1. 18
In: Areman EM, Loper K, eds.
Cellular Therapy: Principles, Methods, and Regulations, 2nd edition
Bethesda, MD: AABB, 2016
2
The FDA Perspective on the Manufacturing,
Production, and Processing of Regulated
Cellular Therapies
Eric Dollins, PhD; Patrick Lynch, PhD;
Prajakta Varadkar, PhD; and Keith Wonnacott, PhD
OMATIC CELLULAR THERAPY IS THE
administration to humans of autologous, alloge-
neic, or xenogeneic living cells that have been
manipulated or processed ex vivo. The Food and
Drug Administration (FDA) asserted27
jurisdiction over these products in 1993.1
Somatic
cellular therapy products are drugs, biological prod-
ucts, and human cells, tissues, and cellular and tis-
sue-based products (HCT/Ps) that are regulated
under Section 351 of the Public Health Service
(PHS) Act.2-4
This means that somatic cellular thera-
pies are subject to current good manufacturing
practice (cGMP) regulations [Code of Federal Regu-
lations (CFR) Title 21 Parts 210 and 211],5,6
biologi-
cal product regulations (21 CFR 600s),7
labeling (21
CFR 201),8
advertising (21 CFR 202),9
and HCT/P
regulations (21 CFR 1271 parts A-D),10
including
general provisions, registration and listing, donor
eligibility requirements, and current good tissue
practice (cGTP). During investigational stages, cel-
lular therapies are also subject to regulations for
Investigational New Drugs (IND) (21 CFR Part
312),11
protection of human subjects (21 CFR 50),12
and Institutional Review Boards (21 CFR 56).13
While beyond the scope of this chapter, there are
certain circumstances in which device regulations
(21 CFR 800)14
could also apply to therapeutic prod-
ucts containing cells, as in the case of combination
products. More information regarding combination
products can be found on the Office of Combination
S
Eric Dollins, PhD, Director, Global Regulatory Lead, Merck Group, Darmstadt, Germany; Patrick Lynch, PhD, Biologist, Center for Drug
Evaluation and Research, Food and Drug Administration, Rockville, Maryland; Prajakta Varadkar, PhD, Visiting Associate, Center for Bio-
logics Evaluation and Research, Food and Drug Administration, Rockville, Maryland; and Keith Wonnacott, PhD, Director, Regulatory
Affairs, Novartis, Washington, District of Columbia
We acknowledge the contributions of Sarah Kennett, PhD; Cynthia Porter, PhD; and Eda Bloom, PhD (now deceased) to previous versions
of this chapter.
The authors have disclosed no conflicts of interest.

2. Chapter 2: FDA Perspective 19
Products (OCP) website.15
In addition, special con-
cerns relevant to xenogeneic cells are not covered in
this chapter, but further information on the regula-
tion of xenotransplantation products can be found
in FDA guidance16
and on the FDA website.17
Because of their complex nature, cellular thera-
pies present both great potential and great chal-
lenges. Specific challenges for the development of
cellular therapies include patient-specific and/or
small lot sizes, maintenance of cell function and via-
bility throughout manufacture and storage, lack of
reference standards, starting material (donor) vari-
ability, limited material for testing, the need for
aseptic processing, and short shelf-life. However,
despite the challenges, these therapies allow manu-
facturers to use living cells that may have the ability
to repair, replace, or restore function in the patient.
This chapter is intended to provide insight for
navigating the regulations during the development
of somatic cellular therapies for clinical use. The
FDA has tried to facilitate an understanding of how
the various regulations apply to cellular therapy
products by issuing guidance documents that are
both directly and indirectly relevant to cellular ther-
apies. This chapter highlights some of the concepts
in those guidance documents that address common
challenges in cellular therapy product and process
development.
Because the vast majority of cellular therapies are
still investigational, this chapter is primarily focused
on product development rather than on product
licensure. After the general tips below, the sections
that follow focus on specific issues and address many
of the common manufacturing challenges encoun-
tered during product development. However, we
conclude with a brief section that discusses some
considerations for products in late-phase develop-
ment and preparation for licensure.
General Tips
The following general tips for product development
are helpful to remember, regardless of the phase of
development or the product being developed.
Tip 1: Be Data-Driven
Good data are an essential ingredient for successful
clinical research studies. This is true not only of the
proof-of-concept and pharmacology or toxicology
data that support the clinical research, but also of
the product development data that support manu-
facturing processes and controls. For example,
answers to questions such as the following should be
supported by data and not suppositions:
• Is the cellular starting material free of infectious
viruses?
• Do the antibiotics in the culture media interfere
with the sterility assay?
Data used to answer these questions should be
complete and directly applicable to the specific
product and manufacturing process, particularly if
the plan is to cite publicly available data.
