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Regulatory Advice on CBER ProducTs is an informal nonbinding meeting intended to pro-
vide guidance early in a product’s development (FDA, 2024c). The INTERACT meeting was
created especially for products with novel aspects and/or no precedent, and does not discuss
IND-enabling nonclinical studies.
Therapeutic applications
Because PSCs can be differentiated into any number of cell types, there is hope that they
can be utilized to treat a wide spectrum of diseases. Diseases that are characterized by a loss
or malfunction of specific cells and tissues may be particularly appropriate for cell therapy.
Some of the more prevalent therapeutic targets amenable to PSC-based therapeutics include
central nervous system (CNS) disorders such as Parkinson’s disease, Huntington’s disease,
traumatic brain injury (TBI), stroke, spinal cord injury, amyotrophic lateral sclerosis (ALS),
age-related macular degeneration (AMD), and epilepsy. Other targets include muscular dys-
trophies, heart disease, endocrine disorders such as diabetes, and skin ailments such as burns,
defective wound healing, and epidermolysis bullosa. PSC-derived cell therapeutics are now
being harnessed to address cancer, blood disorders, and infectious diseases (Zhou et al., 2022;
Rao et al., 2022). While adult stem cells have been clinically tested in some of these diseases,
they have limitations as previously discussed (Zhou et al., 2008; Wagner et al., 2008; Cham-
bers et al., 2007; Bonab et al., 2006; Baxter et al., 2004). The advantage of using PSCs lies in
their higher proliferation capacity, making it easier to obtain a homogeneous source of cells
in unlimited quantities, which can potentially be patient-matched in the case of iPSCs. PSCs
may be differentiated into lineage-specific progenitors or terminally differentiated cells,
depending on the disease target and desired effect.
Early phase clinical studies for PSC-based products are underway and, although limited to
a small number of patients, preliminary safety data have demonstrated no serious adverse
events. Similar to allogeneic (single source) MSCs, a key challenge in using allogeneic
PSC-based therapies is the requirement for immunosuppre ssion to prevent allo-rejection
(analogous to an organ transplant). In contrast, iPSCs offer the potential for production of
an autologous cell line for each intended patient, obviating the need for chronic immunosup-
pression. Yet, a recent analysis reveals that most clinical studies to date use iPSCs as alloge-
neic cell sources, likely due to manufacturing challenges and cost, and only a few are taking
advantage of their value for autologous therapy (Kobold et al., 2023). Additional research is
needed to address potential safety concerns unique to the clinical application of iPSC-based
products, including the potential for mutations due to genomic insertion, the risk of tumor
formation with the transduction of oncogenes such as c-Myc or mutated p53, as well as
the potential for incomplete genetic reprogramming (Hong et al., 2013). Of note, while for
some indications of unclear etiology it may not be desirable to develop an autologous cellular
therapeutic from somatic cells carrying the same disease-state genetic background, for mono-
genetic disorders, the use of autologous iPSC-derived products may be particularly desirable
as the manufacturing strategy of the candidate therapeutic may include “correction” of the
gene defect or its consequences.
Other applications: In vitro toxicology testing and disease modeling
PSCs also represent valuable tools for drug testing, early safety evalu ation, disease
modeling, and investigation into the underlying biology of cells. Differentiated PSCs can
12. Considerations in the development of pluripotent stem cellebased therapies406
III. Specialty biologics and indications
be used to assess and de-risk cardiac (Burnett et al., 2021) and hepatic toxicity (Lu et al., 2015),
two major causes of pharmaceutical-associated side effects, leading to attrition in the drug
development pipeline. In addition, embryoid bodies formed by PSCs represent a novel plat-
form for investigating stage-specific embryotoxicity (Wobus and Loser, 2011). If such assess-
ments could be made reliably in vitro using PSCs, this approach could save precious
development resources. iPSCs are also being evaluated as tools for modeling diseases as
they allow for the creation of “diseased” stem cell lines (Marchetto et al., 2010b; Rivetti di
Val Cervo et al., 2021) as well as three-dimensional multicellular structures such organoids,
assembloids and tissue or organ-on-chip models (Goldrick et al., 2023; Kanton and Pasca,
2022; Novelli et al., 2022) by sourcing the cellular starting material from diseased individuals
(e.g., SMA (Ebert et al., 2009), Parkinson’s Disease (Park et al., 2008; Soldner et al., 2009), ALS
(Dimos et al., 2008), Rett syndrome (Marchetto et al., 2010a), Huntington’s Disease (Jeon et al.,
2012; An et al., 2012) and other neurodevelopmental disorders (Miura et al., 2020)). While
additional work is required before PSC-based assays are routinely used to support drug
development, this area represents one of the fastest growing sectors of research using PSCs.
