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Automation of CAR-T Cell Adoptive

Immunotherapy Bioprocessing:
Technology Opportunities to
Debottleneck Manufacturing
by David A. Brindley, James A. Smith, Brock Reeve, Kim Bure, Zeeshaan Arshad, Franziska Faulstich, David
Pettitt, Georg Holländer, Andrew Ball, Kiara Biagioni, Sunil Mehta, Rajan Choudhary and Hussein Al-Mossawi
Monday, April 18, 2016 12:58 pm View PDF

Continued clinical efficacy

demonstrations of cell-based immunotherapies (iTx) such as chimeric
antigen receptor T cell (CAR-T) therapies has made the prospect
increasingly likely of an immunotherapy product achieving
conditional market authorization in the short term. For example,
Novartis and the University of Pennsylvania’s lead candidate
(CTL019) for treating a range of hematological malignancies received
breakthrough status from the US Food and Drug Administration
(FDA) in 2014, permitting access to an expedited drug development
pathway for high unmet medical needs (1). Then in March 2015, the
European Medicines Agency’s (EMA’s) Pediatric Committee agreed
on a pediatric investigation plan for tisagenlecleucel-T (2).

The “oncoimmunotherapy
wave” includes emerging Figure 1: Methods for producing
genetically modified T-cells for
check-pointinhibitor immunotherapy include (A) T-cell
technology (3) as well as T- receptor obtained from a patient with the
disease; (B) T-cell receptor obtained by
cell receptor (TCR) and immunizing transgenic mice expressing
CAR-T cell technologies human major histocompatibility
complexes (MHCs) with target cancer
(Figure 1). And although it cells; and (C) a mechanism for producing
chimeric antigen receptors.* Following T-
has highlighted the tangible cell engineering, resulting cells are
expanded ex vivo with interleukin 2 (IL-2)
potential for an before administration.
immunotherapy to reach
approval, the attention garnered by these approaches also has
underscored the need for significant improvements in bioprocessing
technologies and capacities to meet potential and expected market
requirements. Critically, commercial-scale production methods — as
opposed to those suited to point-of-care and/or blood-processing
units — are critical to ensuring commercial sustainability of iTx (4, 5).

Because of the autologous nature of most iTx, however, their

manufacturing processes remain highly manual (6), requiring
extensive human-operator interaction and decision-making
throughout production. Labor costs, inter- and intraprocess
consistency, and operator error(s) consequently are major challenges
in the effective translation of such therapies from laboratory to
commercial scale. For these novel products to succeed, technological
innovations will need to enable more seamless transitions between
process steps and allow for automation. To achieve those goals,
developers will need a strong understanding of the rational path to
automation (7) as well as available technology options.

Adoptive iTx Clinical Trial

Landscape Figure 2: Reported adverse events across
29 studies included in analysis; references
No comprehensive, cited from the CAR-T and TCR box
quantitative assessment of
CAR-T/TCR efficacy or safety has been conducted yet. Here, we
provisionally attempt such an assessment by investigating adverse
events, target indications, and outcome rates across systematically
identified published clinical trials. We

began with a provisional

systematic literature review, Figure 3: Clinical outcomes of
patients after TCR or CAR-T cell
identifying 29 studies that would therapy; successful outcomes are
be suitable for inclusion in this complete remission, partial
remission, or negative for minimal
analysis (see the “CAR-T and residual disease (MRD); “disease
TCR” box). Preliminary analysis present” means stable disease or
MRD positive; unsuccessful
indicated that to date more trials outcomes include progressive
disease, end of remission, and
are using CAR-T cell approaches death. Outcomes for all papers
than genetically engineered TCRs (cited from the CAR-T and TCR box)
with reported clinical outcomes are
(Figures 3 and 4). In these trials, categorized by therapy type (bars
grades 3 and 4 adverse events on left axis) and cancer type (bars
on right axis).
appear to be more common with
CAR-T cell therapies than with TCRs (Figure 2), although success
rates do appear to be greater for the former (Figure 3).

Further research is required for a statistical analysis to be conducted.

But it is evident already that demonstrated clinical efficacy is likely to
be sufficient to drive significant demand in biomanufacturing
capacity that cannot be achieved without a quality by design (QbD)
and automated approach. Current biomanufacturing strategies have
limited amenability to commercial-scale biomanufacturing scale-
up/scale-out. As such, those processes represent a major barrier to
industry development that could be alleviated by innovation in
bioprocess automation and downstream processing technologies as
well as in cell population purification and enrichment.