Tip 2: Provide Complete and Accurate
Documentation
In addition to good data, good documentation is
essential during product development. Using an
organized format is helpful in ensuring that all nec-
essary information is included. For example, IND
applications for somatic cell therapies should follow
the same format and contain the same sections as
IND applications for any investigational drug or
biological product, as described in the IND regula-
tions.18
In addition, the organization and content of
the chemistry, manufacturing, and controls (CMC)
section within the IND application may follow the
template described in the Guidance for FDA review-
ers and sponsors: Content and review of chemistry,
manufacturing, and control (CMC) information for
human somatic cell therapy investigational new
drug applications (INDs).19
Flow charts, tables, and
narrative descriptions are helpful, and they contrib-
ute to the clarity of submissions, but they should be
supported with the details. [Readers should remem-
ber that, in the process for IND applications, unlike
that for National Institutes of Health (NIH) grant
applications, there is no limit to the amount of
detailed and relevant information that can be sub-
mitted.]
If documentation for an IND or biologics license
application (BLA) submission is missing, incom-
plete, contradictory, or incorrect, the FDA will not
be able to make an independent assessment of the
product, and the study will not be allowed to pro-
ceed. Complete, well-organized, and internally con-
sistent data facilitate an accurate safety assessment.

3. 20 Cellular Therapy: Principles, Methods, and Regulations
Tip 3: Be Informed
There are many resources available to sponsors who
are developing cellular therapy products. Formal
resources include the laws, regulations, and guid-
ance documents. Many of the relevant resources can
be found on the FDA website.20
In addition, the FDA
staff participates in open public meetings and other
forms of outreach to help inform stakeholders.
Many of these presentations are easily accessible
through the internet.
Tip 4: Communicate with the FDA
The FDA has a defined process for granting formal
meetings.21
There are several specified meetings,
such as pre-IND meetings, end-of-Phase-II meet-
ings, and pre-BLA meetings, but other formal meet-
ings are also possible. In addition to formal
meetings, reviewers at the FDA, when possible, try
to answer questions as part of their outreach to
sponsors.
Tip 5: Plan Ahead
Many of the problems that sponsors may have with
product development can be avoided by having a
better understanding of their product earlier in the
manufacturing process. For example, some charac-
terization testing of a product may not directly affect
safety and, therefore, may not be required by the
FDA for early-phase studies. However, early product
characterization testing, including tests for identity
and potency, can help to avoid potential problems
later in development.
Preparing a Cellular Therapy Product for
Clinical Trials
Translational research often revolves around the pre-
clinical animal studies needed beyond the initial
proof-of-concept studies. However, translational
research is also important in the development of a
clinical manufacturing process and product. A com-
mon deficiency in IND applications is that many
assumptions are made about either the product or
the process to be used in clinical studies. Although
many assumptions may prove to be true, they
should also be supported by data. For example,
studies may be needed to demonstrate that the clini-
cal trial manufacturing process results in a product
that is similar to that used for the preclinical studies
used to demonstrate safety.
Another area where additional preparation is
needed in manufacturing for clinical trials is in the
manufacturing facility and operations. Many aca-
demic institutions have recently built, or are inter-
ested in building, manufacturing facilities to
support clinical research within the institution. It is
not uncommon to hear that these new facilities are
cGMP facilities. In such cases, the term is used very
narrowly, applying only to the design and function
of the building and facility. However, cGMPs include
much more than facility requirements. They provide
for systems that ensure proper design, monitoring,
and control of manufacturing processes and facili-
ties. This includes establishing strong quality man-
agement systems, obtaining appropriate-quality raw
materials, establishing robust operating procedures,
detecting and investigating product quality devia-
tions, and maintaining reliable testing laboratories.22
This formal system of controls, if adequately put
into practice, helps to prevent instances of contami-
nation, mix-ups, deviations, failures, and errors.22
cGMPs for Phase 1 Clinical Trials
Because of their critical importance in ensuring that
products meet their quality standards, cGMPs are
requirements mandated by law. Section 501(a)(2)(B)
of the Food, Drug, and Cosmetic Act [21 U.S.C. 351
(a)(2)(B)] requires drugs, which include IND prod-
ucts, to comply with cGMP. On the basis of this stat-
utory requirement, the FDA issued regulations for
drug and biological product cGMPs (21 CFR parts
210 and 211). However, because certain require-
ments in 21 CFR 211 are directed at commercial
manufacture, they may not be appropriate to the
manufacture of most investigational drugs used for
Phase I clinical trials. Thus, new products entering
Phase I clinical trials are exempt from the more
detailed cGMP requirements outlined in the regula-
tions; however, they are not exempt from the
requirement in the law (FD&C Act) mandating
cGMPs.
The FDA has issued guidance that provides rec-
ommendations regarding appropriate cGMPs for
drugs and biological products in a Phase I clinical
trial.23
This guidance is part of the agency’s effort to
develop an approach to implementing manufactur-

4. Chapter 2: FDA Perspective 21
ing controls during the early stages of development.