Important early decisions: Chemistry, manufacturing, and controls
Due to the complexity of PSC-based therapies, early-stage development requires a care-
fully designed strategy and integrated view of all stages of development as the product,
design of preclinical studies, and clinical plan are inextricably linked. Ideally, preclinical
studies should reflect the proposed clinical study as closely as possible. Selection of the
most appropriate cell type and stage of maturation (precursor cell, progenitor cell, partially
or terminally differentiated cell) to employ is largely dependent upon a better understanding
of the mechanism of action. The ultimate goal of the therapy (i.e., restoration of function, cell
replacement, regeneration, repair, and/or support) as well as the clinically proposed route of
administration and dose level, including the use of any device or encapsulation technology,
will affect both product development (i.e., CMC) and preclinical studies (proof-of-concept
and safety/toxicology).
Early decisions in this process can greatly impact both time to clinic/market and develop-
ment costs. For example, ideally the same cell line would be used for pivotal preclinical and
clinical studies. Should the cell line be changed at some point because some aspect of later
product development had not been considered early on, key studies may need to be repeated,
adding significant delays and costs. Similarly, significant changes made to the manufacturing
process or critical reagents can greatly affect the development path and should be minimized.
Manufacturing process
The key objective for the manufacture of PSC-derived products is to develop a robust pro-
tocol for the differentiation of cells with desired characteristics that lead to clinical effective-
ness and safety, while minimizing undiffere ntiated stem cells and other undesired cell types,
in a consistent, quality-controlled, and reproducible manner. To achieve this objective, a
comprehensive strategy is required with careful attention paid to early decisions such as
Important early decisions: Chemistry, manufacturing, and controls 407
III. Specialty biologics and indications
the choice of starting material, thoughtful and informed selection of reagents used in
manufacturing, and development of appropriate in-process assays, controls, and release tests.
Depending upon the intended application, PSC-based products may consist of a mixture of
cell types, with varying capacities for proliferation or differentiation. See Fig. 12.1 for typical
steps in the manufacturing of PSC-based products. Differentiation of PSCs may be strongly
influenced by processing methods, conditions, and duration of culture. In addition, variation
in adhesion substrates and media composition, in particular the use of signaling molecules,
growth factors, serum or other additives, can significantly alter the composition of the result-
ing cell population. Due to this plasticity, it is important that the manufacturing process is
well-defined and tightly controlled (EMA, 2011). We refer readers to, in addition to applicable
FDA guidance (FDA, 2024a ), A-CELL, a valuable resource describing CMC considerations for
the manufacturing of CAR-T therapies that applies to the development of other cell-based
therapies as well (https://alliancerm.org/manufacturing/a-cell-2022/).
Manufacturing processes should be developed with an emphasis on avoiding the use of
animal products where possible, limiting residual levels of in-pr ocess reagents, and mini-
mizing the formation of degradation products. In-process and final process testing should
be performed to assess the presence of residuals or degradation products and determine
any potential impact on patient safety. When feasible, manufacturing processes should be
divided into discrete, appropriately controlled manufacturing units as this can improve the
quality of intermediates and greatly de-risk production run failure.