Toward Systematic
Automation of an Figure 4: Stages in systematic automation
of an autologous cellular immunotherapy
Autologous Cellular iTx biomanufacturing process
Biomanufacturing Process
To successfully automate a complex biomanufacturing process, a
stepped approach provides the most effective solution (Figure 4). In
the first phase, developers break down their manufacturing process
into separate, sequential steps that each encompass a single function
called a unit operation. Next, the development team assesses
appropriate equipment for those unit operations and connections
between them determine whether linkages are affordable relative to
their benefits or demand unjustified expenditures (8). Often, certain
breakpoints in a process would be exorbitantly costly to bridge
through automation — e.g., morphological assessments and vessel
movement between different platforms — yet are easily
accommodated through minor operator interactions, which allow for
critical-thinking decision points that can be assessed only by human
operators (9). Finally, the timing for each process step and its
associated equipment costs must be considered. It should not
adversely affect equipment use rates, causing integrated
components to sit idle when a staged series approach could be more
efficient (10). Taking into account unit operations, components,
connections, and timelines will ensure that a company ends up with
the most robust and affordable iTx manufacturing system with the
greatest equipment use rates.

A robust system builds on the basic tenets of automation: removing

the human element from bioprocessing and enhancing consistency
for both product and process. Clearly, limiting human-operator
interactions with a product stream assures higher sterility and
reduced opportunities for contamination while also lessening the
potential for errors and expanding production hours beyond
attended operation. But a common misconception in the industry is
that automation confers cost-of-goods (CoG) savings through
expedited processing; cost savings usually come from reduced labor
needs and the benefits of controlled and reproducible handling (11).
Consequently, improved quality and consistency of products made
through automation lead to fewer out-of-specification (OoS) results
and product losses (12). Closed systems and processes also reduce
the risk of contamination.

Taking into account those factors as applied to current iTx processes,

it becomes evident that many challenges lie ahead in automating
them for commercialization. Methods that originally supported
clinician-initiated trials tend to rely primarily if not exclusively on
manual techniques and human assessments for cell characteristics
requiring further study (13). Additionally, such processes merge
equipment from multiple tool providers, making their output
communications and connections vital to continuity. And finally, the
small process volumes of personalized therapies force highly parallel
operations that will be compelled to be cost efficient because of
reimbursement constraints (14).

The advantages of implementing automation for the regenerative

medicine market are evident and uniquely significant in helping to
ensure patient safety. Beyond the improved sterility and consistency
of iTx products, online process analytical technology (PAT) allows for
monitoring and response to irregularities that arise based on patient
differences in a tremendously intricate process. Also, automation
supports quality management systems (QMS) in complying with good
manufacturing, laboratory, and clinical practices (GMPs, GLPs, and
GCPs) and good automated manufacturing practice (GAMP)
requirements by mitigating errors and oversights that could foster
infractions (15). Furthermore, recent technological advancements
are bringing the industry closer to achieving its goal of a more
integrated and automated iTx process. Innovations such as liquid-
phase detachable selection, activation, and purification technologies
are poised to make step-change differences in throughput.

Opportunities to Optimize Immunotherapy Manufacturing

Cell Purification and Enrichment: Purification and enrichment of
specific cell subpopulations from heterogeneous starting populations
is a critical component of multiple life-science workflows spanning
basic cell biology research through clinical-grade cell therapy
manufacturing. In all such workflows, a key objective is to obtain a
highly purified and viable population of cells for downstream
applications, whether in a laboratory or a clinic. For therapeutic
applications such as CAR-T and engineered TCR cancer
immunotherapies, key objectives include manufacturing efficiency,
which requires target-cell purification and processing that are both
cost and time efficient.

Cell purification methods currently in widespread use for clinical iTx

applications can be broadly classified into two main categories:

physical separation based on properties such as size and density,

typically using techniques such as density and counterflow
centrifugation
surface-marker–based separations based on using capture ligands
and techniques such as magnetic cell separation (MACS) or
fluorescence-activated cell sorting (FACS).

Each approach is well established but faces limitations, particularly in

terms of unit-operation throughput and equipment use. So
alternative solutions are being sought.

