Cell
t h e r a p i e s technology
Cell Therapy Bioprocessing
Integrating Process and Product Development
for the Next Generation of Biotherapeutics
by Ralph Brandenberger, Scott Burger, Andrew Campbell,
Tim Fong, Erika Lapinskas, and Jon A. Rowley
T
he past 15 years have seen
approval and commercialization
of the first cell-based
therapeutics, including cartilage
repair products; tissue-engineered
skin; and the first personalized,
cellular immunotherapy for cancer.
Those successes are outnumbered,
however, by all too common product
failures. Notable failures can be
attributed to commercial concerns such
as high cost of goods (CoGs) and
technical hurdles such as inadequate
characterization, high process
variability, and loss of product efficacy
when manufacturing is scaled up (1).
Arguably, the root cause of those
commercial and clinical failures is a
lack of sophistication in developing
living cell-based “drugs” with all the
verifiable consistency of any other
drug class. Many early cell therapy
companies lacked drug development
expertise and pursued products that
were not feasible commercialization
candidates. Some companies, unable
to continue with iterative development
and further characterization, instead
relied on insufficient understanding of
their products and ineffective process
development, which impaired critical
product characteristics and likely set
the stage for clinical failure (1).
For cell therapy products to achieve
the manufacturing success of
biopharmaceuticals, there must be
communication and cooperation
among three groups of people:
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• cell processing professionals
typically working in academic centers
that collectively process hundreds of
cell products for transplantation every
month
• cell biologists who have a strong
foundation in product development
and cell characterization (as it relates
to therapeutic efficacy of cell-based
drugs) and can help identify and
quantify quality parameters and
product specifications
• bioprocess engineers with the
engineering discipline required to
create scalable and robust
manufacturing processes (2) that will
maintain the critical quality
parameters of living cell products
while minimizing manufacturing costs
inherently expensive products.
Only with a cross-functional
approach that encompasses those
skill-sets will it be possible to achieve
an integrated approach to product and
process development that can lead to
clinical and commercial success.
The Process and Product
Development (PPD) subcommittee of
the International Society of Cell
Therapy (ISCT) is a group of industry
and academic cell therapy
professionals aiming to help establish
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and communicate best practices for
integrating the process and product
development aspects of cell-based
therapeutic development. Its work will
include establishing and sharing best
practices in establishing specifications
for identity, potency, purity, and safety
of cell-based therapeutics as well as
development of commercially viable
bioprocesses to manufacture these
products to specifications. These
efforts will include driving
standardization when possible, sharing
best practices within the industry, and
helping position early stage, academicbased therapies for potential
commercialization.
Cell therapy manufacturing is
poised to benefit from know-how and
technical innovation that the protein
bioprocessing field has driven over the
past 20 years (3). Production, storage,
and delivery of living cell-based
pharmaceuticals presents several
unique challenges. Novel, innovative
technologies and strategies will be
required to bring cell therapies to
commercial success.
Major Differences Between
Therapeutic Cells and Proteins
In just a couple decades, the
commercialization of monoclonal
antibodies and other therapeutic
proteins led to the growth of a
multibillion-dollar industry. A great
deal of effort has been applied to the
establishment of methods for efficient
product scale-up and cost reduction of
manufacturing processes (4). In
addition, a tightly controlled
regulatory policy that includes
sterility, purity, and quality of raw
materials (animal-origin–free, and so
on) guidelines has been adopted for
manufacturing biotherapeutic
molecules. The cell therapy industry
has generally been slow to adopt such
practices because of specific
(sometimes prohibitive) differences
between the two product categories.
Recombinant monoclonal
antibodies and other therapeutic
proteins are generally secreted by
genetically engineered cell lines into
their culture medium and then can be
highly purified, sterilized, and
concentrated under well-defined and
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regulated conditions. By contrast, the
product of a cell therapy is the cell
itself. That presents a challenge: The
end product must be harvested
efficiently and cannot be terminally
sterilized. Additionally, many cell
types currently are not amenable to
cell culture systems that exclude
animal serum or other animal-origin
materials. Further development of
serum-free culture systems will be
necessary to reach the level of
regulatory compliance that is required
in the therapeutic protein market.
A major difference between the
protein and cell industries is the
source of their cells. Many
recombinant proteins are produced
from selected clones of mammalian
cells that are fully characterized,
tested, consistent, and stable. Most
cell therapies are derived from primary
cells isolated directly from tissues that
have limited expansion potential. Such
cells cannot be selected for optimal
performance characteristics, and
donor-to-donor variability leads to
substantial variability among
processes. Scalable production
platforms, process validations, and
quality control release thus remain as
daunting challenges.