The recent development of genome editing has widened the opportunity for generation of
truly customized PSC-derived products, and these are mostly derived from autologous or
allogeneic iPSC lines. The list of DNA editing tools is expanding and includes four main types
of nucleases: meganucleases, zinc-finger nucleases (ZFNs), transcription-activator-like
effector nucleases (TALENs) and clustered regularly interspaced short palin dromic repeats
(CRISPR)-associated nucleases (Doudna, 2020; Khalil, 2020). In addition to knocking in or
knocking out specific genes or portions of genes, CRISPR-based editing can also be deployed
to modulate gene expression (CRISPRi and CRISPRa) (Gilbert et al., 2013), excise DNA do-
mains, or change single base pairs (base editing) (Komor et al., 2016). Finally, prime editing
affords more control and precision to insertions, deletions or single base conversions (Anz-
alone et al., 2019; Godbout and Tremblay, 2023; Sun and Roy, 2021). In addition to its appeal
for gene correction, editing is also being used to generate “universal” hypoimmune iPSC
Blastocyst
Skin Biopsy
Directed
Differenaon
Blood
Culture
Fibroblasts
Culture/ Inner
Cell Mass
Reprogram
Autologous: Intermediate
Bank iPSC
Allo: Master and Working
Cell Banks iPSC
Formulate
Fill Finish
Master and Working
Cell Banks hESC
or
Directed
Differenaon
Formulate
Fill Finish
Directed
Differenaon
Formulate
Fill Finish
Source
Material
Primary
Processing
Banking
PSCs
*Drug Substance may require gene eding, and/or be further purified, and/or cryopreserved
Drug
Product
ESC
iPSC
iPSC
Drug
Substance*
Culture
PBMCs
Reprogram
Isolate hESC
colonies
or
FIGURE 12.1 Steps in manufacturing pluripotent stem cell-based products.
12. Considerations in the development of pluripotent stem cellebased therapies408
III. Specialty biologics and indications
starting material from which immune system-evading differentiated cells can be produced
(Hu et al., 2023). Importantly, hypoimmune iPSC-derived products are already in the clinic
(see Clinical translation section later in this chapter).
Raw materials
The source and quality of raw materials used in manufacturing of PSC-based clinical prod-
ucts is critically important. In principle, all raw materials should be of the highest grade avail-
able and, whenever feasible, the same raw materials used to manufacture clinical product
should also be used in the manufacturing of cells intended for testing in pivotal nonclinical
studies. While FDA-approved or cleared, or clinical-grade reagents should be used when
available, many do not fall into those categories. Indeed, the majority of raw materials
commonly used in manufacturing of PSC-derived products are research grade and of those,
only a portion are manufactured following current good manufacturing practices (cGMPs).
While there is some flexibility in using non-cGMP manufactured reagents during early clin-
ical investigation (FDA, 2008), cell therapy developers should endeavor to secure the use of
clinical grade and cGMP-manufactured raw materials as early as possible, and “lock-in” re-
agents to be used in manufacturing with particular attention to availability, quality control,
and documentation during manufacturing, vendor reliability, and reagent lot-to-lot stability.
Ideally, reagents should be available for use throughout clinical development to avoid poten-
tially extensive comparability studies and changes in product quality and biopotency. Theo-
retically, secondary back-up sources can be identified for critical reagents but it is not
uncommon for the unique materials to be single-source; note that even if there are multiple
vendors they may be obtaining a reagent from the same manufacturer.
The use of animal- and human-sourced reagents, if unavoidable, will require additional
testing and/or documentation to minimize the risk of transmission of viruses or certain
communicable diseases. For these reasons, many cellular therapies currently under develop-
ment are manufactured using animal product-free reagents, serum-free media, and small
molecule modulation of signaling pathways to achieve targeted differentiation.
Testing
Testing should be performed at critical stages of the manufacturing process to ensure qual-
ity and safety. In partic ular, testing for genomic stability and tumorigenicity via in vitro
assessment and phenotypic profile is highly advised (Suresh Babu et al., 2023). For iPSC start-
ing material in particular, applicable reprogramming clearance (absence of reprogramming
vectors) must be demonstrated using qualified assays, which are customized to the reprog-
ramming method used. Furthermore, in the case of edited PSC-derived products, additional
testing is critical to ensure edit specificity, lack of off-target effects, translocations or undesir-
able reagent residuals (FDA, 2024b).