Physical separation techniques such as centrifugation are commonly

used for prepurification and cell concentration but have low
specificity because they separate cells based on sedimentation
properties, not by characteristic features or expression of specific
markers (16, 17). Centrifugation cannot separate cells with similar
sedimentation properties.

Cell purification based on surface markers is dominated by MACS

approaches because of their greater scalability in format compared
with FACS technology (18). However, currently MACS leaves large
numbers of paramagnetic particles bound to separated cells, which is
highly undesirable for cell-based therapeutic applications (19).
Minimizing the number of attached or internalized beads represents
a formidable obstacle here because most products based on
magnetic beads lack the ability to readily release bound cells from
capture molecules without altering their viability and phenotype.
Additionally, the resulting isolated cells require a lengthy culture
process to reduce magnetic-bead concentrations to meet clinical
release criteria for patients receiving cell-based therapeutic
infusions.

Hydrogel Technology: A potential solution to those challenges has

come to market recently. A biocompatible phase-change hydrogel
can be easily functionalized with cell-capture agents such as
antibodies, and it can be dissolved rapidly in a buffer containing a
chelating agent (20, 21). (Editor’s Note: See Maribel Rios’s interview
with Sean Kevlahan of Quad Technologies in this issue titled
“Development of a Novel CellSeparation Platform” for more
information.)

As described in the “Fundamental Properties” box, this hydrogel

enables liquid-phase cell separation and might solve multiple
challenges in cell separation and cell processing workflows. The
hydrogel is versatile and can be packed into columns or coated on
different substrates such as paramagnetic beads, polymethyl
methacrylate (PMMA) acrylic, polydimethylsiloxane (PDMS) silicone,
polystyrene, and others, without adverse effect on its differentiate
properties.

Fundamental Properties of Phase-Change Hydrogel

Highly
biologically
compatible and
noncytotoxic

Can be
functionalized
with any
biological
capture moiety

Readily
dissolvable
without damage
to captured cells
Advantages
Enables high-
No chemical (e.g., staining) and/or physical (e.g.,
efficiency
attachment of antibody, lyses of cell membrane)
purification and
cell treatment requiredCells remain unchanged
release of cells
before and after separation, so collected cells can
from complex
be used for further experimental or separation
starting
techniquesSpeed of separation is rapid
populations
Minimum shear stress(es), high recovery viability
Improved purity
and throughput
and viability of
cells compared
with alternative Disadvantages
separation Cannot separate different types of cells with
technologies similar sedimentation properties
Purified cells Limited to cells that can be individually suspended
free of in a buffer solution
paramagnetic
particles

Separation
protocol highly
amenable to
automation

Can also be used

to deliver
stimulatory/
signaling
molecules to a
targeted cell
subpopulation

Liquid-phase cell chromatography is proving to be highly efficient for

the purification and enrichment of specific cell subpopulations such
as T lymphocytes from heterogeneous starting populations (22).
Successful proof-of-concept experiments have established the
potential for functionalized, phase-change, hydrogel-based cell
selection and purification using microfluidic devices with
paramagnetic particles as carriers for the hydrogel. The phase-
change, hydrogel-based approach to cell purification may provide
improved cell enrichment and contribute to an overall simplified
experimental protocol for cell processing in development of cell
therapies. Relative to standard methods, this releasable, liquid-phase
technology could solve many inherent challenges of cell separation
by safely and rapidly eliminating magnetic particles and achieving
high cell recoveries and viabilities through a fast, scalable, and easily
executed separation process. Ultimately, it could form a key part of a
continuous-flow integrated solution for cell bioprocessing.

Counterflow centrifugation
may not be a novel Table 1: Comparing separation
technologies highlights potential benefits
technology itself, but of releasable magnetic particle
commercial equipment that technology; MACS = magnetic cell
separation
could process small-scale
samples such as autologous immunotherapies would be
groundbreaking (Table 1). It would allow for a single system to handle
multiple unit operations unit operation while maintaining the most
beneficial conditions for stable cell viability. Moreover, the same
equipment could be additionally purposed for daily media exchange,
debeading, purification, washing, concentration, and infusion into
final delivery media. As mentioned above, those steps might
otherwise require separation into various suites within a CGMP
production facility. However, simplification to one principal piece of
equipment would enable operator familiarity and further strengthen
process robustness.

