Recommendations for Microbial Vectors Used for Gene Therapy: Guidance for Industry

Administrative guidance, not enacted legislation. This captured publisher edition preserves the listed document and attachments; later developments are not independently consolidated.

Recommendations for Microbial
Vectors used for Gene Therapy
Guidance for Industry
Additional copies of this guidance are available from the Office of Communication, Outreach
and Development (OCOD), 10903 New Hampshire Ave., Bldg. 71, Rm. 3128, Silver Spring,
MD 20993-0002, or by calling 1-800-835-4709 or 240-402-8010, or email ocod@fda.hhs.gov, or
from the Internet at
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guida
nces/default.htm.
For questions on the content of this guidance, contact OCOD at the phone numbers or email
address listed above.
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Biologics Evaluation and Research
September 2016

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Table of Contents
I. 	INTRODUCTION............................................................................................................. 1
II. 	BACKGROUND ............................................................................................................... 1
III. 	PRODUCT MANUFACTURING AND CHARACTERIZATION ............................. 2
A. 	Product Manufacturing – Components .............................................................. 2
1. 	MVGT Seed Stock ............................................................................................ 2
2. 	MVGT Banking System ................................................................................... 4
3. 	Reagents ............................................................................................................ 6
B. 	Product Manufacturing – Procedures ................................................................ 8
1. 	MVGT Production/Purification ........................................................................ 8
2. 	Inactivated/Killed Cells .................................................................................... 9
3. 	Final Harvest ..................................................................................................... 9
4. 	Final Formulation.............................................................................................. 9
C. 	Product Testing ................................................................................................... 10
1. 	Microbial Purity and Sterility Testing for MVGT Products ........................... 11
2. 	Identity ............................................................................................................ 11
3. 	Purity ............................................................................................................... 12
4. 	Potency............................................................................................................ 13
5. 	Other ............................................................................................................... 13
D. 	Drug Substance ................................................................................................... 14
E. 	Drug Product ....................................................................................................... 14
F. 	Stability Testing .................................................................................................. 14
1. 	In-process Stability Testing ............................................................................ 15
2. 	Final Drug Product Stability Testing .............................................................. 15
G. 	Additional Testing............................................................................................... 15
1. 	Antibiotic Sensitivity Tests ............................................................................. 15
2. 	Container/Closure ........................................................................................... 15
3. 	Tests for Residual Moisture Content .............................................................. 16
4. 	Environmental Assessment ............................................................................. 16
5. 	Qualification of the Manufacturing Process ................................................... 16
IV. 	PRECLINICAL STUDIES ............................................................................................ 16
A. 	Animal Species and Models................................................................................ 17
B. 	Safety Evaluation ................................................................................................ 17
C. 	Attenuation Evaluation ...................................................................................... 17
D. 	Biodistribution/ Shedding .................................................................................. 18
E. 	Antibiotic Use ...................................................................................................... 18
V. 	CLINICAL STUDIES .................................................................................................... 18
A. 	General Considerations ...................................................................................... 18
B. 	Prior Human Experience ................................................................................... 19
C. 	Patient Population............................................................................................... 20
D. 	Starting Dose, Dose Escalation, and Dosing Schedule ..................................... 20

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E. 	Treatment Modifications .................................................................................... 21
F. 	Monitoring ........................................................................................................... 21
VI. 	REFERENCES ................................................................................................................ 22
APPENDIX .................................................................................................................................. 24

