D
Xpansion (multilayer)
250
Tangential-flow filtration (multilayer)
10s 50s
Phases 1−3
100s
500s
...
5,000s
Commercial
10,000s
...
Patients treated/year
scale production of traditional 2D cell
cultures. Each plate provides 612 cm 2
of surface for adherent cell growth.
Stacking 10–200 of those plates
provides increasing surface from 6,000
up to >122,000 cm 2 per single unit
without modification to the
bioreactor’s footprint. Only its volume
and weight change. The smallest
version (Xpansion 10 bioreactor) is
normally used for technical
evaluations, whereas the others
(Xpansion 50, 100, and 200
bioreactors) are designed for
manufacturing purposes. The model
numbers correspond to the number of
plates in each unit.
This bioreactor was designed to
provide the same microenvironment as
multitray stacks to ease transition
from traditional open models to closed
and controlled systems. Some
tweaking is necessary, mainly
optimizing parameters such as pH and
dissolved oxygen (DO) as well harvest
protocols. The systems scale up
linearly from 10 to 200 plates.
The same hardware is used across
the bioreactor range. So settings
contribute to maintaining the culture
condition consistent. Parameters such as
stirring speed are adapted according to
bioreactor size for maintaining linear
speed of the culture medium over the
monolayer. You change stirring speed to
scale out. All Xpansion bioreactors have
the same footprint, and they contribute
to reducing manual operations.
Microcarriers: Transitioning a cell
culture system from 2D to 3D and
scaling it up requires time and
expertise. Special attention must be
paid to the selection of microcarriers.
Their density, surface area,
concentration (g/L) in a bioreactor,
and surface coatings all influence
mixing requirements, the amount of
cells obtained per milliliter of culture,
and ultimately product quality.
To increase quantities, cells need to
colonize more microcarriers. To
achieve that goal, you can rely either
on bead-to-bead transfer or on
traditional subcultivation techniques
(e.g., trypsinization).
During growth in a bioreactor,
microcarriers must predominantly
remain in suspension. The vessel
design must ensure proper mixing
with minimal shear stress on cells
regardless of the tank size. Increasing
volumes also need to be managed,
with an ultimate goal of guaranteeing
product quality and its comparability
with cells obtained on a planar
surfaces. All these steps require
process-development efforts that
should not be underestimated. The
timeline has to be carefully
considered. However, microcarriers
are scalable to very high-volume
manufacturing and do represent the
lowest-cost alternative for large
production of cells per lot.
Processing: Once billions of cells
have been generated, they must be
harvested, concentrated, and washed,
then formulated and finally filled into
their final containers — all under time
pressure. Upstream manufacturing
S eptember 2015
13(8)sp
BioProcess International
37
Figure 3: Pall approach — product life cycle
T-Flask, Multilayers
Cost of Goods (CoG)
Microcarriers
Process Development Costs
Multilayer Bioreactor — Xpansion
Product Comparability
R&D
Phase 1
Phase 2
Phase 3
Commercial
! Commercial ! (high volume)
Process Development
Table 1: Downstream process scale-up — available technologies
Continuous
Fluidized
(e.g., Ksep)
400–9,600
100–1,600
+
Parameter
Flow rate (mL/min)
Hold-up volume (mL)
Process development effort
and downstream clarification
operations thus need to be aligned.
The main consideration in large-scale
harvesting include detaching cells
from their substrate (either a planar
surface or 3-D microcarriers). After
that, the cells must be separated from
the microcarriers before concentrating,
washing, formulating, and inline
filling the cell therapy product.
Table 1 lists a few common
technologies used to concentrate cells
along with their indicative flow rates
and outputs. Continuous centrifuge
systems are the closest thing to
laboratory batch centrifuges and thus
require the least effort in process
development. Along with tangentialflow filtration (TFF) systems, they
feature greater flow rates than the
others.
Clinical Considerations: Figure 3
shows different technologies available
for large-scale culture of allogeneic
adherent cells. Throughout clinical
development, an increasing number of
patients will receive cellular doses,
with a significant increase beginning
in phase 3. Development costs will
rise and plateau until phase 1 scale-up
is complete. Not only must a marketed
38 BioProcess International
13(8)sp
Continuous
Pelleted
(e.g., Unifuge)
3,000
1,700
–
Tangential-Flow
Filtration
20–12,500
100s to 1,000s
+++
product demonstrate comparability,
but it also should be manufactured in
an optimized culture system.
Progressing through clinical phases
require time and effort. Pall supports
its customers in this endeavor by
providing innovative single-use
technologies, teams of specialists for
process development assistance,
automation and validation, and a global
manufacturing presence to secure the
supply. When developing an industrial
process from research and development
(R&D) to good manufacturing process
(GMP) production, a company should
focus on market approval. In an
iterative approach to implement
fundamental changes, both upstream
and downstream processes should be
considered as a whole.
Three primary reasons why cell
therapy companies fail are
inappropriate business plans,
overestimated market penetration, and
underestimated costs. Making the
informed and correct process design
decisions will help to align technology
and product roadmaps to bring a highquality product to market at the right
moment — and at the right price.
S eptember 2015
Professor Suzanne Farid leads
research into novel, computer-based
decision-support tools that provide
systematic foundations to help
companies make better decisions
with inevitable uncertainty in process
performance, market projections,
and clinical success rates. Her team’s
research focuses on establishing and
integrating modules on bioprocess
economics, manufacturing logistics,
dynamic simulation, uncertainty
analysis, multiobjective decision
making, and combinatorial
optimization. Below, she presents
insights from the advanced
bioprocess economics model
designed by her team.
An Advanced Bioprocess
Economics Model
Over the past 15 years, cell therapies
have begun to reach the market. Some
companies have faced challenges to
achieving scalable, cost-effective, and
robust cell therapy manufacturing —
leading to notable failures caused by
manufacturing concerns, such as high
CoG. Now the industry is asking,
“How can cell therapies achieve the
manufacturing success of
biopharmaceuticals?”
A significant proportion of cell
therapy products in development are
allogeneic. Obtaining cells from
universal donors is closer to a
traditional, product-driven
biopharmaceutical business model
than patient-specific therapies. “Offthe-shelf ” products and processes can
benefit from scaling up. Yet key
differences remain between
biopharmaceuticals and cell therapies,
creating unique manufacturing
challenges for the latter.
Protein-based biopharmaceuticals
have benefited from the availability of
large-scale technologies and associated
economies of scale. That is not the
case of cell therapies because of their
relative novelty and the inherent
complexities of manufacturing living
cells as products. A further challenge
is the adherent nature of the types of
cells used for allogeneic therapies.
They come from healthy donors and
have limited expansion potential,
complicating matters with large
Sponsored Supplement
Figure 4: Decisional tool for cell therapy manufacturing
Demand
Technology Options
Process/Facility/Cost Parameters
Demand (103 doses/year)
Cell type
Figure 5: Lot size and market demand
Lot Size (doses/lot)
Decisional Tool
Decisional tool
integrated:
C#
• Process economics
• Optimization
• Visualization
Case study scope:
• Allogeneic manufacture
• Optimal USP & DSP kits
• Current technology gaps
• Performance targets
Bioprocess
Economic
Model
Optimization
Algorithm
Origin
MS Access
Visualization
Tools
MS Excel
Data Analysis
Tools
Database
Graphical User Interface
Visual C#
Optimal upstream and downstream process strategy for each demand
Cost of goods per dose (CoG/dose) and CoG breakdowns
Sponsored Supplement
database of process facility and cost
parameters. Key outputs describe the
optimal technologies to use for
upstream and downstream processing,
with details of their CoG structure.
Using a step-by-step approach, we
first looked at the economic
competitiveness of cell culture
technologies and then at volumereduction decisions. Finally, we
explored cost implications of making
process changes at different times
throughout a product’s life cycle.
Case Study — Cell Expansion (1):
We explored multiple scenarios with
different demands, lot sizes, and
50
100
20
10
500 1,000 2,500 10,000
<10 lots/year
5
100 50
10
10
200 100
20
10
100
50
20
>200
200 100
lots/year
40
10
200
50
50
100
500
doses, considering million- to a
billion-cell doses. Figure 5 shows lots
per year for each combination of lot
size and demand. For each of practical
combination of lot size and demand,
we ran an optimization to pick out the
most cost-effective technology.