Processing Laboratories
in Academia
Most clinical cell therapy laboratories
at academic medical centers have
grown far beyond their origins in
processing bone marrow to support
blood and marrow transplant
programs. They have become
powerful resources in development of
novel cell-based products. Academic
cell processing laboratories commonly
serve a range of clinical programs and
are closely linked to their academic
investigators. Their staffs have
developed a valuable breadth of
knowledge as well as cutting-edge
processing techniques and
characterization methods (Photo 1).
The most active laboratories produce
hundreds (in some cases, thousands)
of cell therapy products each year. To
date, it is estimated that tens of
thousands more “doses” of living cells
have been processed in academic
laboratories than have been
manufactured in all of industry, which
represents a remarkable level of
experience.
Focusing on phases 1 and 2, such
laboratories represent an early stage in
the development pipeline. Technology
transfer, characterization, and process
development activities in an academic
processing laboratory thus have great
impact on later stages of clinical
development and commercialization.
It is neither practical nor necessary for
an academic cell therapy laboratory to
develop commercial-scale
manufacturing or testing. It is,
however, essential that early stage
development work in such laboratories
enable rather than impede subsequent
commercialization.
To give cell therapy products the
greatest chance of success, academic
cell therapy laboratory staffs must
understand the needs of their industry
counterparts further along the
development pathway. It is always
useful to begin with the end in mind,
which can be thought of as
establishing a target product profile
while in early clinical development.
Early stage process development
should focus on defining and
optimizing a reproducible
manufacturing process and
establishing a characterization profile
that can support product release and
subsequent development. Raw
materials should be selected and
qualified to simplify sourcing and
regulatory compliance at later stages of
development and commercialization.
In the next decade, commercialization
of cellular therapies will force most
manufacturing from academic
laboratories to industry. Leveraging
the knowledge of those centers will be
important to minimizing wheel
reinvention.
Comparing Autologous and
Allogeneic Product Manufacture
There are two main manufacturing
process paradigms in cellular therapy:
patient-specific/autologous and
universal-donor/allogeneic products
(Figure 1). Historically, most cell
therapy products have been patientspecific due to a need for immunologic
compatibility. Many cell therapy
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Figure 1: Allogeneic processes generally include strategically placed cell banks and large lots of
patient doses. In autologous/patient-specific processing, every lot manufactured is meant to treat
only a single patient, and in some cases much of the product is lost to product release testing.
Autologous = Patient Specific
Allogeneic = Universal Donor
Cell Expansion
or Purification
Master Cell Bank
Lot Tested
Cell Expansion
Working Cell Banks
Lot Tested
Patient Doses
Lot Tested
Testing
Patient or Donor
Cell Ampule or Dose
Submitted for Testing
Figure 2: Challenges to scale up/out for a typical cell therapy manufacturing process
MCB
WCB
Tissue
Acquisition
Primary Cell
Isolation
Cell Culture
Harvest
Volume
Reduction
Washing
Formulate
and Fill
Cryopreservation
Storage and
Inventory
Testing and
Release
Shipping
Logistics
End User
(Handling,
Delivery)
products in clinical development
today, however, are based on cell types
that do not give rise to an immune
response. Such allogeneic unmatched
cell therapies could be more
pharmaceutical-like “off-the-shelf ”
products.
Unmatched allogeneic donor
products are amenable to bulk
manufacturing, and their production
often can take advantage of some
aspects of bioprocess technology.
Manufacturing commonly involves
establishing and qualifying
cryopreserved master and working cell
banks, then producing large lots of
product for release testing. As in
biopharmaceutical manufacturing, the
process is scaled up during clinical
development, and release testing may
32 BioProcess International
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be performed on lots that represent
hundreds or thousands of product
doses. The main challenges in
allogeneic manufacturing are
maintaining a cell product’s critical
quality parameters while scaling up its
manufacture (stressing cell
characterization, as discussed below),
and controlling the cost of goods
(CoGs) for these inherently expensive
products. Although labor and facilities
are the greatest component of CoGs
for allogeneic cell therapy products
during clinical development, it is
expected that culture medium and
supplements will be the largest part
during commercial-scale
manufacturing.
Production of autologous, patientspecific cell therapy products are not
amenable to a scale-up approach, so
their bioprocesses will be scaled out
for commercial manufacturing. To
achieve efficiencies of scale, highthroughput production by parallel
processing of multiple, separate
products in automated, functionally
closed systems should be widely
adopted (5, 6). Functionally closed
process technology provides product
and process isolation, maintaining
each product entirely within its own
separate, presterilized, disposable
processing set. Such systems are
highly amenable to automation and
associated with extremely low rates of
contamination, and they typically
improve product yield.