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Recommendations for Microbial Vectors used for Gene Therapy
Guidance for Industry
This guidance represents the current thinking of the Food and Drug Administration (FDA or
Agency) on this topic. It does not establish any rights for any person and is not binding on FDA
or the public. You can use an alternative approach if it satisfies the requirements of the
applicable statutes and regulations. To discuss an alternative approach, contact the FDA staff
responsible for this guidance as listed on the title page.
I. 	INTRODUCTION
We, FDA, are providing you, investigational new drug application (IND) sponsors, with
recommendations concerning IND submissions for microbial vectors used for gene therapy
(MVGTs) in early-phase clinical trials. MVGTs meet the definition of “biological product” in
section 351(i) of the Public Health Service (PHS) Act (42 U.S.C. 262), when such products are
applicable to the prevention, treatment, or cure of a disease or condition of human beings. This
guidance focuses on the chemistry, manufacturing, and control (CMC) information that you
should submit in an IND for MVGTs and provides an overview of preclinical and clinical
considerations for these products.
This guidance finalizes the draft guidance of the same title dated October 2015 and supplements
the guidance entitled, “Guidance for FDA Reviewers and Sponsors: Content and Review of
Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy
Investigational New Drug Applications (INDs),” dated April 2008 (Ref. 1).
FDA’s guidance documents, including this guidance, do not establish legally enforceable
responsibilities. Instead, guidances describe the FDA’s current thinking on a topic and should be
viewed only as recommendations, unless specific regulatory or statutory requirements are cited.
The use of the word should in FDA’s guidances means that something is suggested or
recommended, but not required.
II. 	BACKGROUND
MVGTs include bacterial vectors such as Salmonella, Listeria, or E. coli genetically modified to
express human tumor antigens, cytokines, growth factors, enzymes, therapeutic proteins, or
nucleotides. For example, the bacterial vectors may be modified to express human tumor
antigens, cytokines, growth factors, enzymes, therapeutic proteins, or nucleotides. MVGTs may
also be generated by the modification (deletion, truncation, or point mutation) of chromosomal
or episomal genes and by the insertion of foreign genetic material into the chromosome, or into
naturally occurring episomes; or by the introduction of one or more plasmids. In addition to the

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genetic modifications that may alter an MVGT’s therapeutic profile, MVGTs may also be
modified to alter their growth characteristics. For example, MVGTs may be live, dead or
replication restricted as a result of specific biochemical requirements or genetic modification.
Gene therapies are defined in the FDA guidance document entitled, “Guidance for Industry:
Gene Therapy Clinical Trials – Observing Subjects for Delayed Adverse Events” dated
November 2006 as “[p]roducts that mediate their effects by transcription and/or translation of
transferred genetic material and/or by integrating into the host genome and that are administered
as nucleic acids, viruses, or genetically engineered microorganisms. The products may be used
to modify cells in vivo or transferred to cells ex vivo prior to administration to the recipient.”
(Ref. 2)
III. 	PRODUCT MANUFACTURING AND CHARACTERIZATION
Under § 312.23(a)(7)(i) (21 CFR 312.23(a)(7)(i)), you must, in your IND, as appropriate for the
particular investigations covered by the IND, submit a section describing the chemistry,
manufacturing and control of the drug substance and the drug product. Under
§ 312.23(a)(7)(iv)(b), you must also include in your submission a list of all components used in
the manufacturing of your product. In addition, under § 312.23(a)(11), if requested by FDA, you
are required to submit any other relevant information needed for review of the application.
In accordance with these regulations, and as described in the following sections, we recommend
that you provide a detailed description of the complete MVGT manufacturing process in your
IND submission. You should also describe all of the components used during the manufacture of
the MVGT product, such as microbial vector, microbial cell bank systems, plasmid(s)/phage(s)
harbored by the microbial cells, reagents used in the manufacture, and any excipients. In
addition, you should describe all procedures used during the manufacturing process. This
information will allow FDA to assess1 the identity, quality, purity, and potency of your product.
A. 	Product Manufacturing – Components
You should provide the following information about your microbial product in the IND:
1. 	MVGT Seed Stock
A description of the history and detailed derivation of the MVGT
including:
1 As a general matter, Office of Cellular, Tissue, and Gene Therapies (OCTGT) reviews clinical studies for MVGTs
and related products such as oncolytic bacteria. The Office of Vaccine Research and Review (OVRR) in FDA’s
Center for Biologics Evaluation and Research (CBER) reviews the clinical studies for live biotherapeutic products
(LBPs), as described in the FDA guidance entitled, “Early Clinical Trials with Live Biotherapeutic Products:
Chemistry, Manufacturing, and Control Information; Guidance for Industry,” dated June 2016.

