Cell Therapy
Cellular raw material
collection in cell therapy:
critical determinant of product quality
For more than two decades, the biotechnology and pharmaceutical industries
have been working to unlock the great potential of cell therapy, which uses
products composed of living, functional cells to mediate the therapeutic effect.
These novel biologics have potential applications in disorders estimated to
affect nearly 130 million patients in the United States alone (Figure 1),
including cardiovascular disorders, cancer, Alzheimer’s disease, Parkinson’s
disease and autoimmune diseases1.
A
wide number of disorders may ultimately
be treatable using products derived from a
broad range of cells and tissues, as shown
in Table 1. Stem cells from blood and bone marrow, liver, muscle, brain and fat are being tested in
structural, metabolic, genetic, neurologic,
orthopaedic and cardiovascular disorders, while
immunotherapies involving dendritic cells, natural
killer cells and lymphocytes are in clinical trials for
a variety of malignancies, autoimmune diseases,
and viral infectious diseases. Islet cells, hepatocytes, myoblasts, chondrocytes and dermal cells
also are being explored as treatments for diabetes,
renal and hepatic failure, orthopaedic disorders
and severe burns2.
The pace of cell therapy product development
has accelerated sharply in recent years. Since 2001,
more than 2,700 cell therapy clinical trials have
been initiated, with approximately 2,000 currently
in progress3. To date, 51 cell therapy products
worldwide have received marketing authorisations4. While the promise of cell therapy has generated much excitement among scientists and the biopharmaceutical industry, the complexity and variability of living cells presents a number of challenges as it relates to controlling the quality of cellbased therapies.
Drug Discovery World Summer 2014
Commercial success driven by quality
Clinical and commercial success of cell-based therapies relies on a deep understanding of the product’s Critical Quality Attributes (CQAs). CQAs of
a product are those characteristics that should be
within specified limits to ensure the product has
the desired efficacy and safety (ie, quality)5,6.
CQAs include elements of safety, identity, purity
and potency characterisation testing. CQAs are
influenced by manufacturing process parameters
(Critical Process Parameters, CPPs) and raw material characteristics (Critical Raw Material
Attributes, CRMs). The manufacturing process
and critical raw materials, as represented by CPPs
and CRMs, respectively, must be sufficiently controlled and consistent to result in a product with
CQAs within the specified limits.
By Dr Scott R.
Burger, Louis Juliano,
and Dr Wenshi Wang
Understanding the biological realities
Cells and tissue are the critical raw material from
which cell therapy products are manufactured.
Both the cell therapy product and the cellular raw
material are composed of living, functional cells,
and therefore are to some extent heterogeneous
and cannot be fully defined. Given these biological
realities, it is essential that the manufacturing
process be rigorously controlled and consistent.
29
Cell Therapy
scale. Treatment histories often differ between
patients, and may impact CRMs, further complicating comparisons of cellular raw material.
Selected types of cellular raw material
– example applications and CRMS
Figure 1
Numbers of patients, in
millions, potentially treatable
by stem cell-based therapies.
Adapted from 1
Unlike the heterogeneity and variability resulting
from the living functional nature of cells and tissues, process-related variability can be greatly
reduced by identification, control and monitoring
of CPPs. Controlled, consistent processes yield
controlled, consistent products.
Cell therapy products are manufactured from
autologous or allogeneic cells or tissue. Autologous
cell therapy products are manufactured from the
patient’s own cells or tissue, while allogeneic cell
therapy products are derived from a donor other
than the patient. Where allogeneic cell therapy is
not personalised, the manufacturing process differs
in that it often involves extensive cell expansion in
culture with master cell banks being generated.
Autologous cell therapy product manufacturing
generally must contend with a higher degree of raw
material variability, as raw material is obtained
from each patient, and used to manufacture product for the same patient, at a one lot per patient
Unmobilised apheresis product (PBMCs)
One of the most common cell collection methods
for cell therapy is apheresis, a centrifugation-based
technology that separates and collects white blood
cells ex vivo, returning the remaining blood components to the donor or patient. Apheresis products collected from untreated (unmobilised) individuals are an excellent source of peripheral blood
mononuclear cells (PBMCs), a category principally
composed of lymphocytes and monocytes. Cell
therapy applications of PBMCs include several
types of cellular immunotherapy products,
described below, and listed in Table 1.
Lymphocytes in PBMCs are used to generate regulatory T-cells (T-regs), chimeric antigen receptor
T-cell therapies (CAR T-cells, CARTs), cytotoxic Tlymphocyte therapy, tumour-infiltrating lymphocytes (TILs) and natural killer cell therapies,
among others. Contaminants when manufacturing
these cell therapy products include granulocytes
(white blood cells that are not mononuclear cells),
monocytes, red blood cells, and in some cases
platelets. CRMs, accordingly, include numbers and
percentages of mononuclear cells, a measure of
mononuclear cell purity and indicator of granulocyte contamination, numbers and percentages of
lymphocytes (desired) and monocytes (contaminants), and hematocrit, a measure of red blood cell
contamination.