The approach described in the guidance is in concert
with the approach of CBER to taking into consider-
ation the type of product and stage of development
in determining the applicable cGMP.
Following are some of the recommendations of
the guidance on cGMPs for Phase I Investigational
Drugs23
:
• Use adequate quality-control (QC) procedures,
including well-defined written procedures, ade-
quately controlled equipment, and accurately
and consistently recorded data.
• Consider the risks posed by various aspects of
the production environment that might
adversely affect the quality of a product, and
give thorough consideration to controls for
aseptic processing, especially when the IND
product is produced in a facility that was not
expressly designed for that function.
• Use disposable equipment and disposable pro-
cess aids, prepackaged water or validated water
for injection, and presterilized containers.
• Use a closed system for manufacturing.
Additional information about complying with
cGMP regulations after Phase I is included later in
this chapter.
Manufacture of the Cellular Therapy Product
The information that is discussed in this section is
designed to assist manufacturers in understanding
common challenges and complying with regulatory
requirements in the manufacture of cellular therapy
(CT) products. The information can also be helpful
in preparing information to submit in the CMC sec-
tion of an IND for cellular therapy products.
Components and Materials
The manufacture of a cell therapy product that
meets the standards set forth in FDA regulations
necessitates beginning with safe and pure compo-
nents and materials. This is often a challenge for cel-
lular therapies, not only because of the risks
associated with the cells themselves, but also because
of the broad range of cytokines, growth factors, and
other materials that are used for cell growth, cellular
differentiation, or cellular activation. To facilitate
regulatory review, it is beneficial to include detailed
information regarding components and materials in
IND submissions. All materials used in the manu-
facture of the cell therapy product should be docu-
mented; the source and all testing should be noted.
Cellular Starting Material
Cells should be classified as autologous or alloge-
neic, and the tissue source, mobilization protocol (if
appropriate), and collection method should be doc-
umented. Donor screening, safety testing, and any
additional characterization should also be docu-
mented.
Donor screening and testing are essential for
ensuring that the cellular starting material is safe;
however, failure to provide complete donor infor-
mation is a common deficiency in many cellular
therapy IND applications. For allogeneic cellular
products, prospective donors must meet the donor
eligibility requirements as described in 21 CFR 1271
part C.24
A complete description of the testing and
screening of the donors should be provided in IND
submissions, and documentation of testing should
be maintained in the study records.25,26
Each pro-
spective donor should be screened for high-risk
behavior and tested for communicable disease
agents as appropriate.27,28
The specific relevant com-
municable disease agents for which donors of HCT/
Ps must be tested are listed in 21 CFR 1271 part 85
and described in an FDA guidance document.28,29
Testing should be performed by using FDA-
licensed, -approved, or -cleared donor screening
tests when they are available.30
An updated list of
licensed, approved, or cleared tests can be found on
the FDA website.31
Donors whose screening tests are
reactive for human immunodeficiency virus (HIV)
type 1, HIV-2, hepatitis B virus (HBV), hepatitis C
virus (HCV), and, for leukocyte-rich HCT/Ps,
human T-cell lymphotropic virus (HTLV) type I or
HTLV-II should be excluded. Detailed information
on donor eligibility is available in an FDA guidance
document.29
Recent draft guidance documents with
current and proposed recommendations for screen-
ing and testing HCT/P donors for syphilis and West
Nile virus are also available.32,33
To have a consistent and stable supply of cells for
manufacturing, it may be desirable to use a cell bank
system for cellular therapy products. Donor screen-
ing, cell characterization, and safety testing are
required in establishing cell banks. Failure to pro-
vide complete information of this sort is a common
deficiency in original IND submissions, and it can

5. 22 Cellular Therapy: Principles, Methods, and Regulations
lead to long delays in the initiation of Phase I stud-
ies. Cell banks should be thoroughly tested for
adventitious agents, including bacteria, fungi, myco-
plasmas, and viruses. This testing includes that for
additional adventitious agents other than those for
donor eligibility, because cell lines are at risk of addi-
tional exposure to microbial agents, for example,
through various culture additives or the culture pro-
cess itself. Cell banks should also be tested to estab-
lish identity, purity, and stability. If a feeder cell line
is used, information describing the characterization
of this cell line should be included. If the feeder line
is of animal origin, the final product falls within the
definition of a xenotransplantation product, and the
relevant guidance document should be consulted.16
Additional information regarding cell bank systems
is available in FDA and International Conference on
Harmonisation (ICH) documents.34-36
Section VIII
of this publication (Biorepositories) provides fur-
ther discussion of cell banking issues.