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a. 	Selection of the MVGT clone
You should include a description of the history, source, and
derivation of the MVGT. The MVGT seed stock should be
derived from a single, well-isolated colony. You should ensure
that antibiotic selection markers, such as beta lactamase, are not
introduced into the MVGT for the purpose of propagation of the
MVGT. Attenuation of MVGT should be through redundant and
robust means, which may be achieved by mutation at more than
one site on the MVGT genome and by more than a point mutation.
b. 	Physical properties and growth characteristics
The IND submission should describe the physical properties of the
organism, such as shape under a microscope, colony morphology,
and staining properties including their ability to take up gram stain
or acid fast staining (staining should be done on a bacterial culture
in a log-phase growth). Growth characteristics include dividing
time under optimal growth conditions, and any specific
characteristics of the organism used to evaluate its growth such as
an evaluation of bacterial motility.
c. 	Defining the genetic make-up of MVGT
You should provide information regarding the MVGT’s
chromosomal genetic marker(s) and distinguishing genomic
restriction pattern(s). If the MVGT contains a naturally occurring
plasmid(s), you should include a description of the plasmid(s), and
a method to assay for the presence of the plasmid(s) in the Master
Cell Bank (MCB) and final product. You should describe the
MVGT’s selection markers/selection method.
d. 	Growth conditions
The IND should include a description of the growth media and
growth conditions, such as media composition, growth
supplements, antibiotics, growth temperature requirements, and
aeration/bioreactor/anaerobic requirements.
e. 	Episomal/Plasmid based foreign gene inserts
If the MVGT contains a plasmid vector, you should describe the
method used to introduce the plasmid vector into the parental cells
to establish the MVGT (e.g., use of an intermediate bacterial
“transfer” strain, and/or the use of techniques such as
electroporation, transformation, conjugation, etc.). In addition,

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you should provide the following information on the derivation of
the MVGT clone in your IND package:
• 	A diagram of the plasmid/phage that identifies the gene
insert, regulatory elements such as the promoter, ribosomal
binding sites/Shine-Dalgarno sequences, and transcriptional
terminators, and pertinent restriction endonuclease sites,
such as multiple cloning sites (MCS);
• 	Information on any codon optimizations that were
performed; and
• 	Details of any signature sequences/linker sequences
introduced into the plasmid/phage vector.
f. 	Chromosomally inserted foreign genes
You should provide a detailed description of the methods used to
insert foreign genes into the chromosome. We recommend that
you provide a flow chart of the derivation of the final clone
containing a chromosomally integrated gene insert, and describe in
the text any intermediate clones that were generated in the process
of derivation of the final clone.
2. 	MVGT Banking System
The IND should contain information, as described below, on the microbial
cell bank system (i.e., master cell bank, working cell bank).
a. 	Master cell bank (MCB)
Your IND should include information regarding MCB
characterization, including testing to adequately establish the
safety, identity, purity, and stability of the cells. The information
should address the following:
• 	Purity of the strain: We recommend that the microbial
MCB be derived from a single isolated colony (sequentially
isolated after at least two rounds of colony selection). All
further amplification of the bacterial products should be
derived from this MCB without further single colony
selection.
• 	Microbial purity2: For an MVGT that is a live bacterial
product, if conventional application of sterility testing as
2 For the purposes of this guidance, the term “microbial purity” is defined as freedom from microbes other than
MVGT. Microbial purity may be evaluated by a monoculture assay (an assay designed to demonstrate the presence
of a single strain of microbe in the test sample).