Because each product represents a
unique donor, autologous and patientspecific products have the greatest
potential for variability, which is a
major challenge. Logistics present a
challenge as well in that
manufacturing each product involves
obtaining living cellular raw material.
Because patient-specific products are
manufactured as one lot per patient,
release testing often accounts for the
bulk of their CoGs. Despite all these
challenges and costs, multiple patientspecific, autologous cell therapy
products are on the market or in
clinical development today. They
generally target applications of major
unmet medical need.
Cell Characterization
and Potency Analysis
Cell-based products present unique
characterization challenges. Producers
of small-molecule and recombinant
biologic drugs use defined
manufacturing and purification
processes that yield relatively
homogeneous final products with little
lot-to-lot variation. Characterization of
purity and identity for such drugs can
be directly related to several measurable
physical traits such as molecular
weight, chemical structure, and purity.
Drug potency is often correlated with
and measured by specific biochemical
or cell-based assays in vitro. However,
most methods used to characterize the
purity and potency of traditional drugs
are not suitable for testing cell-based
products.
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One major challenge to the cell
therapy industry is to develop
characterization and potency
specifications for products with
considerable inherent variability —
particularly for autologous, patientspecific products. Cell-based products
are often derived from donor or
patient tissues that contain many cell
types. Depending on the stringency of
methods used in cell isolation, final
products can contain several other cell
types in addition to the therapeutic
cells of interest. Donor- or patientbased variations in tissue composition
add to variability in the final cell
product purity and yield. Even
products derived from cell lines (e.g.,
hES or iPS cells) may contain several
cell types. Such products require some
level of differentiation and expansion,
and no current protocol is 100%
efficient, so manufacturing produces a
mix of undifferentiated or partially
differentiated cells in the final cell
products.
The most commonly used
technology for characterizing cell
therapeutics is fluorescence-activated
flow cytometry. Multicolor flow
analysis allows phenotypic
determination of antigens on the
surface of a cell. If an appropriate
reagent cocktail is used, then both the
identity and purity of a final cell
product can be determined using a
single assay. Gene expression array
analysis is increasingly being used, as
well. More established analytical
technologies such as automated cell
counting and enzyme-linked
immunosorbent assays (ELISAs) play
major roles too. Assay validation,
including developing robust reference
standards, will have to be addressed as
cell therapy products come to market.
Like with traditional drugs,
potency assays must be established for
each cell-based product before phase 3
clinical trials. However, the
mechanism of action for many such
drugs is either multifaceted or
currently unknown. Developing
potency assays for cell products is
fraught with ambiguity. FDA
guidance suggests including a matrix
of assays including gene expression
profiles, cytokine secretion, or in vitro
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cell-based assays showing biological
activity such as target cell lysis,
inhibition of target cell proliferation,
or pluripotent differentiation potential
(7). With the large variety of potential
cell products under development, the
challenge will be to develop assays
that are reliable, reproducible with low
variance, inexpensive, simple, and
rapid, with appropriate reference
standards — that also have relevance
to the intended clinical activity.
Common Upstream
Processing Platforms
With a wide variety of potential tissue
sources (e.g., blood, bone marrow,
cord blood, placenta, adipose and
other adult tissues, fetal tissue,
embryonic stem cell lines, induced
pluripotent cells), an important step in
the cell therapy manufacturing process
is initial isolation or enrichment of a
cell population of interest from the
tissue source. Several commercial
systems are already in use for bulk
enrichment and have attributes that
facilitate compliance with current
good manufacturing practice (CGMP)
guidelines. Examples include the
CliniMACs system from Miltenyi
Biotec (www.miltenyibiotec.com), the
Sepax system from BioSafe SA (www.
biosafe.ch), and the Elutra system
from CaridianBCT Inc. (www.
caridianbct.com). For processes that
require highly defined cell
subpopulations, fluorescence-activated
cell sorting (FACS) may have
applications, but it presents some
complications and limitations (8).
Unfortunately, no single cell-sorting
instrument platform yet offers a
complete, CGMP-compliant system
for cell therapy use. However, leaders
in the field are pushing forward and
adapting current platforms for clinical
cell sorting. A challenge for cell
product manufacturers will be to find
an isolation process that is costefficient, easy to use, and CGMP
compliant — and that can provide a
cell population of the required yield
and purity.