Manufacturing scale
Manufacturing scalability should also be considered in the early stages of development.
While most PSC-based production is currently performed in two-dimensional culture sur-
faces, this presents challenges when it comes to scaling products for clinical trials that test
large cell doses and more critically, for commercialization. Suspension or three-
dimensional culture formats are better suited for scalability as they can be readily translated
Important early decisions: Chemistry, manufacturing, and controls 409
III. Specialty biologics and indications
to bioreactors. Several protocols have been established for prolonged suspension culture of
PSCs and subsequent differentiation of various cell types (Baharvand et al., 2012; Fluri
et al., 2012; Olmer et al., 2012; Schulz et al., 2012; Chen et al., 2012; Th omas et al., 2022; Cohen
et al., 2023). Establishing such methods early on for ideally the entire manufacturing process,
or at least portions of it, can take time but also could potentially save significant developmen t
time and costs later.
Critical decisions in cell line selection
One of the most critical decisions to be made in the development of PSC therapeutics is
selection of an appropriate starting material cell line. There are thousands of reported ESC
and iPSC lines in registries, although only a fraction are being used in clinical trials (Kobold
et al., 2023). Efforts by CIRM (Califo rnia), CiRA (Japan), WiCell (USA), Answer ALS, and the
European Bank for induced Pluripotent Stem Cells (EBiSC) have led to the generation of
several thousand disease-specific and healthy donor iPSC lines produced under standardized
culture conditions and QC for research purposes (Steeg et al., 2021). Similarly, the number of
iPSC lines being developed for therapeutic purposes has been steadily increasing over the last
5 years, fueling >100 clinical-stage programs worldwide (Kobold et al., 2023). A great
resource for tracking clinical studies using iPSC as starting material is accessible through
the Human Pluripotent Stem Cell Registry Portal (https://hpscreg.eu/).
Many ESC lines are available through public banks such as the UK Stem Cell Bank, insti-
tutions such as the Harvard Stem Cell Institute and the Massachusetts Human Stem Cell
Bank, and commercial banks such as the Wisconsin International Stem Cell (WISC) Bank.
The NIH Human Embryonic Stem Cell Registry lists over 500 ESC lines and these are the
only lines eligible for development using federal funding in the United States. In addition,
there are well-established protocols for deriving new lines. Whether using an existi ng cell
line or deriving a new line, special care must be given to the provenance of the cell line.
Detailed documentation is required on source donors, consent, materials used in derivation
of the line, and subsequent culture history. Table 12.3 provides a summary of the required
documentation. While cell line provenance is not a part of product characterization, the his-
tory of a cell line and culture methods used may have significant effects on the function and
characteristics of cells. Therefore, it is prudent to take special care in selecting an appropriate
cell line from which to develop a candidate therapeutic.
Appropriate screening and testing of donor material or tissues must be performed in accor-
dance with 21 CFR 1271, Subpart C: Donor Eligibility Final Rule. Table 12.4 lists donor
screening and testing requirements. For embr yo donations, screening must be performed
on both male (sperm) and female (egg) donors. In addition to appropriate screening and
testing of donors, proper informed consent of donors is critical. Table 12.5 lists key criteria
for informed donor consent as defined in the 2009 NIH Guidelines for Human Stem Cell
Research (NIH, 2009). Improper or incomplete consent documentation may adversely impact
later stages of development, particularly when approaching market approval. Availability of
such documentation should be taken into consideration when selecting an existing PSC line
for use in preclinical development. In contrast, per 21 CFR 1271.90(a)(1), eligibility and testing
requirements are not required for donors of biological material (such as fibroblasts and blood
12. Considerations in the development of pluripotent stem cellebased therapies410
III. Specialty biologics and indications
TABLE 12.3 Proposed cell line provenance checklist.