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outlined in § 610.12 (21 CFR 610.12) is not feasible
because the live vector strain will show growth, testing for
microbial purity by monoculture assays may be appropriate
(see section III.C.1 microbial purity testing). The test
method should be capable of detecting slow-growing
microbial contaminants.
• 	Identity of the bacterial cells (and plasmids/phages if
applicable): Your development program should include
tests to distinguish the specified cells through physical or
chemical characteristics (i.e., phenotype, genotype,
deoxyribonucleic acid (DNA) sequence, or other markers),
selection marker(s), and antibiotic resistance.
• 	Sequencing of genetically modified plasmids 50kb or less:
You should provide the full sequence, along with a
description of the sequencing method for all genetically
modified plasmids introduced into the microbe to derive the
MVGT MCB.
• 	Annotate sequence information: You should summarize
the plasmid sequence information with appropriate
annotations, identifying all open reading frames (ORF),
whether expected or unexpected, and encoded genes and
their regulatory elements. You should indicate whether
there is sequence alignment between the MVGT derived
plasmid/phage sequences and sequences identified by a
search in a relevant current database.
• 	Sequencing of chromosomally inserted foreign genes: We
recommend that you perform and provide sequence
analysis of the gene insert and at least 0.5kb of flanking
regions, and provide a diagram of the insert and its
regulatory elements and any other regions of the genome
that are modified.
• 	Activity: You should assay for product specific
characteristics such as expression of the introduced gene,
duration of gene expression, localization of the expressed
gene product (such as the percentage of gene products that
are either secreted/membrane bound or intracellular), and
growth rate/dividing time (in vivo and in vitro).
• 	Antibiotic sensitivity: Antibiotic sensitivity tests should
cover a panel of antibiotics, including at least two first line
antibiotics and two second line antibiotics for that class of
microbial agent.
• 	Characterization of attenuating mutation: You should
describe the size and stability of the attenuation. The
attenuation site and at least 0.5kb of sequence on either side
of the attenuation should be sequenced.

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• 	Bacterial growth: You should describe bacterial growth
conditions and include documentation of all media and
reagents/components used during production, with copies
of relevant certificates of analysis (COA). You should also
include timing and duration of any induction conditions
used to drive gene expression, such as change in
temperature, pH, or other growth conditions or addition of
inducing agents.
• 	Cryopreservation, storage, and recovery: The IND should
include details on cryopreservation, storage, and recovery
of the MCB, including information pertaining to cell
density, number of vials frozen, storage temperature, and
cell bank location.
• 	Genetic and phenotypic stability: A description of the
genetic and phenotypic stability of the MCB after multiple
passages, as well as viability of cells after cryopreservation,
should be included in the IND.
• 	Bacteriophage testing: The MCB should be qualified by
culture-based tests under conditions conducive for
production of the bacteriophage lytic cycle to assay for the
presence of bacteriophages in the bacterial product. The
assay(s) should include positive and negative controls to
evaluate test sensitivity. When available, positive controls
for phage testing should include well-characterized
bacteriophages specific to the MVGT.
b. 	Working cell bank (WCB)
The amount of information needed to document characterization of
the WCB is usually less extensive than that needed to document
characterization of the MCB. If there is a two tiered cell bank
system in place, we recommend that you test the WCB for:
• 	Purity
• 	Limited identity testing (e.g., target specific polymerase
chain reaction (PCR), for the presence of deletions or
insertions)
• 	Test for plasmids (in case of MVGT products containing
natural or introduced plasmids)
• 	Antibiotic sensitivity profile
• 	Viability
• 	Cell number
• 	Stability
3. 	Reagents

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You must list in your IND any reagents used in manufacturing the product
(§ 312.23(a)(7)(iv)(b)). For the purpose of this guidance, reagents are
those components that are essential for bacterial growth, selection,
purification, or other critical manufacturing steps, but are not intended to
be part of the final product. Reagents can affect the safety, potency, and
purity of the final product, especially by introducing adventitious agents.
Examples of reagents include media and media components, antibiotics,
and gene expression inducing agents. In addition to the list of reagents,
the following information should be submitted in your IND:
a. 	Tabulation of reagents used in manufacture
For each reagent used in the manufacture of the MVGT, the
following information should be submitted:
• 	Concentration of the reagent.
• 	The manufacturing step at which it is used.
• 	Vendor/supplier information, including the product
identifying number and lot number.
• 	Information on the source of the reagent (i.e., whether it is
synthetic, recombinant, or plant, animal, or human-derived)
(Ref. 1).
• 	Reagent quality: We recommend that FDA-approved
products or clinical grade reagents be used whenever they
are available.
b. 	Qualification of reagents used in manufacture
The COA provided by the vendor of the reagent should be
reviewed for each lot. Additional testing may be needed to help
ensure the safety and quality of the reagent for use in the
manufacture of your MVGT. We recommend that you establish a
qualification program that includes safety testing (purity,
endotoxin, mycoplasma, and adventitious agents), functional
analysis, and assays to demonstrate the absence of potentially
harmful substances (e.g., residual solvent testing). We believe that
the appropriate extent of testing will depend on how the specific
reagent is used in the manufacturing process.