Culture processes for expansion
and/or differentiation of final cell
products (either allogeneic or
autologous) are based on current cell
culture technology and are ripe for
innovations specific to cell therapy
application. In general, cells for
therapeutic applications are either
grown in nonadherent suspension or
adherently through traditional twodimensional (2D) culture. Many cells,
such as hematopoietic stem cells
(HSCs) and T cells, can be grown in
nonadherent suspension culture. That
can involve relatively large-scale
bioreactors such as bag-based and
traditional stirred-tank vessels.
Through such means, large numbers
of cells can be generated with a
relatively small footprint and under
tightly controlled conditions.
Other cell types, such as
mesenchymal stromal cells (MSCs) (9)
from various tissues, are traditionally
grown adherently on tissue-culture–
treated surfaces. Large-scale
expansion of these cell types requires
much surface area using traditional
2D methods. Multilayered flasks have
been developed to reduce the
laboratory footprint per squarecentimeter of culture surface.
Examples include the CellCube and
CellSTACK systems from Corning
Inc. (www.corning.com) and Cell
Factory system from Nunc A/S (www.
nuncbrand.com). But to reach
sufficient cell numbers for commercial
lot sizes (~1010 –1012 cells), large clean
rooms (or even whole buildings) would
have to be filled with incubators of
flasks. Next-generation adherent
culture platforms are under
development specifically for cell
therapy applications, including
bioreactors, hyper-density stacked
vessels, microcarrier-based cultures,
and induced-suspension adaptation
growth methods for primary adherent
cell types. As the amount of tissue
culture surface area per lot increases,
the enzymatic harvest and
downstream processing of hundreds to
thousands of liters of cells will be a
new and challenging process
bottleneck.
Common Downstream
Processing Platforms
Current downstream processing
methods for cells use common
laboratory equipment for
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concentrating, washing (clarifying),
and formulating them before
packaging and storage. Transition to
closed, scalable systems will be
important to scale-up/out that will be
required for commercial production
(10). Many culture processes are still at
relatively small scale (1- to 10-L
harvest), but some midstage allogeneic
processes can reach 20–30 L in scale.
Cell concentration and washing are
currently performed using open
centrifugation tubes (≤500 mL) or
performed using blood processing
equipment.
Recently, bioprocessing
technologies such as tangential-flow
filtration and continuous or
counterflow centrifugation have been
adapted for maintaining very high cell
viabilities and important biological
functions of living products. Cell
therapy is benefiting greatly by the
move of bioprocess suppliers toward
ready-to-use, sterile, single-use
systems, but focused development
effort specifically for cell therapy
applications is greatly needed. The
transition to scalable technologies for
processing cells will be central to cell
therapy success. As processes scale up
to 100-L and even 1,000-L harvests,
open centrifugation and blood
processing equipment will be unable
to accommodate such volumes.
Fill and finish processing of
therapeutic cell formulations is also in
need of standardization. Patientspecific processes typically involve
small volumes that are manipulated in
biological safety cabinets or through
closed bags, tubing sets, and sterile
welders. Automation of such processes
(5, 6) — including automated logistics
and tracking — will ultimately be
required because single facilities will
be processing, releasing, and shipping
hundreds of lots per day. Most
allogeneic and autologous products are
still being filled in blood bags, which
are appropriate for small lot sizes.
However, as allogeneics increase to
hundreds or thousands of doses per
lot, a switch to pharmaceutical vials
and off-the-shelf filling lines is
anticipated (10, 11). Large-scale
cryopreservation and cold-chain
management and logistics will be an
36 BioProcess International
M arch 2011
engineering challenge as the industry
moves forward.
Unique Challenges
for hESC Processing
Therapeutic application of human
embryonic stem cells (hESCs) offers
promising opportunities for addressing
diseases using unprecedented stategies.
This type of allogeneic process comes
with unique challenges, however.
These cells can proliferate indefinitely
in culture and differentiate into
lineage-restricted cells of all three
primary germ types (ectoderm,
mesoderm, and endoderm). That
ability of hESCs to proliferate
indefinitely allows for large,
characterized, CGMP-compliant cell
banks of undifferentiated hESCs to be
manufactured and tested. Cells from
those banks are thawed and expanded
to create large quantities of
undifferentiated hESCs, from which
differentiation cultures are initiated.
Differentiation culture processes
are designed to send hESCs down
specific differentiation pathways
through chemical signals from their
culture microenvironment (e.g.,
soluble signals in their media or
insoluble signaling from an adhesion
matrix) until a therapeutic cell
population of interest is produced.
During differentiation, the cells
typically pass through several
developmental stages and require
many media changes with different
formulations. Because of these
complex processes, differentiation
protocols are only now being
developed that are sufficiently robust
and reproducible to support
therapeutics manufacture.