The monocyte component of PBMCs is used in
cell therapy to generate dendritic cell-based therapeutic vaccines. For these applications, lymphocytes are contaminating cells, as are granulocytes
and red blood cells, while mononuclear cells and
specifically monocytes are desired populations. In
order for cell-based therapy to reach its full
Figure 2
Unmobilised apheresis product
mononuclear cell purity (%
MNC) and red blood cell
contamination (% hematocrit)
for HemaCare apheresis
collections. Adapted from 7
30
Drug Discovery World Summer 2014
Cell Therapy
potential, apheresis collection must be fully controlled in order to minimise operational sources
of variability, and greatly increases the likelihood
of commercial success in manufacturing. In the
US, there are several blood products and services
companies providing human-derived primary
blood cells and tissues for advanced biomedical
research, clinical trials and for use in patient specific therapeutic settings.
A recent study published in the journal
Cytotherapy, titled ‘Human-derived raw materials:
controlled, consistent collection and cryopreservation enable successful manufacturing of cell-based
products’, details the impact of well controlled
training and procedures and the impact access to
an extensive registry of well-characterised repeat
donors can have on obtaining consistently high
quality starting material.
In this study, apheresis collections were completed using qualified processes on validated instruments, with ongoing process control and monitoring in place. The value of applying this process
control can be seen in the quality and consistency
of apheresis products collected. Unmobilised
apheresis products showed consistently high MNC
purity, with 93.8% of products containing ≥75%
MNC, and an average of 85.66% MNC ± 7.1%
(mean ± 1 SD). Red blood cell contamination was
consistently low, with hematocrit averaging 1.78%
± 0.7%7 (Figure 2).
Access to an extensive registry of healthy and
disease state donors with the ability to request
repeat donor collections is essential to delivering
high-quality collections for the most complex cellbased therapy applications. Approximately 85%
of donors included in the study had donated
apheresis products five or more times, contributing
to product consistency, as MNC content of individual donor apheresis products had an average
co-efficient of variation of 3.5%, compared to a
CV of 7.7% for all apheresis products7. These data
demonstrate that with well controlled training and
procedures, consistently high quality starting material can be obtained.
Mobilised apheresis products
Hematopoietic stem and progenitor cells can also be
collected by apheresis, if the donor or patient is
treated in advance with a cytokine, commonly GCSF. As with unmobilised apheresis collections,
important CRMs include measures of content and
purity of mononuclear cells, and of potentially contaminating granulocytes and red blood cells. In
addition, CRMs include number and percentage of
CD34+ cells, representing stem and progenitor cells.
Drug Discovery World Summer 2014
Table 1: Clinical applications of cell therapies. Adapted from
DISEASE STATES
2
CELL THERAPIES
Cancer
Hematopoietic Stem Cell Transplantation
Autologous and allogeneic HSC
Ex vivo expansion of HSC
‘Suicide’ T cells – gene transfer
Mesenchymal stem cell transplantation
Immunotherapy
Chimeric antigen receptor T-cells
Dendritic cells
NK cells
Orthopaedic
Expanded chondrocytes
Mesenchymal stem cells
Neurodegenerative disorders/trauma
Adult stem cell-derived neural cells
Embryonic stem cell-derived neural cells
Cardiovascular disease
Mesenchymal stem cells
Organ replacement
Pancreas (diabetes)
Pancreatic islet cells
Adult stem cell-derived islet cells
Liver (failure, metabolic disorders)
Bioartificial liver
Isolated hepatocytes
Hepatocyte stem cells
Kidney (failure)
Bioartificial kidney
Wound healing
Keratinocytes
Skin stem cells
Infectious diseases
Antigen-loaded dendritic cells
Lymphocyte expansion
Macrophages
Genetic deficiencies
HaemophiliaGene therapy
SCID
Gene therapy
Autoimmune diseases
Immunotherapy
Dendritic cells
Lymphocyte expansion
HSC, hematopoietic stem cells; NK, Natural Killer; SCID, severe combined
immunodeficiency
Bone marrow
Bone marrow is a critical raw material for several types of cell therapies, including mesenchymal
stem cell therapies for a variety of applications,
and hematopoietic stem cells for transplantation. Typical CRMs are similar to those for
mobilised apheresis products but with the addition of megakaryocyte frequency, as a means of
distinguishing actual bone marrow from circulating blood. Example applications and CRMs
31
Cell Therapy
Table 2: Example applications and CRMs for different cell and tissue raw materials
RAW MATERIAL
Unmobilised apheresis product
EXAMPLE APPLICATIONS
Chimeric antigen receptor (CAR)
T-cell immunotherapy
SELECTED EXAMPLE
CRMS
MNC content and
% Lymphocyte content and %
Potential contaminants:
Monocyte content and %
Granulocytes (captured by MNC %)
Hematocrit
Microbial contamination (sterility
culture)
Unmobilised apheresis product
Dendritic cell vaccine
MNC content and %
Monocyte content and %
Potential contaminants:
Lymphocyte content and %
Granulocytes (captured by MNC %)
Hematocrit
Microbial contamination (sterility
culture)
Mobilised apheresis product,
umbilical cord blood
Mesenchymal stem cell therapies
Hematopoietic stem cell
transplantation
MNC content and %
CD34+ cell content and %
Potential contaminants:
Granulocytes (captured by MNC %)
Hematocrit
Microbial contamination (sterility
culture)
Bone marrow
Mesenchymal stem cell therapies
Hematopoietic stem cell
transplantation
MNC content and %
CD34+ cell content and %
Megakaryocyte frequency
Potential contaminants:
Granulocytes (captured by MNC %)
Hematocrit
Microbial contamination (sterility
culture)
for several cell and tissue raw materials are
shown in Table 2.