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c. 	Determination of removal of reagents from drug product
You should test the drug product for residual manufacturing
reagents with known or potential toxicities. The IND should
describe the test procedures used to detect residual levels of these
reagents in the final product. We recommend that you determine
whether a qualification study is sufficient to document their
removal, or whether lot release testing is appropriate prior to
initiation of clinical trials.
d. 	Other concerns
We recommend that you not use antibiotics for bacterial selection.
Specifically, beta-lactam antibiotics such as penicillin should not
be used during the manufacturing of a therapeutic product for
humans. If antibiotics are used, we recommend that you quantitate
the amount of residual antibiotics present in the final product and
describe precautions to prevent hypersensitivity reactions.
B. 	Product Manufacturing – Procedures
You should include in the IND a detailed description of all procedures used during the
derivation, production, and purification of an MVGT product. We believe that inclusion
of a schematic of the production and purification process, and in-process and final
product testing, will help to provide this information more clearly.
1. 	MVGT Production/Purification
You should describe the procedures used during the production of the
MVGT, including:
• 	Culturing procedures, media components (including animal
derived components) used, and antibiotics used during MVGT
propagation.
• 	Cell growth conditions, cell density, method of monitoring cell
growth, pH adjustments, bioreactor specifications, etc. Include a
description of culture systems (shake flasks, bioreactors, etc.), and
state whether the system is closed or open. Describe any
defrothing agents used during culture, cell harvest conditions (e.g.,
membrane filtration, centrifugation, etc.).
• 	Harvest conditions, including the volume, timing (duration of
culture) and optical density of the culture at the time of harvest.
• 	All purification steps, such as centrifugation, column purification,
and density gradients, listed in the order of conduct.
• 	Process timing and intermediate storage: We recommend that you
report the approximate time elapsed for each step from the end of

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cell growth to filtration/wash steps and final harvest. It is
important to know the time limit of each step in production to
determine what, if any, in-process testing to perform.
2. 	Inactivated/Killed Cells
If the final product is an inactivated/killed MVGT, you should describe the
method and conditions of cell inactivation/killing. The IND should also
include data to demonstrate that the bacteria are rendered replication
incompetent, but still maintain their desired characteristics after
inactivation. You should evaluate cell killing by sterility assays that
include media and growth conditions used in the manufacture of the
MVGT product and by sterility assays, as described under § 610.12.
3. 	Final Harvest
You should provide a detailed description of the final harvest. You should
describe whether the final MVGT harvest is concentrated prior to final
formulation, and if so, describe the wash conditions and buffers/media
used. If the MVGT is lyophilized prior to storage, describe the
lyophilization conditions and stabilizers used for lyophilization. If the
MVGT Drug Substance (DS) or Drug Product (DP) is cryopreserved, you
should include this information along with any stability studies initiated
(see section III.B.4 of this guidance). You should also describe the time
and conditions of storage (if any) between cell growth and final harvest
and between final harvest and lyophilization. If the manufacturing process
includes intermediate storage steps between final harvest and final
formulation, the storage conditions and the length of storage should be
described.
4. 	Final Formulation
You must describe the formulation of the final product as prescribed under
§ 312.23(a)(7)(iv)(a). In addition, the IND should describe whether
excipients such as growth factors, buffers and salt stabilizers are included
in the final formulation and state their source (see section III.A.3 of this
guidance). You should also identify the vendor and final concentration of
these excipients.
The MVGT cell density or concentration (live cells vs. killed cells) used in
the final product should be described. If the final product is delivered to
the clinical site frozen, you should include a description of how the
product will be shipped and data to show that the product can be thawed
with consistent results. If the final product is to be reconstituted, you
should describe the volume and composition of the reconstitution