We considered planar technologies
(e.g., 10-layer vessels and multilayer
bioreactors) and microcarriers in singleuse bioreactors. The tool identified
that, for low-dose scenarios, planar
technologies are feasible (blue in Figure
6). We extended our analysis across all
dosages (106 –109 cells). The figure
shows where 2D technologies cease to
be feasible and it’s better to switch to
3D culture: at the high doses of 108
and 109 cells with four large lot sizes
(pink in Figure 6). The gray area in the
bottom right-hand corner of the figure
for very high-demand, high-dose
scenarios indicate production scenarios
Figure 6: Allogeneic cell expansion decisions — optimal technologies across demand/lot-size
matrix and dosage requirements; blue indicates where planar technologies are feasible, pink where
microcarriers in bioreactors are the only option, and gray where no upstream technologies exist.
Blue indicates where planar technologies are feasible, pink where microcarriers in bioreactors are
the only option, and grey where no technologies exist.
Lot Size (doses/lot)
Demand (103 doses/year)
50
Demand (103 doses/year)
commercial demand. Another key
difference is that single-use
technologies are considered essential
for cell therapies because of sterility
concerns, putting further constraint
on technology options available.
At University College London
(UCL), we investigated production
processes used for cell therapies in both
clinical and commercial processes and
found that they tend to rely on T-flask
or 10-layer trays. However, based on
typical dosages and market demand
projections, we estimated that
commercial therapies could require lot
sizes of over a trillion cells. To meet a
maximum demand of 10-trillion–cell
lot sizes, you would need 100,000
10-layer vessels. That is a very large
number, but operators can handle only
about 50–100 vessels per lot. This
points to a need for alternative
technologies that can support sufficient
cell numbers for commercial lot sizes.
We created a decisional tool for
identifying the most cost-effective
technologies for manufacturing
commercial allogeneic cell therapies.
We tested it across a range of different
scales and identified technology gaps
and technical innovations required to
fill them. Our tool combines bioprocess
economics with optimization and
visualization (Figure 4).
Key costs captured in the tool cover
materials (e.g., media and single-use
components), labor, quality control (lot
release testing), and fixed overhead.
Key inputs include cell type and
demand, technology options, and a
1
1
100
Lot Size (doses/lot)
500 1,000 2,500 10,000
50
100
500 1,000 2,500 10,000
Dose = 10 cells
Dose = 107 cells
Dose = 108 cells
Dose = 109 cells
6
5
10
50
100
500
1
5
10
50
100
500
S eptember 2015
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Figure 7: Allogeneic cell expansion decisions — technology S-curve for cell therapy manufacture
Target: 10,000 billion cells/lot
(e.g., lot size = 10,000 doses,
dose = 109 cells)
10,000
5,000
Performance (109 cells/lot)
1,000
500
Microcarriers
Automated multilayers
Multilayered bioreactors
100
50
Compact flasks and multilayers
10
5
1
0.5
commercial lot sizes, where even
microcarrier technologies would need
to double their performance. The
team then looked at how this could be
achieved. Doubling the cells/lot can
be achieved through different
combinations of cell concentrations
and number of single-use bioreactors
(Figure 8, left). The right side of
Figure 8 shows combinations of
microcarrier densities and surface
areas required to achieve the desired
cell concentration.
Multilayers
Case Study — Volume Reduction
(2, 3): Trillion-cell lot sizes pose
challenges for volume reduction and
downstream processing. The current
approach is to use bench-top
centrifuges. But to meet the maximum
demand for very high lot sizes would
require a ridiculous number of those
instruments (25,000). So we explored
the potential of more scalable
technologies, determining that they
would also need to be single use. The
choices were TFF and single-use,
fluidized-bed centrifugation. Again,
our goal was to identify the most costeffective technology and look for
bottlenecks.
Figure 9 overlays downstreamprocessing bottlenecks with the
optimal cell culture technology
matrices from the previous case study.
T flasks
0.1
R&D Effort and Investment
that cannot be met by any existing
technologies because the number of
units required per lot exceeds the
maximum allowable.
We created a technology “S” curve
to visualize performance trajectories
and limits in cell output for each
technology (Figure 7). For each one,
we plotted the maximum number of
cells per lot against the R&D effort.
Each technology covers one log of
performance (in billions of cells per
lot) before it must be replaced by a
newer technology. For example,
T-Flasks will allow users to reach a
billion-cell lot size, then 10-layer
vessels will reach up to 10-billion–cell
lot sizes. The output of all planar
technologies caps at ~500 billion cells
per lot, above which microcarriers
become the only feasible option.
The target was to reach 10 trillion
cells per lot. But a technology gap
exists for meeting the largest
Figure 8: Allogeneic cell expansion decisions — future performance targets for microcarrier applications
8
Cell harvest density
20,000 cells/cm2
6
Microcarrier surface area
5
Microcarrier density
4
3
10,000
Production target
10,000
A
2
B
1
0
1
2
3
4
5
6
Number of Single-Use Bioreactors
7
LEFT: Base Case: 0.5M cells/mL
Production target can be achieved through different
combinations of cells/ml and #SUBs: (A) 2.6 million cells/mL
with three SUBs per lot; (B) 1.3 million cells/mL with six
SUBs per lot
8
Microcarrier Surface Area (cm2/g)
Million Cells/mL
Characteristics of point X:
Billion cells/lot
7
8,000 cm2/g
16 g/L
it y 2 20,000
ns )
de /cm
s t e lls
e
r v (c
Ha nge
25,000
ra
30,000
9,000
8,000
y
si t 2 )
en m 20,000
t d lls/c
s
e e
r v (c
25,000
Ha nge
ra
30,000
1.3
7,000
6,000
5,000
But currently:
360–5,500 cm2/g
(literature)
X
2.6
4,000
3,000
2,000
Million cells/mL
5
RIGHT: Windows of Operation:
10
15
Microcarrier Density (g/L)
20
Desired number of cells/mL can be achieved through different
combinations of microcarrier density, surface area, and harvest density.
40 BioProcess International
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S eptember 2015
Sponsored Supplement
Sponsored Supplement
Figure 9: Allogeneic cell downstream process decisions — downstream processing bottleneck
identification across demand/lot-size matrix and dosage requirements; blue indicates where planar
technologies are feasible, pink where microcarriers in bioreactors are the only option, and gray
where no upstream technologies exist.
Lot Size (doses/lot)
Demand (103 doses/year)
Demand (103 doses/year)
50
100
Lot Size (doses/lot)
500 1,000 2,500 10,000
1
50
100
500 1,000 2,500 10,000
Dose = 10 cells
Dose = 107 cells
Dose = 108 cells
Dose = 109 cells
6
5
10
50
100
>200
500 lots/year
1
5
m
ea
tr n g
ns si ck
w ce s n e
D o pro t tle
bo
A bottleneck occurs downstream in
the pink region, as you move to large,
single-use bioreactors with
microcarriers (often >1,000 L in
volume). Those large, single-use
bioreactors would need to be
staggered, and only fluidized-bed
centrifugation would be an option.
We also explored whether CoG is
dominated by upstream or downstreamprocessing costs. It is also important to
consider cells/lot because the ratio of
upstream to downstream processing
costs varies with scale. For planar
technologies, as scales increase, the ratio
of upstream to downstream costs moves
from being a 50–50 split to one that is
dominated by upstream processing
costs. By contrast, for microcarrier
processes, the cost of goods is
dominated by downstream processing.
This type of analysis can help identify
where to focus R&D efforts.
We then investigated CoG targets
as a percent of sales (in relation to
reimbursement values) by plotting
CoG/dose for different multilayer
vessels and microcarrier processes
against typical selling prices. For the
high-dose scenario of 109 cells/dose,
we assumed that allogeneic therapies
would have gross margins in line with
those of other biologics. If that target
is for a CoG as 15% of sales, only
microcarrier processes would meet the
target and allow for a successful
business model. If the selling price is
even lower, as some reports suggest,
then not even microcarriers could help
a company meet that target gross
margin without process improvement.
More Work to Be Done: In the
examples above, results predict that for
the cell therapy industry to be
sustainable in high-demand cases,
technical innovation and optimization
are still required to make costeffective and scalable manufacturing
processes possible. However, recent
research and offerings from some
vendors to bridge the gaps that are
currently restraining large-scale
commercialization suggest that this
will be achievable.
Our model is being extended in
on-going collaborations: e.g., how
uncertainties affect robustness of
manufacturing processes, as well as
10
50
100
500
their CoG and reimbursement
economics. We’re also working to
provide a framework for reconciling
conflicts between financial and
operational benefits for different
technologies. And in parallel, the
UCL team is exploring how to design
feasible business models for autologous
manufacture and providing a detailed
economic evaluation of centralized
and decentralized facility
configurations.
Questions and Answers
The industry faces many challenges to
the manufacture of allogeneic cell
therapies. With the end goal of market
approval in mind, a stepwise approach
to fundamental changes is critical.