Other Materials
Certain materials may be essential for critical manu-
facturing steps but may not be intended to be part of
the final product. Whenever possible, FDA-licensed
products should be used in the manufacturing pro-
cess. If a material is not FDA-approved, a certificate
of analysis (COA) should be supplied, or, if a master
file exists in the FDA, a letter of cross-reference from
the manufacturer should be provided, and addi-
tional testing may be important to ensure the safety
and quality of the material. The FDA recommends
that a qualification program be established for all
critical materials and that this program consist of
appropriate safety tests, an analysis for purity, and a
functional assay to ensure that these materials are
performing as desired in the manufacturing process.
These tests should be performed each time a new lot
is qualified. Program functions may include testing
each lot after receipt from the vendor or reviewing
certificates of analysis supplied by a qualified vendor
that document all testing results. After the materials
have been deemed adequate, they can be released for
use in the manufacturing process.
Some materials that are frequently used in pro-
duction of cellular therapies are the subject of spe-
cific regulatory concerns. Many of the potential
concerns they raise can be avoided if the proper
documentation is provided. However, when the
supplier of the material has not performed all the
required testing, additional testing is needed.
Examples of materials that warrant additional
consideration include materials derived from
human plasma (and that are not FDA-licensed
products), animal-derived materials [such as fetal
bovine serum (FBS)], and proteins produced in
mammalian expression systems such as monoclo-
nal antibodies and growth factors (including cyto-
kines). Antibiotics used during cell culture also
generate regulatory concerns because they may
confound the results of sterility testing, and some
patients may be sensitive to beta-lactam antibiot-
ics. Sponsors should consult appropriate regula-
tions and guidance documents for more detailed
information on the use of antibiotics and animal-
derived materials during product manufac-
ture.19,37,38
Additionally, an FDA points-to-
consider document for the use of monoclonal
antibodies is available.39
Manufacturing Procedures
A common misconception among sponsors of cell
therapy IND submissions is that the FDA is inter-
ested only in starting materials and final product
testing and not in the details of manufacture. For
biological products in general, and cellular therapy
products in particular, the manufacturing process
itself plays a significant role in defining the product.
This principle is reflected in the commonly used
phrase, “the process is the product.” Because of the
critical role the manufacturing process itself plays,
the FDA requests detailed descriptions of all proce-
dures used during the production and purification
of the cellular therapy product.
To ensure product safety, the procedures, in
addition to the assays used for product release
testing, should be adequately developed by the
time of an original IND submission, and their
descriptions should be provided in that submis-
sion. A schematic of the production and purifica-
tion process, in-process testing, and final product
testing, with timing, should be provided in the
IND submission. To adequately monitor the prod-
uct quality during extended culture periods, the
FDA recommends that in-process testing be per-
formed at various time points. This testing may
include safety, product characterization, and
purity of the cellular therapy product, including

6. Chapter 2: FDA Perspective 23
the types of cells present and the percentage of
each cell type. It is expected that the procedures
used in manufacture will be refined during early-
phase studies, but the manufacturing process
should be well established before pivotal studies
are initiated, and changes to the process should be
minimized once the pivotal trials are initiated.
Because the clinical trial results could be con-
founded by manufacturing changes, changes in
manufacturing procedures during or after the piv-
otal studies may require demonstration of product
comparability. Validation of analytical methods,
systems processes, and facilities must be com-
pleted by the time of submission of a BLA. (See
section below on later phase development and
marketing.)
Preparation of Autologous or Allogeneic Cells
Procedures describing the preparation of the autolo-
gous or allogeneic cells should include information
about the method of cell collection, processing, and
culture conditions. Cell numbers and concentra-
tions or volumes of solution or medium should be
documented, as well as mechanical or enzymatic
digestion steps or the use of a cell-selection or
-separation device, including density gradients,
magnetic beads, or fluorescence-activated cell sort-
ing (FACS). If irradiation procedures are used, the
irradiation dose and qualification of equipment
should be supported by data.
Sterility, an important aspect of safety, is of great
importance in cellular therapy manufacture.
Because it is not possible to sterilize cellular therapy
products before administration, documentation
should be provided to demonstrate that aseptic pro-
cessing steps are adequate. It is beneficial to conduct
a process simulation using bacterial growth media to
demonstrate that processing is performed in a man-
ner that will maintain sterility of an aseptically
processed IND product. The FDA has published
guidance that details considerations related to asep-
tic processing.40
Tracking and Labeling
Systems for tracking, labeling, and segregation
should be followed from collection of the cells until
administration or other disposition of the product.
Among cellular therapy products, descriptions of
these systems are common omissions from the CMC
section of IND submissions. The systems for track-
ing, labeling, and segregation should be designed to
ensure unambiguously that a patient receives the
correct product. The labeling should include two
unique patient-specific identifiers, and segregation
procedures should be combined with appropriate
cleaning and control procedures to prevent cross-
contamination among products produced in the
same facility.