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medium/or buffer and provide data to show bacterial viability when
reconstituted. The viability data should be from at least two different
product batches tested independently in triplicates.
C. 	Product Testing
Product testing for MVGTs includes microbiological tests to help ensure safety, and
assessments of product characteristics such as identity, purity (including endotoxin),
viability, and potency.
You should perform testing throughout the manufacturing process, including the
manufacture of MCB/WCB, to evaluate the manufacturing process itself and to ensure
the quality and consistency of the product. You should describe the specifications used
for intermediate acceptance criteria and final product release criteria. Specifications are
the quality standards (i.e., tests, analytical procedures, and acceptance criteria) that
confirm the quality of products and other materials used in the production of a product.
Acceptance criteria mean numerical limits, ranges, or other criteria for the tests
described. Specifications should be appropriate to the stage of product development,
because release criteria are generally refined and tightened as product development
progresses toward licensure (Ref. 1). Table-1 in the Appendix of this guidance provides
examples of suggested testing for MVGT products.
You must, under § 312.33(b)(7), provide an annual report to your IND that includes a
summary of any significant manufacturing or microbiological changes made during the
past year. Your annual report should contain manufacturing updates that include results
related to stability testing of MCB/WCB, and results of characterization testing and lot
release testing for lots manufactured during the reporting period. The results may be
submitted in a tabular form and should include the lot number or identifier, date of
manufacture, test used, test method, the sensitivity and specificity of test methods when
appropriate, release criteria, and test results. All assay methods should be adequately
sensitive, specific, and reproducible for the intended purpose.
Under § 312.23(a)(7)(i), you must provide a section describing the composition,
manufacture, and control of the drug substance and the drug product. Although in each
phase of the investigation, you are required to submit sufficient information to assure the
proper identification, quality, purity, and strength of the investigational drug, the amount
of information needed to make that assurance will vary with the phase of the
investigation, the proposed duration of the investigation, the dosage form, and the amount
of information otherwise available. As such, you must provide information in your IND
for MVGT products to assure their: (1) freedom from other contaminating organisms
(microbial purity and sterility); (2) identity; (3) purity (freedom from extraneous process
related material); and (4) potency. In accordance with these requirements, we
recommend the following testing:

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1. 	Microbial Purity and Sterility Testing for MVGT Products
• 	Live MVGT products should be assayed for freedom from causative
agents of other diseases or conditions. The bacterial growth medium,
growth conditions and amount of DP used to evaluate purity should be
described and justified.3 Tests of microbial purity and test
components (growth promoting medium, conditions) should be
validated to demonstrate that the test is capable of reliably and
consistently detecting the presence of viable contaminating
microorganisms. For examples of common bacterial agents for which
the assay(s) should be capable of detecting, please refer to the Centers
for Disease Control and Prevention (CDC) list of invasive bacterial
pathogens (Ref. 3), and specific pathogens described in the United
States Pharmacopeia, Chapter 62,4 as appropriate. We also
recommend evaluating for environmental agents isolated from the
manufacturing facility. For early-phase clinical trials, when a
dedicated live microbe manufacturing facility is not feasible, tests for
microbial purity should include tests for other microbial products
manufactured in the same manufacturing facility.
• 	For killed MVGT product or an MVGT product that is incapable of
host-free growth, such as bacteriophages or empty bacterial envelopes
(mini cells), you should perform sterility testing of the DP. Sterility
testing should include assays for both aerobic and anaerobic bacterial
contaminants and fungal contaminants. For these products, the MCB,
DS and in-process samples should also be tested to demonstrate
microbial purity.
2. 	Identity
You should test for identity with respect to the MCB and DP by assays
that will be specific for each product in a manner that will adequately
identify the MVGT (§ 312.23(a)(7)(i)) and distinguish it from any other
products being processed in the same manufacturing facility. For the final
product, identity testing is important to ensure that the contents of the vial
are labeled appropriately.
3 Additional information can be found in the US Pharmacopeia (39 USP <61>) Microbiological Examination of
Non-sterile Products: Microbial Enumeration Tests.
4 The specific pathogens and detection methods described under US Pharmacopeia (39 USP <62>) may not be
appropriate for all MVGT products. We recommend that you discuss with FDA the appropriateness of test methods
and test organisms during the Pre-IND meeting.