Both upstream and downstream
processes should be considered as
parts of the whole manufacturing
process. Cost, scalability, and
flexibility are some key considerations
to take into account when choosing
the best manufacturing platform for a
cell therapy. Dose, patient population,
desired scale, and number of lots
required are critical determining
factors in technology selection. The
UCL decisional tool can help you
identify optimal technologies for
commercial cell therapy bioprocesses
and technical innovations required to
move these therapies forward.
Here are some questions posed by
clients interested in these topics.
What about porous microcarriers as
an alternative to surface-only
microcarriers? (TB) Porous
microcarriers increase the surface
available for cell growth, but they also
create physical gaps in some portions
where cells can grow but the
microenvironment is modified, which
could affect cell quality. So although it
may be good that you have more cells,
their quality may not remain
consistent and comparable to that of
early cell cultures.
What factors do companies need to
consider when switching from planar
to microcarrier technologies? (SF)
Microcarriers can offer >50% CoG
savings at commercial scale. Consider
whether the CoG savings at
commercial scale would outweigh the
higher cost of development. Also
consider the best time to switch,
whether early or late in development
or after product approval.
Analyses have been extended to
capture the consequences of switching
from planar to microcarrier
technologies on drug development,
clinical and commercial manufacture,
and clinical trials. Considerations
included their effects on process
development, tech transfer,
comparability studies, and process
S eptember 2015
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validation or qualification batches.
And from a clinical trial perspective,
it’s important to consider extra
bridging studies and whether you may
have parallel arms in your clinical trial
studies.
So the outcome depends on
whether you take a drug-development
or product life cycle perspective. That
will affect the weightings you assign
to costs (of drug development and
commercial CoG savings). Go–no-go
decisions are also influenced by the
potential market size, dose, and
therapeutic selling price.
What cell types have been
successfully grown in the Xpansion
bioreactor? (TB) The bioreactor was
developed after we were contacted by
a potential user who wanted to grow
stem cells and maintain them in the
same microenvironment as in a
multilayer stack. So the default use is
for stem cells of different origins. But
there have been some successful
attempts to grow mammalian cell
lines (e.g., VERO) and others for gene
therapy, as well.
How would you sum up the current
state of cell therapy manufacturing?
(SF) There are bottlenecks in both the
upstream and downstream processes,
particularly for the high-demand,
high-dose scenarios. This offers
innovation potential for technology
providers to adapt existing technologies
for cell therapies and help bridge the
gaps constraining large-scale
production. Existing characterization
methods make comparability difficult
and can act as a disincentive to process
changes once human trials have begun.
So choosing the best technology early
in development can be critical to
success. And to pick the right
technology early on, you need to have
an outlook toward the potential
commercial scale and consider the
whole life cycle of your drug. That will
help ensure success of cell therapies
overall, to push them through
development to market.
What is the primary driver for
choosing planar or 3D culture
platforms in cell therapy applications?
(TB) That would be the ultimate
production scale. Current planar
systems can achieve only 5 × 1011 cells
42 BioProcess International
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at maximum. If you need to go higher
than that, then you need to use a 3-D
system. Some people are discussing
microfibers and hydrogels, but they
present some significant problems and
have their own limitations of scale.
So cost and scale of manufacturing
are the main drivers and space as
well. With classical multitray, you
would need a huge space and many
technicians to manage so many units.
That’s becoming really problematic.
What is the best time to begin
process development for scaling up to a
microcarrier bioreactor platform to
volumes required in phase 3 and
beyond? (TB) You have to look at the
number of cells you need to produce for
the market. You should know how
many cells will be needed per patient
during or at the end of phase 2. Some
people focus on getting to market as
quickly as possible with as few process
changes as possible just because they
are running short of cash and want to
move on and raise additional funding.
They would have to switch to 3-D a bit
later if they need to scale it up. Others
to consider going 3-D immediately or
as early as possible and really scale up
during clinical testing, thus avoiding
the switch and maintaining product
comparability. It’s always the client’s
choice, and our recommendation is to
transition whenever you can, knowing
that it will represent hard work and
eventually additional clinical trials
unless you start as early as possible
using microcarriers — if that will be
necessary in the end.
Quality control is critical. You need
to ensure that the comparability of the
cells remains consistent across the
different technologies that you use. So
quality control really needs to be cast
in stone.
What are your general
observations regarding cell culture
media requirements for the different
approaches to cell expansion? (TB)
The Xpansion bioreactor was
designed to consume the same
amount of medium as in a regular
cell stack or multitray stack. If you
use ~100 mL/layer, then for 20
10-layers you would use 20–30 L.
When it comes to microcarriers,
you’re going to get more cells per
S eptember 2015
volume, so you may consume more
medium per batch. But if you
consider the amount of medium
consumed by each cell, then it
remains roughly the same as the
planar technologies.
Costwise, as you scale up processes
and move toward higher demand,
you’ll see that material cost becomes
a much more significant proportion
of CoG. With planar technologies,
consumables dominate over media
costs; in 3D microcarrier processes,
media costs are more dominant than
consumables. In all cases, industry
needs to move towards a less
expensive medium free of animalsourced ingredients, which will
follow the media development that
we’ve seen for mammalian cell
culture.
What key questions does the
sector need to address for successful
commercialization of cell therapies?
(SF) It will be underpinned by a
number of factors. First, we need a
cost-effective and robust
manufacturing process at large scale
— and in particular, downstream
technologies must catch up to the
upstream. In addition, transportation
and logistics infrastructure will be
critical to cell therapy success.
Companies need to ensure that they
have feasible business models early on
to help them decide whether a
process is cost effective or requires
innovation. They also need to think
about that in relation to
reimbursement levels. Successful
companies will have the know-how
related to manufacturing logistics,
reimbursement, management of
clinical trials, and marketing to
create that successful business model.
Many people are still using
traditional systems such as multiplate
stacks for development and early
stage clinical trials. What do you
believe is the biggest challenge to
using those systems as cell therapies
move through the clinical pathway to
commercialization? (TB) First, they
are very labor-intensive just to
manipulate large numbers of
multitray stacks. And you can
imagine the space needed to incubate
hundreds of multitray systems. You
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have to manipulate them and protect
them in cleanrooms, validate them,
and so on. The volumes are critical.
Traditional systems are still very
open and not well controlled.
Basically, you put the trays under an
incubator and rely on that to control
the environment in which cells grow.
GMPs are about control, so moving
toward bioreactors is a major step
forward and a critical process
improvement.
Finally, multitray systems will not
be able to support large-scale
production at industrial levels.
Producing some allogeneic therapies
would require the equivalent of one
or two Empire State buildings just
for incubation space, which is pretty
unrealistic.
Acknowledgments
UCL’s cost modeling outputs shown here
were part of a project funded by the UK
Technology Strategy Board with Lonza as
the lead partner. Financial support from the
Technology Strategy Board (UK) and Lonza
is gratefully acknowledged. Constructive
feedback and technical advice from
industrial experts at Lonza and vendors is
gratefully acknowledged.
References
1 Simaria S, et al. Allogeneic Single-Use
Cell Expansion Decisions. Biotechnol. Bioeng.
111(1) 2014: 69–83.
2 Hassan S, et al. Allogeneic Single-Use
Volume Reduction Decisions. Regen. Med.
2015 (in press).
3 Hassan S, et al. Process Change
Evaluation Framework for Allogeneic Cell
Therapies (in preparation). •
Thierry Bovy is global product manager of
Xpansion multiplayer bioreactor systems
(thierry_bovy@pall.com), and Alain
Fairbank is director of marketing for cell
therapies at Pall Life Sciences (alain_
fairbank@pall.com). Suzanne Farid is a
professor in bioprocess systems engineering
at University College London’s department
of biochemical engineering; s.farid@ucl.
ac.uk.
Sponsored Supplement
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Features: The Xpansion single-use
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Contact Pall Life Sciences
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S eptember 2015
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BioProcess International
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S e c t i o n T h r e e CELL THERAPY MANUFACTURING
Production of Viral Vectors
Using the iCELLis® Fixed-Bed
Bioreactor System
Beyond Mesenchymal Stem Cells:
Gene-Modified Cell Therapy, Gene Therapy, and Exosomes
by Matt Kremer
I
n the past few years, the resurgence
of cell-based immunotherapies —
and, by extension, gene therapies
— has accelerated as products
move rapidly from academic research
laboratories into commercial
development. A successful clinical trial
by St. Jude Children’s Research and the
University College London of a gene
therapy for hemophilia B was a seminal
translational event (1), as was the
licensing of the University of
Pennsylvania’s gene-modified chimeric
antigen receptor T-cell technology
(CAR-T) for leukemia by Novartis (2).