Process Timing and Intermediate Storage
Process timing can be critical in cellular therapy
manufacturing. Storage time and conditions
between cell collection and final harvest should be
documented. Adequate procedures should be in
place to ensure stability of the cellular product dur-
ing fresh and frozen storage.
Final Formulation
Both the procedures for preparing the product and
the final product formulation are critical to product
safety and quality. Information regarding these final
steps, including centrifugation, buffers, and media
used for washes, cell concentrations, and excipients,
should be included in the IND submission.
Storage and Shipping
The storage and shipping conditions of cellular ther-
apy products can be as varied as the products them-
selves, so it is important to properly define and
document them. For example, if the final product is
delivered to the clinical site in a frozen state, ship-
ping information and data to show that the product
can be consistently thawed and can retain its critical
quality attributes should be provided.
Delivery of the Cellular Product
An item commonly omitted from IND applications
relates to the device used to deliver the cells. For cel-
lular therapies, the delivery device could be simple
or complex depending on the route of
administration. No matter how simple or complex
the delivery device, it should be tested with the final
cellular product to ensure that the intended clinical
product can be successfully delivered.
The types of questions a sponsor may ask when
considering the use of a delivery device follow:

7. 24 Cellular Therapy: Principles, Methods, and Regulations
• Are the cells recovered, and do they maintain
their viability after being passed through the
device?
• What is the regulatory status of the delivery
device, and has it been modified?
• How much volume is to be delivered and over
what length of time?
• How much product is actually delivered, and
how much leaks back out of the injection site?
Delivery device considerations such as these
should be addressed in the initial IND submission.
Retention Samples
Retrospective analysis of retention samples may be
very useful when unexpected situations arise. The
FDA recognizes that, for some cellular therapy prod-
ucts, traditional methods for collection and storage
of retention samples may not be possible because of
lot size and stability issues that would render the
samples inadequate for future testing. However,
cGMPs require that samples be retained, as appro-
priate to the product, and the FDA has been flexible
in the types and ways that samples are collected and
stored.
Considerations for Manufacturing Stem Cell-
Derived Products
There is current interest among sponsors of cellular
therapy INDs in developing products derived from
or composed of stem cells, such as embryonic stem
cells or induced pluripotent stem cells (iPSCs).
Because of their capacity to self-replicate and give
rise to other types of cells upon stimulation, stem
cells represent potential source material from which
to generate large numbers of specialized cell prod-
ucts on demand. Unlike many somatic cell therapies,
the production of stem cell-based products typically
involves transitions between different cell states, and
thus the potential for unwanted cell types in the final
formulated product represents a unique challenge.
These nontarget cell types are considered impurities,
and they may arise during manufacturing because
of spontaneous differentiation, inefficiencies in
directed differentiation methodologies, and instabil-
ity of the final differentiated state. Different types of
cellular impurities include residual pluripotent stem
cells, which represent a substantial safety concern
because of their potential to engage in undesired
growth or differentiation. In certain instances when
a partially differentiated state is desired, such as for
some tissue-specific progenitor cell products, termi-
nally differentiated cellular impurities may nega-
tively affect product quality. The type, source (such
as the manufacturing step), and prevalence of
potential impurities are important considerations to
take into account during the production and purifi-
cation of stem cell-derived products. To ensure the
purity of stem cell-based products, special consider-
ation should be given to testing for and assessing the
profile of potential contaminating cell types. In-
process testing may be of particular importance in
identifying potential sources of heterogeneity during
manufacturing and setting appropriate specifica-
tions to limit their presence in the final product.
Product Specifications
Specifications are the quality standards (eg, tests,
analytical procedures, and acceptance criteria) that
confirm the quality of products and materials used
in production. They should be based on experience
with the specific product and similar products. Tests
required for cellular therapy products include those
for safety (microbiological testing, including steril-
ity, mycoplasma, and adventitious viral agent test-
ing, as appropriate), identity, purity (including
endotoxin), potency, and other relevant parameters,
such as viability. These tests are described in 21 CFR
Part 610 and in FDA guidance documents.6,7,23
Final
product-release specifications are required, although
the actual specifications may evolve during product
development. Specifications used for other interme-
diates should also be reported, as appropriate to the
product. In-process testing should provide mean-
ingful insight concerning process and product qual-
ity and should contribute to the safety and quality of
the final product. For licensure, a complete set of
specifications that is based on information collected
during development using validated assays must be
in place.41
It is expected that certain specifications, such as
those related to product safety, be in place before
initiating Phase I clinical studies. As product devel-
opment proceeds, additional specifications for prod-
uct quality and manufacturing consistency should
be implemented. Specifications for Phase I studies
should be based on data from lots used in preclinical
studies and should include analytical procedures
that are based on CFR methods or appropriate alter-

8. Chapter 2: FDA Perspective 25
native methods. For Phase II, the specifications
should be refined and tightened on the basis of data
generated during Phase I. Phase III specifications
should be based on further information collected
during product development, and validation of ana-
lytical procedures should be ongoing or complete
and consistent with data generated during clinical
studies.