Donors’ age, sex, and status (living or
dead)
Donors’ medical history and results of
screening and testing for communicable
diseases
IRB approval protocol and consent forms
SCRO Committee approved protocol and
consent forms
Culture history: Methods used in derivation of cell line and stage of tissues/embryo at
time of isolation
Passage history, including media and reagents used, use of animal
feeder layers, etc., from derivation up to time of cell bank
establishment
Results of identity testing
Results of all tests for adventitious agents
TABLE 12.4 Donor screening and testing requirements.
a
Test Screening Testing
b,c
HIV-1 and HIV-2 UU
Hepatitis B UU
Hepatitis C UU
Treponema pallidum (syphilis) UU
Chlamydia trachomatis UU
d
Neisseria gonorrhoea UU
d
Transmissible spongiform encephalitis, including Creutzfeldt
eJakob disease
U
West Nile virus U
Communicable risks associated with xenotransplantation (i.e.,
have they ever had a xenotransplant?)
U
Sepsis (at time of oocyte retrieval) U
Vaccinia (recent smallpox vaccination) U
a
21 CFR 1271, Subpart C: Donor Eligibility Final Rule; 21 CFR 1271.75, 1271.80, 1271.85. Not required for cells or tissues for autologous
use (CFR 1271.90).
b
Testing must be completed by a CLIA certified lab using FDA-licensed, approved or cleared donor screening test for diseases listed.
c
Testing must be completed within 7 days before or after collection of tissues from donors.
d
Testing required only for donors of reproductive tissues.
Important early decisions: Chemistry, manufacturing, and controls 411
III. Specialty biologics and indications
mononuclear cells) intended to generate autologous products. Nevertheless, testing for select
transmissible diseases may still be conducted to inform and safeguard manufacturing activ-
ities and avoid propagation of pathogens to other cellular products. In the case of blood
collection, elective testing can be guided by the recommendations of the CDC and the Amer-
ican Association of Bloo d Banks (Jha et al., 2021). Individual US states, such as California, or
institutions and granting agencies may have additional regulations for the conduct of PSC
research and procurement of donor material. Many US institutions require review by a
Stem Cell Research Oversight (SCRO) committee prior to the procurement of embryos/tis-
sues, derivation of lines or commencement of any research involving PSCs. The Natio nal
Academy of Science (NAS) suggests that the SCRO committee include a mix of individuals
with expertise in developmental biology, stem cell research, molecular biology, assisted
reproduction, and ethical and legal issues in human PSC research, as well as representatives
of the lay public. The role of the SCRO committee is to provide oversight over all issues
related to derivation and use of PSC lines. The SCRO committee will review donor records,
informed consent documents, cell line information, research plans, and protocols to ensure
that PSC research is carried out in accordance with ethical and legal guidelines. Some insti-
tutions have their own SCRO Committees, akin to institutional review boards (IRBs). There
are also a limited number of private, third-party SCRO Committees. SCRO committee review
is performed in addition to, and does not replace, IRB review of consent forms and protocols
for procurement of donor tissues, or institutional animal care and use committee (IACUC)
review and approval of animal protocols. The SCRO committee will review IRB and IACUC
documentation and vice versa.
Cell line characterization
Once a cell line has been established or selected, confirmation of its pluripotency and sta-
bility is important. Pluripotency can be assessed by the expression of molecular markers of
TABLE 12.5 Recommendations for informed donor consent for pluripotent stem cell research.
• Donor material may be kept for many years
• Embryos may be destroyed during the derivation process (ESCs only)
• The cells and donor’s medical information may be shared with other researchers to benefit medical research
and society
• The research is not intended to provide direct medical benefit to the donor (not applicable to autologous iPSC
donors)
• The results of the research may have financial or commercial potential and that the donors would not receive
any financial or other benefits from any such commercial development
• Donors may be re-contacted for additional samples, medical information, or to consent to additional PSC uses
not originally anticipated, but they do not need to oblige; a provision should also be included for donors to opt
out of being re-contacted by researchers for any reason
• Pediatric re-consent is incorporated to allow children to be informed upon reaching adulthood of the ability to
review the previous consent and continue to participate in the study or not (iPSC only)
• Withdrawal at different stages of the research process is allowed. A request can be made to destroy any
leftover original samples; however, any ESC or iPSC already derived cannot be destroyed and any shared with
other researchers cannot be retrieved, though the codes that link the sample to the donor can be removed
From 2009 NIH Guidelines for Human Stem Cell Research [35]; Lowenthal et al. (2012).