CAR-T cells and gene therapies are
now among the most dynamic areas for
biotechnology investment and
development. Both gene therapies and
gene-modified cell therapies require
large amounts of viral vectors, either
for direct delivery to patients or for ex
vivo modification of cells that are later
dosed to patients.
Another exciting area of research
and development is that of exosomes,
by which stem cells are cultured with
the intent of generating secreted
factors that can be administered for a
therapeutic effect. Instead of
administering cells that will provide
growth and trophic factors in situ, the
potential exists for those factors to be
produced ex vivo, then harvested and
subsequently administered for
therapeutic purposes.
44 BioProcess International
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Figure 1: The iCELLis bioreactor (Pall Life Sciences) is a scalable, single-use, fixed-bed bioreactor.
Many processes for the production
of viral vectors require the large-scale
culture of adherent cells. Although
more traditional two-dimensional
(2D) culture technologies are
sufficient for producing phase 1 and 2
materials, they may not be sufficiently
scalable when companies move down
the industrialization pathway toward
phase 3 clinical trials and commercial
production.
Single-Use, Fixed-Bed Bioreactors
Some companies are attempting to
develop suspension culture processes,
but fixed-bed bioreactors have emerged
as an ideal technology to support large-
S eptember 2015
scale production of viral vectors — e.g.,
>1016 viral particles (vp) per batch. One
primary advantage that comes with the
use of fixed-bed bioreactor technology
is the large surface area to which
adherent cells can attach, expand their
numbers, and then be infected or
transfected for viral-vector production.
Other major advantages include the
ability to achieve higher density
cultures; real-time control of
parameters such as pH, dissolved
oxygen (DO), temperature, perfusion,
and agitation; and low shear stress for
shear-sensitive cells (3).
Fixed-bed technologies are
currently used for commercial
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biologics production. Until recently,
what has been lacking is a large-scale,
single-use system. The iCELLis
bioreactor (Figure 1) is a fully
integrated, high–cell-density
bioreactor designed to simplify
adherent-cell culture processes by
combining the advantages of singleuse technologies with the benefits of a
fixed-bed system. Designed for rapid
implementation and ease of use, this
compact system represents a new
generation of single-use bioreactors.
The fixed bed in an iCELLis
system is based on 0.62-cm × 2.5-cm
macrocarriers made of hydrophilized,
nonwoven, medical-grade polyethylene
terephthalate (PET) microfibers,
ensuring excellent cell growth in a
3-D environment (Figure 2, Figure 3).
Contained in a housing through
which media flows from bottom to
top, the macrocarriers remain fixed in
place. The iCELLis system comes in
two formats: the benchtop iCELLis
Nano system and the large-scale
iCELLis 500 bioreactor. The height
of their fixed beds can vary (2, 4, and
10 cm), as can the density of carrier
packing. Table 1 lists available
configurations for both models.
Customers can choose among three
bed heights and two compaction
densities for a total of six fixed-bed
configurations. So the iCELLis Nano
ranges from 0.53 m 2 to 4.0 m 2 of
surface area for cell growth; the
commercial scale ranges 66–500 m 2 of
surface area. Table 2 compares
iCELLis surface areas with those of
other commercial systems. The scaling
factor between iCELLis Nano and
iCELLis 500 systems is 125×. Both
are fully functional bioreactor systems
that include monitoring and control of
pH, DO, temperature, and agitation.
Notable is the constant bed height
and compaction density for scaling.
Each small-scale iCELLis Nano
bioreactor has a corresponding largescale iCELLis 500 unit with the same
bed height and compaction. The
cellular microenvironment is the same
in an iCELLis 500 as in an iCELLis
Nano system, so a process developed
in the latter can be transferred directly
to the corresponding iCELLis 500.
Additionally, significant biomass
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Figure 2: The iCELLis fixed-bed macrocarrier substrate: polyethylene terephthalate (PET) fiber carriers
Table 1: iCELLis bioreactor configurations at small and manufacturing scales
Bioreactor
iCELLis Nano
iCELLis Nano
iCELLis Nano
iCELLis 500/100
iCELLis 500/200
iCELLis 500/500
Fixed-Bed Bioreactor
Diameter Height
Volume
Volume
110 mm
20 mm
0.04 L
1L
110 mm
40 mm
0.08 L
1L
110 mm 100 mm
0.20 L
1L
860 mm 20 mm
5.00 L
70 L
860 mm 40 mm
10.00 L
70 L
860 mm 100 mm
20.00 L
70 L
Surface Area
Low
High
Compaction Compaction
0.53 m2
0.8 m2
2
1.06 m
1.6 m2
2
2.65 m
4.0 m2
2
66.00 m
100.0 m2
2
133.00 m
200.0 m2
2
333.00 m
500.0 m2
multiplication can occur in the fixed
bed, which simplifies seed-train
development. It is feasible to inoculate
an iCELLis bioreactor at low cell
density (e.g., 5,000 cells/cm 2 or even
lower) and then amplify cells in the
fixed bed to easily reach up to
500,000 cells/cm 2 or higher.
iCELLis System for
Viral Vector Production
Both gene therapies and genemodified cell therapies require viral
vectors to introduce a gene of interest
into target cells. Gene therapy viral
vectors are administered to patients.
For gene-modified cell therapies (e.g.,
CAR-T cells), a patient’s own target
cells are isolated, then modified by a
gene introduced by a viral vector,
expanded in vitro, and finally returned
to the same patient. Several viral
vector types have been used for such
applications: e.g., adenovirus,
adenoassociated virus, retrovirus, and
lentivirus. Some of those are produced
in stable producer cell lines; others
require transient transfection.
The iCELLis system has emerged
as a premier platform for viral vector
production, with successful academic
Figure 3: Micrograph of cells growing on
iCELLis Microfiber substrate.
and commercial operations using such
standard adherent cell lines as human
embryonic kidney (HEK 293) cells,
adenocarcinomic human alveolar basal
epithelial (A549) cells, and mouse
leukemia (PG13) cells.
Retrovirus: A recent issue of the
Journal of Immunotherapy included
an article by researchers at Memorial
Sloan Kettering Cancer Center
(MSKCC) (4). They reported results
from comparing retrovirus production
by stable packaging PG13 and HEK
293Vec cell lines in an iCELLis Nano
system and a Nunc Cell Factory
S eptember 2015
13(8)sp
BioProcess International 45
Table 2: Comparing surface areas for iCELLis Nano and 500 bioreactors with those of other systems for adherent cell culture; scaling factor between
iCELLis Nano and iCELLis 500 systems is 125×.
Bed Height
(Compaction)
2 cm (C1.0)
2 cm (C1.5)
4 cm (C1.0)
4 cm (C1.5)
10 cm (C1.0)
10 cm (C1.5)
iCELLis Nano
850-cm2
Roller
10-Layer Cell
Bottles
Stacks (CS10)
6.2
0.8
9.4
1.3
12.4
1.7
18.8
2.5
31.8
4.3
47.1
6.3
Surface
Area
0.53 m2
0.80 m2
1.06 m2
1.60 m2
2.65 m2
4.00 m2
system (Thermo Scientific), which is a
familiar 2D multiplate system.
High-titer vector stocks were
harvested over 10 days, representing a
much broader harvest window than
the three-day harvest window
afforded by cell factories. For PG13
and 293Vec packaging cell lines, the
average vector titer and the vector
stocks’ yield in the bioreactor were
higher by 3.2- to 7.3-fold and 5.6- to
13.1-fold, respectively than those
obtained in cell factories. The vector
production was 10.4 and 18.6 times
more efficient than in cell factories
for PG13 and 293Vec cells,
respectively. Furthermore, the vectors
produced from the fixed-bed
bioreactors passed the release test
assays for clinical applications.
Therefore, a single vector lot derived
from the 293Vec is suitable to
transduce up to 500 patients cell
doses in the context of large clinical
trials using chimeric antigen receptors
or T-cell receptors. These findings
demonstrate for the first time that a
robust fixed-bed bioreactor process
can be used to produce γ-retroviral
vector stocks scalable up to the
commercialization phase. (4)
Total vector production by 293Vec
cells was reported at 2.53 × 1012 in an
iCELLis Nano 200-mL bioreactor,
which features a surface area of
iCELLis 500
HyperStack 36
0.3
0.4
0.6
0.9
1.5
2.2
Surface
Area
66 m2
100 m2
133 m2
200 m2
333 m2
500 m2
850-cm2
Roller Bottles
776
1176
1565
2353
3918
5882
26,500 cm2 (2.65 m2), compared with
production at 1.93 × 1011 in a 6CF10 with
a surface area of 38,160 cm2 (3.816 m2).