Toward Commercialization
CAR-T cell therapies are extraordinarily efficacious and are
progressing rapidly through clinical trials, supported by significant
investment and adaptive regulatory pathways in both the United
States and the European Union. And although the iTx revolution has
been a clinician-led “cottage industry” to date, such an environment
cannot continue if the field is to realize its commercial promise and
reach the maximum number of patients for the greatest possible
benefit to society.

Life-science tools and technology industries have understandably

sought to respond to the needs of fundamental scientists in
academia, who typically work toward integrating all bioprocess steps
into self-contained, small-scale manufacturing units. They have
predominantly adopted scale-out rather than scale-up purification
technologies. Ultimately, however, few of these technologies will be
commercially feasible either from a capital expenditure, equipment
use, or CoG perspective. In a commercial bioprocess, it is simply
infeasible to have a large number of low-throughput and low-use
upstream and/or downstream process technologies within a facility.
The footprint and calibration burdens are both prohibitive. Online
bioprocess control systems including PATs are critical to minimizing
process variability and meeting ever-more-stringent regulatory
expectations pertaining to current GxPs.

The time is now for applying proven commercial-scale bioprocess

equipment and expertise. Building on the work of many clinicians and
scientists who have made revolutionary therapies such as CAR-T,
TCR, and other iTx possible, the global bioprocessing community
must now step forth and respond through robust innovation and
quality to enable their availability to all patients in need.

Acknowledgments
We express sincere thanks to the following organizations that have
contributed to the CASMI Translational Stem Cell Consortium
(CTSCC) as funding and events partners, without whom the
consortium and the benefits it will bring to stem cell translation
would be constrained: GE Healthcare, the Center for
Commercialization of Regenerative Medicine (CCRM), Sartorius
Stedim Biotech (formerly TAP Biosystems), Lonza, the California
Institute for Regenerative Medicine (CIRM), the Strategies for
Engineered Negligible Senescence (SENS) Research Foundation, UK
Cell Therapy Catapult, NIH Centre for Regenerative Medicine, the
New York Stem Cell Foundation (NYSCF), ThermoFisher Scientific,
Eisai, Medipost (US), Medipost (Korea), Celgene, Roche and Oxford
BioMedica (UK) Ltd. David Brindley gratefully acknowledges
personal funding from the Oxford Musculoskeletal National Institute
for Health Research (NIHR), the Saïd Foundation, and the SENS
Research Foundation. David Pettitt and James Smith gratefully
acknowledge support from the CASMI Translational Stem Cell
Consortium (CTSCC). And Smith also gratefully acknowledges
support from the UK Medical Research Council and Professor
Andrew Carr at the Nuffield Department of Orthopaedics,
Rheumatology, and Musculoskeletal Sciences in the University of
Oxford’s medical services division.

Conflicts of Interest
This text represents the authors’ individual opinions and may not
necessarily represent the viewpoints of their employers. Brindley is a
stockholder in Translation Ventures Ltd. (Charlbury, UK) and IP Asset
Ventures Ltd. (Oxford, UK), companies that among other services
provide cell therapy biomanufacturing, regulatory, and financial
advice to pharmaceutical clients. Smith is a consultant with IP Asset
Ventures Ltd. Brindley also is subject to the CFA Institute’s codes,
standards, and guidelines, so he must stress that this piece is
provided for academic interest only and must not be construed in any
way as an investment recommendation. Finally, at time of
publication, he and the organizations with which he is affiliated may
or may not have agreed to and/or have pending funding
commitments from organizations named herein.

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Further Reading
Cruz CRY, et al. Infusion of Donor-Derived CD19-Redirected Virus-
Specific T Cells for B-Cell Malignancies Relapsed After Allogeneic
Stem Cell Transplant: A Phase 1 Study. Blood 122, 2013: 2965–2973.

Davila ML, et al. Efficacy and Toxicity Management of 19-28z CAR T

Cell Therapy in B Cell Acute Lymphoblastic Leukemia. Sci. Transl.
Med. 6, 2014: 224ra25.

Grupp SA, et al. Chimeric Antigen Receptor-Modified T Cells for

Acute Lymphoid Leukemia. N. Engl. J. Med. 368, 2013: 1509–1518.