Lot Release Testing
Cellular therapies pose a number of challenges to
final product testing. These challenges are often
unique to cellular therapy. They may include the fol-
lowing:
• Short product shelf life due to cellular viability.
• Small sample volumes.
• Heterogeneous populations of cells.
• Variability in composition, activity, or potential
of the cells.
• Inability to create appropriate reference materi-
als.
• Complex or unknown mechanisms of action.
• Ability of the cells to differentiate after adminis-
tration.
Because of these challenges, the FDA tries to be
flexible and science-driven in applying the regula-
tions for product testing. However, failure to provide
procedures for adequate final product testing
remains one of the primary deficiencies in new IND
submissions. Table 2-1 describes the most common
reasons that final product testing has been found
deficient. This table highlights the challenges in
applying final product testing regulations to cellular
therapy products. Additional discussion of the
expectations for lot release testing can be found in
many of the FDA guidance documents cited in this
chapter.
Microbiological Testing
Sterility (bacterial and fungal) testing. Sterility
testing must be performed on each lot of each bio-
logical product’s final container material or other
material, as appropriate.42
Previously, prescriptive
methods to be used for sterility testing were speci-
fied in regulation. However, in 2012, the FDA
amended the sterility testing requirements for bio-
logical products (21 CFR 610.12).43
These changes
are intended to promote improvement and innova-
tion in the development of sterility test methods by
allowing manufacturers the flexibility needed for
sterility testing of some novel products that may be
introduced to the market, enhancing sterility testing
of currently approved products, and encouraging
manufacturers to utilize scientific and technological
advances in sterility test methods as they become
available.43
Regardless of the manufacturer’s choice
of sterility test, the method used for testing should
be validated and described in detail.
Although it is not recommended, manufacturers
of cellular therapies may use antibiotics during cell
culture. The presence of antibiotics in test samples
may confound sterility results. Therefore, bacterio-
stasis and fungistasis testing, as described in US
Pharmacopeia (USP) <71>, should be performed to
ensure that any residual antibiotic does not interfere
with the sterility testing. Samples for sterility testing
should be obtained after final product manipula-
tion—ie, after all washing procedures—and, prefer-
ably, as the final formulation.
When sterility test results are not available at the
time of lot release, the following precautions and
testing should also be included:
• A sample should be taken for sterility testing
approximately 48-72 hours before the final har-
vest and an interim reading of this sterility test
should be taken at the time of product release.
• A rapid microbial detection test, such as a Gram
stain, should also be performed on the final
product before administration, and it should be
found negative before product administration.
• Written procedures to be followed in the event
that any of the sterility tests reveal that a con-
taminated product was administered to a
patient should be established. These procedures
should include physician and patient notifica-
tion, identification and sensitivity testing of the
contaminant, additional patient monitoring,
Table 2-1. Common Product Testing
Deficiencies
Test not performed
Inappropriate timing of sample collection
Inadequate description of test method
Unacceptable or unqualified test method
Inadequate or inappropriate specification

9. 26 Cellular Therapy: Principles, Methods, and Regulations
investigation to determine potential sources of
the contamination and corrective actions, and
reporting of the incident to the institutional
review board (IRB) and the FDA as an adverse
event within 15 calendar days.
Mycoplasma testing. If product manufacture
includes culture for more than 48 hours, the product
should usually also be tested for mycoplasma con-
tamination.
Identity Testing
For licensed products, the identity of the final prod-
uct must be verified by assays that will identify the
product for proper labeling and will distinguish the
product from other products being processed in the
same facility.44
Purity Testing
Product purity can be defined as freedom from
extraneous material, except that which is unavoid-
able in the manufacturing process.45
Testing for
purity includes an assay for pyrogenicity or endo-
toxin and may also include assays for unintended
cell populations (eg, distinguished by phenotypes),
residual proteins or peptides used to stimulate or
pulse cells, and materials used during manufacture,
such as cytokines, growth factors, antibodies, and
sera.
Potency Testing
Potency is the specific ability or capacity of the prod-
uct to effect a given result, as indicated by appropri-
ate laboratory tests or by adequately controlled
clinical data obtained through the administration of
the product in the manner intended.46
Ideally, a
potency assay is a quantitative bioassay that mea-
sures biological function of the clinical mechanism
of action. These assays may be by in vivo or in vitro
tests.47
If direct measure of a biological function is
not feasible, data must be provided to justify use of a
test or combination of tests to ensure product
potency. FDA guidance is available for potency test-
ing of cell and gene therapy products.48
Other Testing Concerns
Viability. For most cellular therapy products, the
cells are intended to be viable. In some cases, cellular
debris or cell clumping may affect the safety of the
product. The FDA recommends an acceptance crite-
ria of at least 70% viable cells. If this level cannot be
achieved, data should be submitted to support an
appropriate level.