If scaled to the corresponding iCELLis
500 bioreactor with 333 m2 of available
surface area, a theoretical vector yield
could reach 3.2 × 1014/batch.
Reported vector production in PG13
cells was 2.9 × 1010 for the iCELLis
bioreactor and 3.96 × 109 in a Cell
Factory system. At the corresponding
iCELLis 500 scale, vector production
could reach 3.6 × 1012/batch. Yields
could be further increased 1.5× by
scaling to higher-compaction bioreactors
that were not available at the time of
MSKCC’s evaluation.
Adenovirus: Finvector, a contract
manufacturer based in Finland,
presented its work with the iCELLis
system at industry conferences including
the March 2015 ISBiotech Spring
Meeting (5) and the annual conference
of the American Society for Gene and
Cell Therapy (6). The company scaled a
transient transfection adenovirus process
using HEK293 cells from an iCELLis
Nano system to a 100-m2 iCELLis 500
bioreactor, achieving a reported
6 × 1015 viral particles/batch. Additional
scaling to a 500 m2 iCELLis could
increase the yield up to fivefold.
Recombinant Adenoassociated Virus
(rAAV): At the 2013 European Society
for Gene and Cell Therapy (ESGCT)
meeting, researchers from the University
10-Layer Cell
Stacks (CS10)
104
157
209
314
524
786
HyperStack 36
37
56
74
111
185
278
College London (UCL) presented
results of three plasmid rAAV
transfections into HEK 293T cells,
again comparing an iCELLis Nano
bioreactor with a Cell Factory control
(7). Conclusions reached in this study
included that the
. . . iCELLis Nano system appears to
be suitable for the development of
industrial scale production of rAAV
vectors by transfection. Optimization
of the culture parameters and
harvesting method will likely allow for
a similar productivity compared to
conventional cell culture platform
(CF10 harvested by freeze/thaw
technique). Linear scalability is
extrapolated to produce viral yields of
at least 2–5 × 1015 vg (viral genomes)
per run at large scale in a 133 m2
iCELLis bioreactor.
In a poster at the same conference,
Rentschler Biotechnologie presented
results of an initial experiment with a
two plasmid rAAV construct in HEK
293T cells. That team observed similar
cell counts and metabolite profiles
between iCELLis and their control,
concluding,
Overall, this first set of data is very
promising, especially when
considering that the underlying
process was optimized for 2D
culture vessels. The controlled
iCELLis bioreactor system offers
Table 3: Comparing iCELLis results with yield extrapolation to iCELLis 500-m2 fixed-bed
Vector
Retrovirus
Adenovirus
rAAV
Group
MSKCC
MSKCC
Finvector
UCL
Rentschler
46 BioProcess International
Cell Line
HEK 293Vec
PG13
HEK 293
HEK293T
HEK293T
13(8)sp
Scale
nano
nano
100
nano
nano
S eptember 2015
Surface Area
2.65 m2
2.65 m2
100.00 m2
0.53 m2
0.53 m2
Vector Yield
2.53 × 1012
2.9 × 1010
6 × 1015
2.3 × 1013
2.2 × 1010
Projected Yield
at 500 m2
4.78 × 1014
5.48 × 1012
3 × 1016
2.17 × 1016
2.07 × 1013
Scaling Factor
189×
189×
5×
943×
943×
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many possibilities for process
development. For instance the results
of the growth experiments
. . . show that lower seeding densities
could facilitate the seed train for
large-scale production. Moreover,
the fact that the cells could be grown
to more than 2E+06 cells/cm 2 by
simple medium exchange indicates
the potential for process
optimization and, therefore, possible
improvement of productivity.
Lentivirus: Viral-vector production
in iCELLis with lentivirus is currently
being pursued by several commercial
entities for which publicly disclosed
information is not yet available. At the
2015 ASGCT meeting, however,
Finvector did present initial lentivirus
results, indicating successful
transfection and virus yield (8).
Exosomes: An Emerging
Therapeutic Approach
Therapeutic application of exosomes is
an emerging area of research and
development. Exosomes are lipid
bilayered nanovesicles secreted by cells.
First described in the 1970s, exosomes
are attracting recent commercial
interest due to the recognition that
they play important roles in paracrine
and autocrine signaling (9). Exosomes
secreted by stem cells may provide the
same functional benefit as their parent
cells but with reduced immunogenicity,
simplified handling (e.g., long term
stability without cryopreservation) and
biomanufacturing through traditional
cell culture techniques (10). Capricor
Therapeutics Inc. (Los Angeles, CA)
and ReNeuron Group plc (Guilford,
UK) have both announced exosomedevelopment programs. For production
of exosomes, the iCELLis bioreactor
can provide a continuous process
environment for long-term cultivation
of stem cells and harvest of exosomes
through perfusion culture.
Conclusion
The varied nature of cell therapies —
including gene-modified cell therapies
— requires a range of manufacturing
technologies to address different
process and scale requirements for
commercial-scale production volumes.
Specifically, viral-vector production
Sponsored Supplement
processes require large surface areas
for adherent cell culture. Fixed-bed
bioreactors represent an ideal
technology for supporting large-scale
production of viral vectors. Pall Life
Sciences’ iCELLis fixed-bed
bioreactor is already being used
worldwide for vector and virus
production applications. The system
provides surface areas up to 500 m 2 in
a compact, closed, and single-use
format, with possible yields reaching
>1016 viral particles per batch.
References
1 Nathwani, et al. Adenovirus-Associated
Virus Vector–Mediated Gene Transfer in
Hemophilia B. New Engl. J. Med. 2011: 2357–
2365.
2 University of Pennsylvania and Novartis
Form Alliance to Expand Use of Personalized T
Cell Therapy for Cancer Patients. University of
Pennsylvania: Philadelphia, PA, 6 August
2012; www.uphs.upenn.edu/news/News_
Releases/2012/08/novartis.
3 Rowley J, et al. Meeting Lot-Size
Challenges of Manufacturing Adherent Cells
for Therapy. BioProcess Int. 10(3) 2012: S16–S22.
4 Wang, et al. Large-Scale Clinical-Grade
Retroviral Vector Production in a Fixed-Bed
Bioreactor. J. Immunother. April 2015: 127–135.
5 Lesch. Process Development and Clinical
Production of Viral Vectors for Phase I and Beyond.
IS Biotech: Washington, DC, 2015.
6 Lesch, et al. iCELLis Fixed-Bed
Technology for Adherent Cells in an Efficient,
Scalable System for Viral Vector Production.
American Society for Gene and Cell Therapy, New
Orleans, LA, 2015.
7 Lennaertz A, et al. Adeno Associated
Virus Production Using a Disposable, FixedBed Bioreactor from Bench-Scale to Industrial
Scale. BMC Proceedings 7(S6) 2013: 59; www.
biomedcentral.com/1753-6561/7/S6/P59.
8 Pegel A, et al. Setting Up TransfectionBased Recombinant AAV Production in the
iCELLis Single Use Fixed-Bed Bioreactor.
Rentschler Biotechnologie GmbH: Laupheim,
Germany, 2013; http://rentschler.de/fileadmin/
Downloads/Poster/14760_REN_POS_Wissen_
Poster_841x1189mm_4c_4_Ansicht.pdf.
9 Ibrahim, et al. Exosomes As Critical
Agents of Cardiac Regeneration Triggered By
Cell Therapy. Stem Cell Rep. 2014: 606–619.
10 CTX-Derived Exosomes. ReNeuron
Group plc: Guildford, UK, 2015; www.reneuron.
com/products/ctx-derived-exosomes. •
For copies of posters cited in this article,
please contact the author: Matt Kremer is
sales manager for new markets in cell culture
technologies at Pall Life Sciences, 1-215-7565837; matthew_kremer@pall.com.
Featured
Products
iCELLis® Fixed-Bed Reactor
Applications: Efficient, scalable viral
vector production for gene therapy
Features: The iCELLis fixed-bed
bioreactor technology has been
validated from bench to process
for production of human and
animal vaccines, viral vectors,
and recombinant proteins. These
systems are available in two scalable
platforms: the large-scale iCELLis
500 and the scaled-down iCELLis
Nano bioreactor. They are different
from any other platform for adherent
cell cultures (e.g., roller bottles, cell
factories, or stirred-tank reactors with
microcarriers).
With a compact, fixed bed of
proprietary macrocarriers made of
polyester microfibers, these systems
provide ≤500 m² of growth surface
area in just 25 L of fixed bed. They can
be inoculated at very low cell density,
simplifying seed-train operations with
no intermediate reactor required.