12. Considerations in the development of pluripotent stem cellebased therapies412
III. Specialty biologics and indications
undifferentiated PSCs and the ability of the line to form cells from all three germ layers (In-
ternational Stem Cell, 2018). The stability of the cell line is assessed by culturing the PSCs for
many passages (or population doublings) to determine the upper limit of passage numbers
prior to loss of pluripotent properties or genomic instability (e.g., aneuploidy as assessed
by karyotype).
Once pluripotency and stability of the cell line have been confirmed, a master cell bank
(MCB) and working cell banks (WCB) should be established, particularly in the case of allo-
geneic therapies. Cell lots to be used in the conduct of nonclinical studies should be derived
from these banks. The probability of acquiring genetic mutations or chromosomal abnormal-
ities increases with increasing passage number in cell culture, and therefore the MCB should
be established from early passage cells (Hanson and Caisander, 2005). WCBs are established
from the MCB and are naturally from slightly later passages. For autologous products
derived from patient iPSC, derivation of the standard two-tiered banking system is not a
strict requisite, however, it is still critical to generate well-characterized and appropriately
tested intermediate cell banks (Jha et al., 2021).
In addition to the initial screening and testing of donors and tissues for viruses and
communicable diseases (discussed above), extensive testing of cell banks for adventitious
agents must be carried out to ensure patient safety. A list of tests performed as part of
MCB characterization is provided in Table 12.6 and a list of tests performed as part of
WCB charac terization is provided in Table 12.7.
For cell products that derive from edited PSC, whether allogeneic or autologous, the gen-
eration of a carefully characterized, clonal edited cell bank is recommended, with critical
attention placed on safety testing via next generation sequencing (NGS) and other sophisti-
cated genomic assays. In addition to ensuring edit specificity by target locus sequencing (con-
firming that the correct sequences are inserted or removed and flanking sequences are
appropriate), and lack of off-target effects, it is critical to test for potential changes in onco-
genes (for example, OncoPanel by Eurofins), and chromosomal integrity (karyotype, chromo-
somal microarrays such as array comparative genomic hybridization (aCGH) and optical
mapping) (Steeg et al., 2021; Kitano et al., 2022; Neumayer et al., 2023). This level of testing
ensures that subsequent differentiation step(s) are conducted with cells that are genomically
stable and carry no, or only benign, mutational burden and are therefore acceptable for
downstream manufacturing. Additionally, the CMC package should include a toxicity risk
assessment of all the tools/reagents used in the editing process and determine if the presence
of trace amounts of these tools/reagents might pose a risk for patients.
Product characterization
Cell products should be manufactured using a robust process under stringent quality con-
trol to ensure a safe, consistent, and reproducible final product. Unlike drug compounds,
which have precise chemical makeup, cell therapy populations have a complex biological
structure and composition, making it a challenge to fully characterize cell products and estab-
lish specifications for identity, purity, and potency. Key markers and attributes of certain cell
types are still being defined. Adding to the complexity is the need to describe the appropriate
developmental/maturation stage of PSC-based products, as well as the level of mitotic
Important early decisions: Chemistry, manufacturing, and controls 413
III. Specialty biologics and indications
activity desired for the clinical effect. While progenitors or precursors may be beneficial in
some indications, terminally differentiated cells may be more suitable in others (Aboody
et al., 2011). Due to our limited understanding of the early stages of biological development
regarding mechanism of action and requirements for therapeutic effect, characterization dat a
should be collected throughout the product development life cycle with continual feedback
from in vitro and in vivo assays to inform process optimization and product characterization.
In addition, cell-based therapies require stringent safety assays if animal products are used in
the manufacturing process, and it is necessary to evaluate the potential for an immune rea c-
tion to the cell product or transgene(s) expressed by the cell product, track any genomic insta-
bility of cultures over time, and assess the risk of tumorigenicity especially as undifferentiated
TABLE 12.6 Master cell bank characterization of pluripotent stem cell lines.