Importance of cell and tissue raw
material, risks associated with
excessive variability
Cells and tissue are critical raw material for manufacturing cell therapy products. This living biological raw material can be a source of significant
variability, however. Biological variability is
inevitable due to the nature of the products and
raw materials, but other aspects of the process can
be rigorously controlled to limit, whenever possible, sources of variability. A robust process that is
well controlled, properly developed and validated
32
will be far more capable of producing acceptable
products, and will protect the patient and the clinical product.
Cellular raw materials (CRMs) are major determinants of both cell therapy product’s CQAs and
of clinical outcomes. If the apheresis product, for
example, does not contain sufficient PBMCs, lymphocytes, monocytes or CD34+ cells, or is excessively contaminated with granulocytes, modified
manufacturing pathways may be necessary, or the
process may fail outright. This in itself is not surprising – this is part of the definition of CRMs
and CQAs. Cell therapy manufacturing process
failure, however, can have substantially different
consequences in compared to pharmaceutical
Drug Discovery World Summer 2014
Cell Therapy
manufacturing. For autologous cell therapy products in particular, a failed manufacturing process
means failure to treat the patient.
Therefore, it is critical for autologous cell therapy collections to be well controlled and highly consistent time and time again. Controlled collection
procedures yield optimal consistent products.
Understanding CRMs, CPPs, CQAs and other key
quality indicators, and being able to consistently
achieve high standards of performance are
absolutely critical in an apheresis peripheral blood
collection. CPPs, CRMs and CQAs for optimal cell
collections should include:
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l Donor testing, screening and infectious disease
testing, in compliance with Good Tissue Practices
(GTPs).
l Controlled, qualified or validated procedures
and suppliers, trained and qualified operators.
l Product QC testing, including safety, purity,
identity and potency parameters, such as sterility
testing, cell counts and 5-part WBC differential.
l Tracking and trending: donor reactions, deviations, exceptions, etc.
l Internal and external audits.
Summary
Human cells and tissue are critical raw material for
cell therapy manufacturing. Quality of this cellular
raw material is a major determinant of final product characteristics – Critical Quality Attributes.
Rigorous operational controls and quality systems,
however, enable optimal collection of high-quality,
consistent cellular material. Implementing and consistently employing rigorously controlled,
GMP/GTP-compliant collection procedures minimises operational sources of variability, resulting
in highly consistent, high-quality cells for use in cell
therapy product development and manufacturing.
Dr Carolyn Compton, who previously held the
position of Director of the Office of
Biorepositories and Biospecimen Research at the
National Cancer Institute, has been very vocal in
recent years about the quality of biospecimens in
translational research, using the well-known computer industry phrase ‘Garbage In, Garbage out’.
Never has this been more applicable than in cell
therapy, in which cells derived these biospecimens
are themselves the drug substance8.
As the field of cell therapy matures, more and
more emphasis will be placed on the necessity to
deliver high quality biospecimens at the start of
the pipeline, ensuring that the final drug product
is a success and ultimately results in improved
patient health.
DDW
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Cell Therapy
References
1 Perry, D. Patients’ voices: the
powerful sound in the stem
cell debate. Science
2000;287:1423.
2 Prince, HM, Wall, DP, Stokes,
KH, Wood, R, Burger, SR,
Coghlan, P, Boyce, N. Cell
processing for clinical trials
and commercial manufacture.
Cell & Gene Therapy
2004;1:15-21.