The iCELLis systems were designed
for efficient and innovative media
circulation and oxygenation. A builtin magnetic drive impeller evenly
circulates media through the fixed bed
to ensure low shear stress and high cell
viability. These systems achieve and
maintain high cell densities that equal
or exceed the productivity of larger
stirred-tank units. With cells trapped
inside the fixed bed, no perfusion
equipment is required.
Contact Pall Life Sciences
bioreactors@pall.com
S eptember 2015
13(8)sp
BioProcess International 47
S e c t i o n F o u r PERSPECTIVES
Ask the Experts
Core Technologies Expand Opportunities
for Cell Therapy Manufacturing
by S. Anne Montgomery
P
all Life Sciences has long been
known for its expertise in
processing and filtration
equipment for the
biopharmaceutical industry. In recent
years, the company has broadened its
offerings in upstream manufacturing
by expanding its core capabilities in
the single-use, bioreactor, and
microcarrier arenas, with unique and
innovative technologies for cultivation
of cells to be used as therapies.
To explain how its scientific and
technology expertise is a logical fit
with this burgeoning area of the
industry, three experts from various
Pall business segments were asked to
comment on the company’s process
development capabilities, microcarrier
technologies, and the Xpansion
bioreactor system, specifically.
Contributing their insights to this
discussion are Fabien Moncaubeig
(head of bioprocess development
services at Pall Life Sciences in
Brussels, Belgium), Mark Szczypka,
(senior director, applications and new
product development, Pall Life
Fabien Moncaubeig
54 BioProcess International
13(8)sp
Europe
Brussels, Belgium — Process
Development, Application
Support
Portsmouth, UK — Research
and Development
USA
Westborough, MA — Process Development,
Research and Development, Application Support
Ann Arbor, MI — Process Development, Research and Development
Sciences, Ann Arbor, MI, USA ), and
Thierry Bovy (global product manager,
Xpansion multiplate bioreactor systems,
Pall Life Sciences, Brussels, Belgium).
Process Development Capabilities
— with Fabien Moncaubeig
Fabien Moncaubeig introduces Pall Life
Sciences’ bioprocess development services
and Pall’s expansion into cell therapy
bioprocessing. He then offers thoughts
about cell therapy manufacturing
technologies in general and the future of
microcarrier-based platforms in particular.
Pall Life Sciences approaches its cell
culture and downstream bioprocess
development services in two ways. Our
on-site support uses a team of
specialists who are experienced
scientists and assist our customers at
their sites in their labs. The team offers
extensive training on our technology
but also acts in an advisory capacity to
S eptember 2015
our partners throughout the entire
development process by analyzing
results and designing adequate
experimental plans with them.
A second option is to outsource our
customers’ complete process
development (from upstream to
downstream) to our labs. We offer
contract development services in
Europe and in the United States in
four different locations: Brussels,
Belgium; Portsmouth, UK; Ann
Arbor, MI; and Westborough, MA.
Historically, our process
development activity in the cell
therapy field has been driven by
collaboration to codevelop our
bioreactors (2D and suspension) and
our microcarriers. Thanks to our
partners, we have acquired significant
expertise in optimizing and scaling up
stem cell processes in 2D and 3D for
both autologous and allogeneic
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therapies. Given the demand from the
field for bioprocess experts to help
industrialize processes, we continued
offering this service as a contract
development organization (CDO).
In the past few years, we have
worked with a number of cell types
from various sources, including
mesenchymal stem cells (MSCs),
human embryonic stem cells (hESCs),
induced pluripotent stem cells (iPSCs),
and hepatocytes.
We are also industrializing viral
vector production processes using
adherent or suspension cultures at
scales up to a thousand liters.
How different are technologies for
cell therapy bioprocessing from those
of more traditional bioprocessing?
The main difference resides in the
product of interest. In cell therapy
processes, the cell is your product,
whereas for vaccines, viral vectors, or
monoclonal antibodies (MAbs), cells
are used only to secrete the product of
interest. As a consequence, the cell
therapy industry has been trying to fill
a technology gap to allow efficient cell
amplification and recovery whether
cells are grown in suspension or
adherent cultures. Also, unlike the cell
lines used in traditional bioprocesses
that have been selected and screened
for years, stem cells are far more
sensitive to their environment. This is
why we have adapted these cell culture
technologies.
How do you see Pall‘s role in
advancing cell therapies to
commercialization? Does Pall have a
complementary or competitive
relationship with contract
manufacturing organizations that focus
on cell therapies? Our motivation is to
increase process efficiency to help our
partners reduce production costs. We
are in an ideal position, thanks to a
wide portfolio of innovative
technologies from which we can guide
our customers and give neutral
recommendations. Whatever type of
cells they cultivate and the culture
mode required, we have a suitable
platform for them: adherence in 2D or
3D or suspension in rocking or stirred
bioreactors. Then we can support them
to minimize their learning curve during
process development and shorten their
timing to enter clinical trials.
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Pall’s intention is not to enter the
CMO business, but to facilitate
implementation of our bioreactors and
downstream solutions. Our contract
manufacturing organization (CMO)
partners take over our process
development once it’s optimized and
scaled up to turn it into a GMP
compliant process ready for clinical
phase productions.
Arguably, the most promising
large-scale technologies for expansion
of stem cells use microcarriers. What
are key considerations for choosing a
microcarrier-based cell-expansion
platform? Companies that choose to
use microcarrier platforms should
determine the bioreactor-scale
requirements for each clinical trial
phase to ensure that process
development milestones are
compatible with demand for clinical
material. A key factor for success is to
acknowledge the level of expertise
required for industrialization of
microcarrier-based processes.
When choosing the best
bioreactor for a cell therapy process,
what are key considerations? What
role do you think automation can
play? One of the key considerations
is the type of culture: whether it is
suspension or adherent. The next is
to understand what the final scale
targeted for each clinical and
commercial phase will be and
determine how you will produce
your product. Manufacturers of
allogeneic therapies will look for
bioreactors that can produce a high
number of cells, even if that requires
more complex systems such as stirred
tank bioreactors and microcarrier
combinations. By contrast, makers of
autologous therapies will lean toward
simple, automated systems requiring
small footprints and minimizing the
number of operations per patient.
The level of automation of largescale stirred-tank bioreactors is already
quite high. On the other hand, there
is a lot to do for autologous therapies
in terms of automation. This will be
key for minimizing production costs
and batch failure rates while not
compromising the costs of these future
therapies.
Other key considerations are the
time and resources a company can
afford to spend on its process transfer
into its new platform. Not all
bioreactors require the same amount
of effort to reach desired cell doubling
and quality.
Bioprocessing has almost
completely shifted to serum-free,
protein-free, animal-free, and
chemically defined media. This is much
less the case for cell therapy processes
in general. What are your thoughts
regarding serum/serum-free (and so
on) for cell therapies? Serum-free
processes are very likely to become a
standard in the cell therapy market;
first because of regulatory pressures,
and also because they offer better lotto-lot reproducibility. Finally, using
animal sera introduces limitations such
as significant supply and pricing
fluctuations. Although pricing of
serum-free media for stem cells might
be considered prohibitive for industrial
uses today, it’s fair to anticipate that
increasing demand will drive prices
down, resulting in scenarios similar to
vaccine or monoclonal antibody
markets, where costs of serum-free and
serum containing media have become
comparable.
As an alternative to serum, platelet
lysate is gaining popularity because it
could help transition from serum to
serum-free processes with its economic,
regulatory, and ethical advantages.
Mark Szczypka
Focus on Microcarriers — with
Mark Szczypka
Traditional stem cell culture involves
two-dimensional (2D) surfaces under
static conditions. Mark Szczypka
S eptember 2015
13(8)sp
BioProcess International 55
responds to questions about this
technology’s suitability for expanding cells
to larger scales.
Two-dimensional flatware systems
have traditionally been the initial
platform of choice for most
investigators. There is an intrinsic
familiarity with these systems because
they have been in use for many years,
and a significant body of cell
characterization data has been
collected in 2D systems.
The industry has been somewhat
reluctant to transition into threedimensional culture systems because
cells are exposed to conditions not
encountered in flatware. Cells are
exposed to shear stress in dynamic
cultures, and the configuration for
growth can be different from what is
observed on flat surfaces. For example,
aggregates can form when fibroblastlike, adult stem cells are grown on
microcarriers to high cell densities.
Fortunately, significant amounts of
data collected over the past decade
demonstrate that adult stem cells can
be grown successfully on
microcarriers. This is beneficial
because conventional 2D systems
suffer from significant limitations
when expanding large numbers of
cells.