Test
a
Method
Microbiological characteristics
Sterility 21 CFR 610.12
Bacteriostatic/fungistatic activity 21 CFR 610.12
Mycoplasma Presence of agar cultivable and nonagar cultivable mycoplasma
Human pathogen testing
HIV 1 and 2, HTLV 1 and 2, CMV,
EBV, HP-B19HCV, HBVHHV
(Herpes virus 6, 7, and 8)
PCR
Virus testing
In vitro assay for adventitious
viral contaminants
Culture on MRC-5, VERO and Hs68 cells 28 day culture with 3
indicator cell lines: MRC-5, VERO and HeLa cells
In vivo assay for adventitious viral
contaminant
Adult and suckling mice, chicken eggs (3 injection sites), and Guinea
pigs
Murine adventitious agents
b
Murine antibody testing (MAP)
Murine retroviruses: Mus Dunni coculture with PG4 S þ L-assay
Ultrastructural evaluation of cell
cultures for viral particles (VLPs)
c
Transmission electron microscopy
Reverse transcriptase assay
c
PCR
Cell identity
Characterization of cell
morphology and growth; genetic
fingerprint of ESC line
G-banding
Cell line species identity Isoenzyme analysis by gel electrophoresis
a
Tests should be conducted under GLP (21 CFR 58).
b
Murine testing may be required if murine reagents are used in derivation of cell line or creation of MCB. Additional animal (murine, bovine,
porcine, etc.) virus testing may also be required depending on reagents used in cell line production.
c
Retroviral testing may be required if cells or reagents are exposed to materials that may contain retroviruses.
12. Considerations in the development of pluripotent stem cellebased therapies414
III. Specialty biologics and indications
cells could persist in the final product. Table 12.8 provides a summary of the key themes of
product characterization and potential evaluation criteria.
Definitive preclinical studies should be carried out using a manufacturing process that is as
representative as possible of the methods and materials intended for generation of the clinical
product. Therefore, adequate characterization of the product along with the implementation
of cGMP practices, if possible, should take place prior to the initiation of definitive IND-
enabling animal studies.
Pluripotency and the potential for tumorigenicity
Assessing the stability of a PSC line and the tumorigenic potential of a PSC-derived candi-
date cell product are important parts of product characterization, as described above. Yet in a
related but dis tinct concern, regulatory agencies are requiring a demonstration that a PSC-
derived product is largely free from residual PSCs, which may not have responded to the dif-
ferentiation cues in the process, can remain hidden in the bulk population, and may give rise
to teratomas. For the purpose of demonstrating the absence of residual PSCs, standardi zed
assays have not been established, allowing investigators to individually define how these as-
sessments are made. In the past, significant effort was placed on developing depletion stra-
tegies (genetics, antibodies, small molecules, etc.) to eliminate any residual stem cells from
products during the manufacturing phase (Conesa et al., 2012; Tang et al., 2012; Rong
et al., 2012; Schuldiner et al., 2003). Now, it is becoming evident that tighter control over
the differentiation process may provide an effective more robust solution than depletion.
Certainly, this was publicly recognized by the FDA in 2018 at the International Conference
on Manufacturing and Testing of Pluripotent Stem Cells in the statement “Tumorigenicity
risk represents a continuum that is dependent on the inherent biological properties and
manufacturing processes for stem cell-based products .” (Abbot et al., 2018).
TABLE 12.7 Working cell bank characterization of pluripotent stem cell lines.
Test
a
Method
Microbiological characteristics
Sterility 21 CFR 610.12
Mycoplasma Presence of agar cultivable and nonagar cultivable
mycoplasma
Virus testing
In vitro assay for adventitious viral contaminants Culture on MRC-5, VERO and Hs68 cells 28 day culture
with 3 indicator cell lines: MRC-5, VERO and HeLa cells
Cell identity
Characterization of cell morphology and growth
a
Tests should be conducted under GLP (21 CFR 58).
Important early decisions: Chemistry, manufacturing, and controls 415
III. Specialty biologics and indications
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