Cell count. A specification for the number of
cells should also be established. In many cases, the
total cell number defines the dose. In other cases, the
dose may be defined by the number of cells of a spe-
cific cell type within the total cell population.
General safety testing. Cellular therapy products
are exempt from general safety testing.49
Stability
The characteristics of the final cellular therapy prod-
uct should remain within the specification limits
until the product is administered to the patient. Sta-
bility testing ensures that products retain consistent
safety, purity, and potency through the expiration
date of the product. Documentation of stability test-
ing should increase as product development pro-
ceeds. Stability testing in early phases of clinical
trials should show that the product is sufficiently
stable during intervals of holding and shipping and
for the period required by the study (for cryopre-
served products). During later phases, stability test-
ing should be expanded to develop an expiration
dating period for the product. By the end of Phase
III, validation studies, including those using condi-
tions that stress the system, should be completed.50,51
During later development, or after licensure, it may
be useful to develop formal stability protocols that
are reviewed by the FDA. These protocols may then
be a basis for ongoing or recurring stability testing.
Results of stability studies, and the protocols used
for the studies, should be provided in IND and BLA
submissions.51
The protocols should include analy-
ses for product potency, product integrity, and ste-
rility. In-process stability testing should be
performed as appropriate (eg, prefreeze and post-
thaw testing of cryopreserved cells used in the man-
ufacturing process).
Final product stability testing should demon-
strate that product integrity, potency, and sterility
have been maintained under the proposed shipping
conditions, in addition to having been maintained
under holding conditions. Further information
regarding stability testing and protocols is available
in ICH guidance documents.52-55

10. Chapter 2: FDA Perspective 27
Later-Phase Development and Marketing
As the field of cellular therapy has continued to
develop, we have seen more and more products
entering late-phase clinical development and licen-
sure. We thought it would be of general interest to
provide a snapshot of product approvals issued by
the Office of Cellular, Tissue and Gene Therapies
(OCTGT) at the time this chapter was written (Table
2-2).
We believe that this list will continue to grow
steadily. As more manufacturers move into later
development and prepare for licensure, additional
considerations related to product manufacturing
will arise. Ideally, they will involve a more robust
implementation of cGMPs as well as greater under-
standing and control of product manufacturing.
This section discusses a number of product manu-
facturing considerations for products in late-phase
development and in preparation for licensure.
Validation in Manufacturing Cellular Therapy
Products
All of the facilities, equipment, processes, systems,
and methods used in the manufacture of cellular
therapies should be shown to perform as expected.
Validation of these parameters is required for licen-
sure; the requirement for validation is set forth in
the cGMP regulations for finished pharmaceuticals.6
Validation is a documented program that provides a
high degree of assurance that a specific process,
method, or system will consistently produce a result
meeting predetermined acceptance criteria. Valida-
tion provides documented evidence, acquired across
the full range of operating conditions, that a process,
method, or system does what it is supposed to do. It
takes into account operating differences, such as
changes in operator, conditions, materials, or oper-
ating times, or other changes that might impact per-
formance. Experience during development can often
help to define those changes that are most likely to
affect the product and thus can inform the design of
the validation studies. More information on process
validation can be found in Guidance for Industry:
Process Validation: General Principles and Prac-
tices.56
An independent element of process validation is
the aseptic process validation. The basis for aseptic
process validation is in the cGMP regulations in 21
CFR 211, and specific recommendations can be
found in the Guidance for Industry: Sterile Drug
Products Produced by Aseptic Processing—Current
Good Manufacturing Practice.57
Aseptic processing
is typically validated through process simulation or
Table 2-2. Product Approvals Issued by the OCTGT
Product Manufacturer Year
Carticel (autologous cultured chondrocytes) Genzyme BioSurgery 1997
Provenge® (sipuleucel-T) Dendreon Corporation 2010
Laviv™ (azficel-T) Fibrocell Technologies, Inc. 2011
Hemacord® [HPC(CB)] New York Blood Center 2011
Gintuit™ (allogeneic cultured keratinocytes and
fibroblasts in bovine collagen)
Organogenesis, Inc. 2012
Ducord [HPC(CB)] Duke University School of Medicine 2012
HPC(CB)* Clinimmune Labs, University of Colorado Cord
Blood Bank
2012
Allocord [HPC(CB)] SSM Cardinal Glennon Children’s Medical Center 2013
HPC(CB)* LifeSouth Community Blood Centers, Inc. 2013
Bloodworks 2016
*The manufacturer chose not to give these products a proprietary name.