Microcarriers provide the most
economical and practical solution for
generating multibillions to trillions of
cells. Even the most optimized 2D
systems — such as Cell Cube,
HYPERstack, and Cell Factory
systems as well as hollow-fiber devices
— require significant labor efforts
because of the large surface-area
requirement for generating sizable
numbers of cells. This requirement
necessitates a significant number of
units to generate the requisite cell
numbers. As unit numbers increase,
consumables and labor costs also
increase significantly. Additionally an
associated increase in the number of
manual interventions that are “open”
handling operations increases the risk
of contamination of the cultures.
Microcarriers used in standard or
single-use bioreactors provide an ideal
solution for these challenges. A single
200-L bioreactor containing
microcarriers can replace thousands of
cell factories and several hundred
hollow-fiber units. This advantage
affords the opportunity to
significantly decrease the number of
production runs required and
associated labor to produce large
numbers of cells.
Some problems reported with
microcarriers include difficulties with
adherence of cells to their surfaces
and with dissociating cells from them
later. How are these issues being
addressed? Cell attachment,
adherence, growth, and harvest are
influenced by multiple parameters
including microcarrier surface
composition, media formulation,
vessel configuration used for culture,
methods used for dissociation of cells
from microcarriers, and processes used
to separate cells from microcarriers.
Multiple avenues are currently being
pursued to address these perceived
challenges.
Microcarriers with unique surface
characteristics that promote rapid cell
attachment and tight adherence are
commercially available. New
microcarriers equipped with novel
surfaces are under development.
Additionally, media developers have
recognized the need for optimization
Figure 1: Estimated time periods for microcarrier process development and optimization
Cell
Characterization
Microcarrier
Screening and
Selection
Weeks 2–4
56 BioProcess International
4–6
13(8)sp
Small-Scale
Spinners
10–20
S eptember 2015
Bioreactor
Process
Development
Scale-Up
(20 to 200
Liters)
12–24
12–36
of medium formulations to support
robust cell attachment and growth of
stem cells in dynamic cultures. Simple
and robust methods for cell
dissociation from microcarriers are
also being pursued. Microcarrier
surfaces that facilitate cell detachment
without enzymatic harvest are being
developed, and standard
methodologies for cell harvest from
microcarriers are being optimized.
Some commercially-available
separation technologies for separating
detached cells from microcarriers are
available, and additional processes for
efficient separation of dissociated cells
from microcarriers are under
development by several groups —
including Pall Life Sciences. It is
worth noting that separation
technology at the thousand-liter scale
has been accomplished by multiple
groups both for stem cells and
standard cell lines.
How comparable are cell yields
using microcarrier-based systems with
yields achieved with other
technologies such as stacked trays?
Cell yields/numbers achieved in
microcarrier-based systems are
comparable to yields achieved through
other technologies. Under optimized
conditions it is possible to eclipse
yields achieved in other systems;
however, when optimizing these
conditions, it is imperative that stem
cell identity and functionality be
keenly scrutinized. The overall impact
of expanding stem cells needs to be
further evaluated, but a significant
amount of data have been generated
by many groups to indicate that cell
identity and function appear to be
unchanged when cells are propagated
under these conditions.
Can you explain the differences in
porosity among microcarriers that are
available on the market today? Some
suppliers and users advocate
macroporous options, but you prefer
rigid surfaces. Why? Microcarriers can
be manufactured to be nonporous,
nanoporous, microporous, and
macroporous. Various options are
commercially available. Nonporous and
nanoporous microcarriers are typically
rigid substrates that can undergo
surface modifications to promote cell
Sponsored Supplement
attachment, adherence, and growth.
Such microcarriers are ideal for cell
harvest because standard enzymatic
methods used to harvest cells from
flatware can be ineffective for cell
dissociation. Cells can be separated
using simple methods based on
differential settling, which depends on
the differences in relative density and
size of cells and microcarriers.
Standard screening techniques also
can be used because the rigid
properties prevent microcarriers from
obstructing or fouling screens.
Microporous microcarriers have
micron-sized pores that allow for
efficient cell attachment and growth.
But this property also can impede or
inhibit efficient, reproducible cell
harvest from microcarriers. These
properties are likely to be due to the
ability of cells to extend filapodia into
a microcarrier surface. Although this
phenomenon can promote efficient
cell adherence, it can also inhibit cell
dissociation from the microcarrier
surface. Many microporous
microcarriers are also elastic and
sticky. Microcarriers with these
inherent properties tend to readily
adhere to exposed surfaces and cause
sieving screens to foul, complicating
cell harvest.
Macroporous microcarriers have
pores large enough to allow cells to
grow within the interior of the
microcarrier. The potential benefit of
this configuration is that cells are
protected from shear stress
encountered in dynamic cultures.
However, because cells are propagated
within “niches” for long periods of
time, the maintenance of appropriate
cell identity and function can be
compromised. Harvesting cells from
microcarrier pores can also be quite
problematic. There are reportedly
methods for entirely digesting the
microcarriers and liberating cells, but
such processes are messy. By-products
generated from the digestion of
microcarriers need to be removed
through downstream processing so
that unwanted substances do not
accompany cells when they are
injected into patients. This can cause
significant problems with cell harvest
and final product purity.
Sponsored Supplement
Pall’s SoloHill microcarriers are all
fabricated of rigid polystyrene
materials — the same base material
from which traditional 2D
cultureware is constructed. This
facilitates adaptation and results in
easier cell dissociation, and thus
enables more reproducible and
efficient removal of cells from
microcarriers using standard
enzymatic harvesting coupled with
sieving methods.
Using microcarriers to expand cells
for cell therapy is a great mechanism
for providing larger surface areas for
growth of such cells. What do you see
as other advantages of using this
technology? What, if any, are the
downsides? A significant benefit in
using microcarriers to expand cells for
cell therapy is that the process is
amenable to closed systems and can be
performed cost effectively. Labor costs
and the number of manufacturing runs
needed per year to meet the therapeutic
need are significantly lower because lot
sizes are larger. Process validation and
equipment maintenance can be
streamlined, and flexibility is realized
by using single-use systems in GMP
manufacturing suites. Once a
microcarrier manufacturing platform is
validated, the process can be
implemented for other stem cells types
after some optimization.
The greatest potential risk
associated with using microcarriers in
large reactors is for failure of a
manufacturing run. When scaling
with microcarriers, the loss of a single
run due to operator error, equipment
failure, or facility problem can
represent significant loss of revenue.
Therefore, it is important to design
multiple redundancies within the
manufacturing setting and use
thorough risk assessment processes
before implementing this technology.
These measures will allow
manufacturers to anticipate and
prevent failures and carefully plan and
construct efficient facilities.
How amenable are microcarriers to
implementation in single-use systems?
How disposable, or reusable, are they,
themselves? Microcarriers are a
natural fit for single-use systems
because they can be used as single-use
It is important to
design multiple
redundancies within
the manufacturing
setting and use
thorough RISK
ASSESSMENT
processes before
implementing
microcarrier technology.
components in cell manufacture.
However, reasons not to reuse
microcarriers include regulatory
concerns, maintenance of GMP
manufacturing compliance, potential
impact on manufacturing
reproducibility, challenges of process
validation, and unknown microcarrier
stability after repetitive use.
Additionally, nonporous, rigid
microcarriers can be gamma-irradiated
without discernible loss of function.
This enables direct, closed-system
transfer of microcarriers into sterile
reactors for immediate use and makes
them ideal partners for single-use
systems.
One perception in the industry
today (right or wrong) is that using
microcarriers requires many, many
months of process development to get
a truly optimized process. What has
led to that perception? And do you see
the paradigm changing? Process
development to meet large-scale
manufacturing needs in any platform
is by nature a fairly lengthy process.
This is especially true for cell therapy
products. Difficulties with cell
harvest, handling of large numbers of
units, and time constraints required
for maintenance of cell health and
identity are challenges encountered in
all platforms. Processes that appear to
be straightforward can manifest
unforeseen challenges. Manufacturers
often encounter difficulties even when
they use seemly straightforward
processes such as 2D cultures.
Microcarrier development can be
streamlined like any other process
development activity. Small-scale
studies help expedite identification of
S eptember 2015
13(8)sp
BioProcess International 57
conditions that are optimal for cell
growth and harvest. Fortunately,
many process parameters are scalable.
Although optimization is needed as
scale is increased, unit operations that
are optimized at each scale can be
used in subsequent process
development activities. Using a
carefully planned, methodical strategy
for development of a microcarrierbased process is the best way to ensure
success. Paying close attention to
details used for cell propagation also is
important. Parameters used for cell
harvest before seeding dynamic
cultures should be optimized to ensure
reproducibility and efficiency.
Although it is good practice to
consider optimizing these parameters
in standard systems, investigators
rarely perform detailed
characterization of cell harvest in 2D
systems because the level of detail is
usually not required for successful cell
propagation in static cultures.