Currently hESCs are cultured in
2D adherent systems on animalderived surfaces. Unlike many
adherent cultures, they are grown as
colonies of tightly clustered cells
(Photo 2). Cell–cell adhesion is
important in chromosome stability,
and cells are in many cases passaged as
colonies using mechanical methods to
remove them from surfaces. The
passaging technique limits expansion
in multilayered vessels.
Technology development in the
emerging field of hESC bioprocessing
has focused on addressing technical
and raw-material challenges. For
example, several defined surfaces
including peptides and purified
recombinant proteins have been
identified recently that support
undifferentiated expansion and
differentiation of hESCs (12–15),
which marks an important step toward
animal-origin–free cultures.
Nonmechanical methods for
harvesting such cultures from flasks,
while maintaining the hESCs in
colony form, will make multilayer
vessel expansion a possibility. Proofof-concept work has used suspension
culture on either microcarriers or as
cell aggregates in suspension, which
would enable scalable bioreactor
production (2). Establishing
comparability of such scaled processes
will be critical, which emphasizes the
importance of analytical methods for
cell characterization and potency.
Because hESC production remains at
relatively small scales, little work has
been performed on scalable
downstream processing systems for
their postculture manipulation. Cell
separation technologies for reducing
cellular impurities in final products
may need to be developed and scaled,
as well.
The Future of
Cell Therapy Bioprocessing
Two decades ago, biotherapeutic yields
were beginning to reach 100 mg/L,
and today’s 10 g/L yields would have
been almost unimaginable. Cell
therapy bioprocessing is still in its
early stages, and innovation focused
Supplement
Therapy Position Statement. Cytother. 8(4)
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10 Rowley JA. Developing Cell Therapy
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(SBE Stem Cell Engineering Supplement)
November 2010: 50–55.
11 Woods EJ, et al. Container System for
Enabling Commercial Production of
Cryopreserved Cell Therapy Products. Regen.
Med. 5(4) 2010: 659–667.
12 Melkoumian Z, et al. Synthetic
Peptide-Acrylate Surfaces for Long-Term SelfRenewal and Cardiomyocyte Differentiation of
Human Embryonic Stem Cells. Nat. Biotechnol.
28(6) 2010: 606–610.
13 Rodin S, et al. Long-Term SelfRenewal of Human Pluripotent Stem Cells on
Human Recombinant Laminin-511. Nat.
Biotechnol. 28(6) 2010: 611–615.
14 Villa-Diaz LG, et al. Synthetic Polymer
Coatings for Long-Term Growth of Human
Embryonic Stem Cells. Nat. Biotechnol. 28(6)
2010: 581–583.
15 Klim JR, et al. A Defined
Glycosaminoglycan-Binding Substratum for
Human Pluripotent Stem Cells. Nat. Meth.
7(12) 2010: 989–994. •
References
Ralph Brandenberger is director of
process sciences at Geron Corporation;
Scott Burger is principal at Advanced Cell
and Gene Therapy, LLC; Andrew Campbell
is a senior staff scientist for Life
Technologies; Tim Fong is technical
director for cell therapy at BD Biosciences;
Erika Lapinskas is in business
development at Sartorius-Stedim; and
corresponding author Jon A. Rowley is
director of cell therapy research and process
development services at Lonza Walkersville,
Inc., 8830 Biggs Ford Road, Walkersville, MD
21793-0127; 1-301-898-7025 x2620; jon.
[email protected]; www.lonza.com.
All are members of the Process and Product
Development subcommittee of ISCT.
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Therapy Products: Models, Methods, and
Process Development. Cell Therapy
Manufacturing: Stem Cell and Immunotherapies,
London, UK, December 2010. Informa Life
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7 CBER. Draft Guidance for Industry:
Potency Tests for Cellular and Gene Therapy
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downloads/BiologicsBloodVaccines/
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BioProcess Int. 8(6) 2010: 44–53.
9 Dominici M, et al. Minimal Criteria for
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To order reprints of this article,
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154. Download a low-resolution PDF online
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on addressing specific challenges will
bring similar advances. To succeed,
commercial success of at least a few
late-stage products currently in
development will be needed to fund
development of next-generation tools
and technologies for this field.
The Process and Product
Development subcommittee of ISCT
is working within the precompetitive
space of this field; driving home the
importance of integrating product and
process development; and stressing the
importance of cell characterization,
process reproducibility, and
standardization. Our end goal is a
sustainable industry based on robust,
scalable processes that are developed
to maintain the critical quality
parameters and important biological
functions of this new class of livingcell products to address many diseases
that currently are untreatable, all at
reasonable manufacturing costs (3).