11. 28 Cellular Therapy: Principles, Methods, and Regulations
media fill studies. Aseptic process validation (APV)
is usually done by performing all of the manufactur-
ing steps using a microbial growth medium in lieu of
the product. During a successful validation, the
growth medium used as surrogate product must
remain sterile while an accompanying growth pro-
motion test shows that the medium is capable of
supporting the growth of selected organisms found
in the facility.
Test Method Validation
The purpose of test method validation is to demon-
strate that a given assay is suitable for its intended
purpose.58
Similar to manufacturing process valida-
tion, test method validation begins with a prospec-
tive validation plan that is approved by the quality
unit before execution.
Two important considerations for the design of
the validation protocol are choosing appropriate val-
idation parameters and setting appropriate accep-
tance criteria. Validation parameters should be
chosen to demonstrate that the assay method is
appropriate for measuring a given attribute in a reli-
able and reproducible manner. Acceptance criteria
are typically based on data generated from qualifica-
tion studies and past manufacturing experience, and
they will depend on the type of assay and validation
parameters. Considerations for acceptance criteria
could include sample size to allow for meaningful
statistical analysis, replicates to be measured, maxi-
mum allowable variance (eg, % CV), minimum cut-
off for correlation coefficient, and others. The
validation report is separate from the validation
plan, and it generally includes information regard-
ing validation parameters, acceptance criteria, sam-
ple size, raw data and data analysis, conclusion (pass
or fail), and deviation information if applicable. If a
method fails validation, an assignable cause for the
failure must be investigated and resolved, and the
method must be revalidated.
Compendial methods or accepted reference
methods, if unmodified, do not have to be separately
validated [21 CFR 211.194(a)(2)]. However, the
“suitability of all testing methods used shall be veri-
fied under actual conditions of use.”59
This is com-
monly known as compendial or reference method
qualification. Examples include USP general chap-
ters such as USP <71> (Sterility) or USP <85>
(Endotoxin), but could also include reference meth-
ods from an approved marketing application,
including those for FDA-cleared or -approved test-
ing kits. More information on analytical methods
validation is available in FDA Guidance.60
cGMP Compliance and Prelicense Inspections
cGMP requirements for biological products are
defined in the regulations. These include:
• For drugs and biological products, the require-
ments are described in 21 CFR part 210/211.
• General biological product standards are
described in 21 CFR part 610.
• For HCT/Ps, cGTP requirements apply; they are
found in 21 CFR part 1271.
While this aspect of cGMPs is beyond the scope of
this chapter, we note for completeness that there are
also cGMP requirements described for medical
devices, collectively referred to as Quality System
Regulations (QSR), described in 21 CFR Part 820. In
addition, there are cGMP requirements for “single-
entity” and “copackaged” combination products
that have recently been finalized in 21 CFR Part 4.
A prelicense inspection (PLI) is a normal part of
the BLA review process, and it serves as one mecha-
nism to ensure that manufacturing establishments
and processes meet the appropriate requirements
and comply with cGMP regulations. PLIs are
announced in advance, and they ideally occur
around mid-cycle of the BLA review, when the
establishment is in operation and product manufac-
turing is at or near capacity. Major objectives of the
PLI include the following:
• Verify the firm’s compliance with applicable
laws and regulations.
• Evaluate the consistency of manufacturing oper-
ations as supported by process validation.
• Interview personnel and review/verify docu-
ments and records.
• Visual observation of the actual manufacturing
processes.
• Bring to the attention of the manufacturer any
significant deficiency observed during the PLI.
• Collect evidence (exhibits) to support observa-
tions.
• Document objectionable conditions by issuing
FDA Form 483.
There are many resources that can help manufac-
turers comply with cGMPs and prepare for a PLI.
One resource that manufacturers may not be aware

12. Chapter 2: FDA Perspective 29
of is the Center for Biologics Evaluation and
Research Compliance Program Manual, Chapter 45
(CP 7345.848), which is available on the FDA web-
site.61
This manual provides general information on
what FDA inspectors look for to ensure compliance
with cGMPs when they inspect a facility. In addition
to the compliance manual, there are many guidance
documents that could help a manufacturer prepare
for FDA inspection. For example, many resources
exist with more detailed information about clean
rooms and clean room classifications.62-65
Guidance
for Industry: Q7A Good Manufacturing Practice
Guidance for Active Pharmaceutical Ingredients66
contains many principles that are also helpful in
complying with cGMPs, covering a broad range of
cGMP topics. It is particularly useful in helping to
define the responsibilities of the quality unit, in
explaining what should be in Master Production
Instructions and in a Master Batch Record, describ-
ing general expectations for facilities and equip-
ment, and describing a general approach to
validation and validation documentation.
Conclusion
This chapter has summarized current regulatory
advice, regulations, and practices that pertain to the
manufacture of cellular therapy products. However,
as cellular therapy products are rapidly evolving, so
must the regulation of these products. The reader is
cautioned to investigate appropriate FDA websites to
determine pertinent rules, guidance documents, and
current practices.
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