What are the major industry
limitations to implementing
microcarrier technology at large
scales? This is difficult to assess
accurately. From my perspective there
are few major limitations for adopting
the technology. Each of the perceived
or real logistical challenges associated
with large-scale microcarrier cultures
has been addressed by manufacturers
of vaccines and biologics. Additionally,
at least one group has scaled adult
stem cell cultures into 1,000-L stirred
vessels. Therefore, widespread
adoption of the technology for
expansion of stem cells should be
eminently possible. Development of
internal expertise may be perceived as
one possible impediment to
implementation of the technology, but
cell culture scientists, engineers, and
manufacturing associates can acquire
the skills needed to perform
microcarrier culture. As mentioned
earlier, acquiring the skills is a matter
of diligence and attention to detail. To
facilitate this, Pall has developed
SoloHill Microcarrier Training
Courses, designed to help researchers
get the most out of their SoloHill
microcarrier processes. Each course
can be customized to the specific
needs of an organization and includes
58 BioProcess International
13(8)sp
such areas of focus as adapting
flatware and roller bottle processes to
microcarriers, handling microcarriers
and optimizing attachment conditions,
small scale microcarrier processes in
spinner flasks, glass bioreactor
microcarrier processes from 2 L to
15 L, single-use microcarrier processes
from 2 L to 20 L, and strategies for
process optimization.
From a regulatory perspective, data
need to be generated that demonstrate
functionality of cells grown in
meaningful potency assays, and cell
identity tests need to be established for
cells generated in any platform.
Increasingly stringent regulatory
requirements for controlling
particulate load injected into patients
present another unique challenge for
cell therapy companies. This
requirement is the same for all
manufacturing platforms, so it is not
unique to microcarrier cultures.
Regardless, a solution for controlling
particulates that conforms to
regulatory requirements must be found
for all platforms.
Thierry Bovy
Cell Therapy Manufacturing: The
Xpansion System — with Thierry
Bovy
Thierry Bovy was previously responsible
for cell-therapy manufacturing at a
contract manufacturer. He comments here
on the importance of the role CMOs are
playing in this burgeoning industry and
on the role of automated platforms in
driving commercial cell therapy
manufacturing.
S eptember 2015
CMOs partner with cell therapy
companies along the development
pathway with the goal of reducing
time to market. They are dependable
and provide GMP accredited
infrastructure, qualified equipment,
and skilled personnel during a period
that is mutually agreed upon. They
can represent a more cost-effective
alternative to building or acquiring
brick and mortar as well as hiring and
paying personnel that would be less
occupied between manufacturing
campaigns.
From a financial perspective, this
transforms the CAPEX investments
in expenses, which is easier to defend
in front of investors — especially in
the early phases of a clinical trial.
With time, the CMOs accumulate
experience that future partners can
benefit from. They also provide
regulatory expertise that is required to
file dossiers to the regulatory agencies.
What major hurdles did you
experience in that work? Have those
earlier experiences helped you in your
work now at Pall? My department was
manufacturing autologous and
allogeneic fresh cell therapy products
for the European market. Each
process was different and had different
challenges, but every process had a
very short shelf life, usually only a few
hours. This meant that less than a half
day was allotted to filling the primary
container and returning it to a patient
for injection, including the release of
the product and shipment. The
process had to be robust and safe
enough in absence of sterility testing
to provide confidence to a pharmacist
authorizing human injection.
Also, shipping logistics was a major
hurdle that had to be cleared.
Manufacturing of the cells had to be
aligned to the airline schedules to
make sure that physicians would
receive the cellular doses during
regular business hours.
Scale-out or scale-up: What are the
major drivers when choosing which
path to take in industrializing cell
therapies? The batch size and demand
ultimately dictates the manufacturing
strategy used for a given therapy. The
increase in production for autologous
therapies corresponds to a scaling out,
Sponsored Supplement
whereas allogeneic treatments allow
for scaling up, provided that the cell
types used are amenable to it. In the
case of autologous treatments, the full
process costs are borne by the unique
treatment. The quality control tests
have the largest contribution in the
final product cost, with limited
possibilities to reduce their burden.
All the efforts to industrialize an
autologous process should focus on
automation of the process to reduce
labor costs.
The allogeneic model is the closest
to the classical pharmaceutical one. A
“universal” donor will provide tissue
from which stem cells will be isolated
and multiplied in vitro to such extent
that clinical doses of cellular material
will be made available off the shelf for
a large population. The optimization of
the cost will be linked to the balance
between batch size, shelf life, demand,
and technology. Indeed, market
demand will dictate the number of
cellular doses to generate, the shelf life
and size of the lots to manufacture to
avoid scrapping expired material, and
the technology costs achievable at the
various scales. Current planar
technologies could generate up to 5 ×
1011 cells per lot, and three dimensional
systems such as microcarriers would
push this limit to 1013 cells per lot (1).
The cost per million of cells produced
will therefore be a combination of all
these parameters.
What are the major platforms for
industrializing autologous and
allogeneic cell therapies? What about
the upside and downside of each? Scale
up for adherent cells is associated with
the extension of the culture surface or
of the volume of the vessels for cells
grown in suspension. Autologous
therapies typically require fewer cells
per dose and therefore involve classical
culture vessels of limited surface or
volume, such as T-flasks or small-scale
multitray stacks. Automation represents
an interesting avenue to explore to
support the intensification of these
therapies.
Most allogeneic processes still
involve the use of planar technologies,
mainly multitray stacks. The surface
per single unit quickly becomes a
limitation, and multiple containers
Sponsored Supplement
to traditional 2D platforms. What
benefits of the Xpansion system have
made it so attractive? What are its
scaling options? The Xpansion
Even the BEST
TECHNICIAN still
represents a major
source of
CONTAMINANTS
and variability in a GMP
environment.
have to be manipulated to increase the
culture surface for the cells. This scale
up is very labor intensive. It requires
extensive class A and B working and
incubation spaces because some of the
process steps are considered open and
performed under laminar flow hoods.
Automation possibilities are limited
with this type of culture vessel.
Hollow-fiber devices can help cope
with some perceived limitations of
open systems, but they are not scalable
either and require numerous
automated systems to generate
multibillions of cells.
More and more cell therapy
companies consider microcarriers to be
economically sound alternatives to
traditional flatware systems when
trillions of cells have to be generated
per lot.
There are of course challenges
associated with scale up and
downstream processing, but this
technology is fast becoming one of the
readily available, favorable technologies
that can overcome the obstacles
associated with planar technologies.
Can you offer some thoughts
regarding automation’s role in cell
therapy bioprocessing? Even the best
technician still represents a major
source of potential contaminants and
variability in a GMP environment. A
robot would mitigate these risks, will
work 24/7, and will therefore
dramatically reduce labor costs.
Automation also represents the best
avenue to explore to reduce costs in an
autologous process.
When growing adherent cells (e.g.,
stem cells), a surface must be provided
on which they can attach and grow.
Xpansion technology seems to be
gaining acceptance as an alternative
multiplate bioreactor is part of Pall
Life Science’s single-use bioreactor
family, designed for shear-sensitive
adherent cell applications such as
cultivation of stem cells. It was
developed for the safe, large-scale
production of traditional 2D cell
cultures, providing the same
microenvironment to the cells as in
traditional multitray stacks.
The Xpansion bioreactor’s
multiplate structure comprises a large
cell growth surface area (up to
122,400 cm 2). The 80-cm high, 200plate bioreactor provides the same
growth surface as a 4-m high set of 10
traditional stacks. The compact design
enables elimination of the gas phase
between the plates. This gas phase is
replaced by an automatically controlled
aeration system that provides advanced
gas diffusion. Control is automatic
through disposable pH and dissolved
oxygen (DO) sensors. Temperature
monitoring and agitation control are
also included. Xpansion bioreactors
offer the capability of monitoring cell
morphology and density through use
of an optional digital holographic
microscope.
Scalability is another key feature.
The Xpansion multiplate bioreactors
are available in four sizes, providing
an increasing surface for cell growth
from 6,120 cm² to more than
122,000 cm² per single-use unit,
without modification to the footprint
of the bioreactor. The Xpansion
10-plate bioreactor, the smallest
version, is normally used for technical
evaluation whereas the 50-, 100-, and
200-plate versions are generally used
for manufacturing. Once the
parameters such as pH and dissolved
oxygen are optimized using the
smallest size, the scale-up from 10 to
200 plates is fairly linear and
straightforward. c
S. Anne Montgomery is cofounder and
editor in chief of BioProcess International,
amontgomery@bioprocessintl.com.
S eptember 2015
13(8)sp
BioProcess International 59
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