Hollyman D, et al. Manufacturing Validation of Biologically

Functional T Cells Targeted to CD19 Antigen for Autologous
Adoptive Cell Therapy. J. Immunother. 32, 2009: 169–180.

Jensen MC, et al. Antitransgene Rejection Responses Contribute to

Attenuated Persistence of Adoptively Transferred CD20/CD19-
Specific Chimeric Antigen Receptor Redirected T Cells in Humans. J.
Am. Soc. Blood Marrow Transplant. 16, 2010: 1245–1256.

Jensen MC, et al. Human T Lymphocyte Genetic Modification with

Naked DNA. Mol. Ther. J. Am. Soc. Gene Ther. 1, 2000: 49–55.

Johnson LA, et al. Gene Therapy with Human and Mouse T-Cell
Receptors Mediates Cancer Regression and Targets Normal Tissues
Expressing Cognate Antigen. Blood 114, 209: 535–546.
Kalos M, et al. T Cells with Chimeric Antigen Receptors Have Potent
Antitumor Effects and Can Establish Memory in Patients with
Advanced Leukemia. Sci. Transl. Med. 3, 2011: 95ra73.

Kalos M, June CH. Adoptive T Cell Transfer for Cancer

Immunotherapy in the Era of Synthetic Biology. Immunity 39, 2013:
49–60.

Kochenderfer JN, et al. Eradication of B-Lineage Cells and Regression

of Lymphoma in a Patient Treated with Autologous T Cells
Genetically Engineered to Recognize CD19. Blood 116, 2010: 4099–
4102.

Laport GG, et al. Adoptive Transfer of Costimulated T Cells Induces

Lymphocytosis in Patients with Relapsed/Refractory Non-Hodgkin
Lymphoma Following CD34+Selected Hematopoietic Cell
Transplantation. Blood 102, 2003: 2004–2013.

Lee DW, et al. T Cells Expressing CD19 Chimeric Antigen Receptors

for Acute Lymphoblastic Leukaemia in Children and Young Adults: A
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Kim Bure is director of regenerative medicine, and Franziska

Faulstich is regenerative-medicine product manager at Sartorius
Stedim (Göttingen, Germany). Andrew Ball is vice president of
research and development, and Kiara Biagioni is a marketing product
manager at Quad Technologies (Woburn, MA). Sunil Mehta is
president and chief executive officer of kSep Systems (Morrisville,
NC). Rajan Choudhary is an undergraduate researcher at the
Oxford–UCL Centre for the Advancement of Sustainable Medical
Innovation (CASMI). Zeeshaan Arshad is an undergraduate student
at CASMI, at the University of St. Andrews School of Medicine (St.
Andrews, UK). David Pettitt is a doctoral researcher at CASMI and in
the medical sciences division’s department of pediatrics at the
University of Oxford, UK. Georg Holländer is an action research
professor in the medical sciences division’s department of pediatrics
at the University of Oxford and the Weatherall Institute of Molecular
Medicine at John Radcliffe Hospital (Oxford, UK). Hussein Al-
Mossawi is a lecturer in rheumatology and musculoskeletal sciences
at the University of Oxford’s Nuffield Department of Orthopedics
(Oxford, UK). Brock Reeve is executive director of the Harvard Stem
Cell Institute (Cambridge, MA). Corresponding author James Smith
is a CASMI research associate and professor in the University of
Oxford’s Nuffield Department of Orthopedics, Rheumatology and
Musculoskeletal Sciences; worcjamessmith@gmail.com. And David
Brindley is founder and academic director of CASMI’s Translational
Stem Cell Consortium, senior healthcare translation research fellow
in the medical sciences division’s department of pediatrics at the
University of Oxford, Cooksey-Saïd fellow in healthcare translation
at the University of Oxford’s Saïd Business School, honorary senior
research associate at the University College London School of
Pharmacy’s Centre for Behavioral Medicine (London, UK), a research
fellow in cell therapy commercialization at the Harvard Stem Cell
Institute, and regenerative medicine regulation and risk management
lead at the USCF-Stanford Center of Excellence in Regulatory
Science and Innovation (CERSI) in Stanford, CA.
 Categories: 2016, April 2016 Supplement, Cell Therapies, Emerging Therapeutics,
Personalized Medicine, Pre-Clinical and Clinical Trials, Regulatory Affairs

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