3 Culme-Seymour, EJ, Davie,
NL, Brindley, DA, EdwardsParton, S, Mason, C. A decade
of cell therapy clinical trials
(2000-2010). Regenerative
Medicine 2012;7(4):455-462.
4 IOM (Institute of Medicine)
and NAS (National Academy
of Sciences). 2014. Stem Cell
Therapies: Opportunities for
Ensuring the Quality and
Safety of Clinical Offerings.
Washington, DC: The National
Academies Press.
5 ICH Q8(R2) Guideline –
Pharmaceutical Development
(http://www.ich.org/fileadmin/P
ublic_Web_Site/ICH_Products/
Guidelines/Quality/Q8_R1/Ste
p4/Q8_R2_Guideline.pdf).
6 ICH Q8/9/10 –
Implementation
(http://www.ich.org/fileadmin/P
ublic_Web_Site/ICH_Products/
Guidelines/Quality/Q8_9_10_
QAs/PtC/Quality_IWG_PtCR2
_6dec2011.pdf).
7 Human-derived raw
materials: controlled,
consistent collection and
cryopreservation enable
successful manufacturing of
cell-based products
Cytotherapy 2014;16(4):S103.
8 Compton, C. Getting to
personalised cancer medicine.
Cancer 2007;110:1641-1643.
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Dr Scott R. Burger is the Principal of Advanced
Cell & Gene Therapy and Scientific Advisory
Board Chair for HemaCare (Los Angeles, Ca.) An
expert in cell therapy, Dr Burger works with
clients in industry and academic centres worldwide, providing assistance in process development
and validation, GMP/GTP manufacturing, GMP
facility design and operation, regulatory affairs,
technology evaluation, and strate gic analysis. He
received his MD from the University of
Pennsylvania School of Medicine and completed
postgraduate training in Laboratory Medicine, as
well as a clinical fellowship in Transfusion
Medicine and a postdoctoral research fellowship
at Washington University in St Louis. Dr Burger
served as Medical Director of the Cell Therapy
Clinical Laboratory and Molecular and Cellular
Therapeutics Facility at University of Minnesota,
where he was responsible for process development, validation and GMP production of a broad
range of novel cell and gene therapies, in support
of more than 75 clinical trials. Dr Burger also was
Vice-President of Research and Development at
Merix Bioscience, a biotechnology company
focused on dendritic cell immunotherapy. He
serves on the USP Cell, Gene and Tissue Therapies
Expert Committee, the advisory boards of several
cell therapy biotechnology companies and has
served as Editor of the International Society for
Cellular Therapy Telegraft, and on the ISCT
Executive Committee. A frequently invited speaker at industry and academic conferences, he is the
author of more than 100 scientific publications
and presentations and the recipient of numerous
honors and awards.
University in 1996. He is a licensed pharmacist and
nuclear pharmacist in New York and Florida.
Dr Wenshi Wang is Senior Scientist at HemaCare.
Prior to this role, Dr Wang served as a Research
Scientist for the Melanoma Comprehensive
Researcher Center at H. Lee Moffit Cancer Center
at the University of South Florida where she was
the co-investigator for a clinical trial to validate
gene signatures that predict clinical response and
development of immune-related adverse events
associated with ipilimumab treatment for
advanced melanoma. In addition, she was part of
a study on the pharmacodynamics and predictive
biomarkers on T cells from stage III and IV
melanoma patients receiving anti PD-1 antibody
treatment. Prior to that, Dr Wang was a Research
Associate for the Division of Medical Oncology at
the Keck School of Medicine at the University of
Southern California and a Research Fellow for the
Division of Immunology at the City of Hope
National Medical Center and Beckman Research
Institute. Dr Wang is a seasoned scientist with
more than 20 years of research experience. Dr
Wang received her Masters from Tianjin Medical
College and her PhD in Microbiology and
Immunology from Tongji Medical University.
Lou Juliano is Senior Vice-President, Research
Products and Cell Therapy for HemaCare. From
2009-11 Mr Juliano served as a General Manager
for Walgreens Infusion and Respiratory Services in
New York. Prior to that he worked for Cardinal
Health for more than 26 years. In his last role
there, Mr Juliano was a Senior Vice-President leading Operational Excellence/Lean Six Sigma teams
within the Healthcare Supply Chain Services division. Before that, he held a succession of positions
with increasing operations and sales leadership
responsibilities including Area Vice-President,
Director of Operations, General Manager,
Regional Manager, and Pharmacy Manager. Mr
Juliano received his Bachelor of Science degree in
Pharmacy from Purdue University in 1984 and
completed a Cardinal Health-sponsored executive
MBA programme affiliated with Pepperdine
Drug Discovery World Summer 2014