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For bacterial strain identification, we recommend at least two
complementary methods5 of identity assays, such as the following tests:
• 	Gram staining.
• 	Colony morphology/cell culture nutrient dependent colony
identification.
• 	DNA based PCR assays/Restriction Fragment Linked
Polymorphism (RFLP) of the chromosomal DNA/16s ribonucleic
acid (RNA) based analysis/analysis of house-keeping genes by
sequencing.
• 	The host genotypic and phenotypic properties should be assayed
using DNA based assays, biochemical and/or phenotypic assays to
detect the presence of genetic markers and or deletions.
• 	PCR or similar sensitive method to identify the number, size, and
sequence of native and introduced plasmids (if any).
3. 	Purity
Product purity is defined as freedom from extraneous material, except that
which is unavoidable in the manufacturing process described in the
approved biologics license application (BLA) (21 CFR 600.3(r)). Product
purity must be described in the IND (§ 312.23(a)(7)(i)). Purity testing for
MVGT products should include assays for pyrogenicity/endotoxin and a
test for monoculture to demonstrate that the product is free from
unintended bacterial and fungal organisms. Tests for purity should be
included as a part of seed stock, MCB and WCB qualification tests, and as
a release test for the final DP.
Suggested purity tests specific to MVGT products include the following:
• 	Residual host cellular protein/DNA content (for
bacteriophage/mini cells).
• 	Strain purity: Presence of colonies of a single strain of bacteria
biochemically identifiable as similar to each other. If the final
product is a mixture of more than one type of MVGT, each MVGT
component should be tested for purity.
• 	A minimum of 10 random colonies (number based on an estimate
of 100 isolated colonies per plate) should be evaluated to confirm
purity of the final product.
• 	Endotoxin assay with a specification of < 5 EU/kg/dose is required
for all parenterally administered MVGT products.
5 Depending on the MVGT product, additional identity tests may be required. We recommend that you discuss your
specific product and the types of identity tests you propose to conduct during your Pre-IND meeting with FDA.

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4. 	Potency
You should describe and justify the assay(s) you will use to measure
potency. We recommend the use of a quantitative assay(s) to measure
potency. An alternative approach using a matrix of assays, including
quantitative and qualitative measures of potency, may be considered (Ref.
4). Potency assay should be included as a part of the MCB, qualification
and DP release tests. For Phase 1/Phase 2 studies, an assay to quantify
transgene expression, along with assays to quantitate the amount of live
and dead organisms, may be acceptable as a surrogate measurement of
potency. For a Phase 3 study, you should include in vivo or in vitro
assay(s) that measure an appropriate biological activity of the MVGT.
Note that potency assays must be validated prior to licensure
(21 CFR 211.165(e)).
5. 	Other
a. 	Viability
For live bacterial products, we recommend that you establish
minimum release criteria for viability and a live to dead bacterial
cell ratio. We recommend that the DP specification be set at
> 60% live/viable cells6 (Ref. 5). Viability should be tested at the
MCB, WCB, DS and DP stages.
b. 	Cell number/dose
If the final MVGT product is administered parenterally, we
recommend that you establish a dose level based on the number of
live and dead bacteria in the final product considering dead
bacteria can also contribute to antigen-specific and vector-specific
immune responses.
c. 	Tests for aggregates
The presence and size of microbial aggregates is a potential safety
concern in parenterally administered MVGT products. The final
product should be tested for the presence of aggregates at the time
of manufacture and at periodic intervals as a part of the stability
protocol. A lot release specification with acceptance limits should
be set for the presence and size of particulates in the DP.
6 If your specific MVGT product is unable to meet this specification, you are encouraged to discuss the issue with
FDA during Pre-IND discussions.

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D. 	Drug Substance
Drug substance is an active ingredient that is intended to furnish pharmacological
activity. An MVGT that is processed further (e.g., heat killed, lyophilized, encapsulated,
made into a tablet, etc.) to obtain the final DP, should be assessed for purity, the presence
of plasmids (if applicable), viability, cell number etc., and this information should be
included in the IND, along with test specifications, acceptance limits and a description of
test methods. Some of the other testing parameters to be considered for inclusion in the
IND are described below in section III.F.2 of this guidance. This information may be
obtained by testing the DS, or if the final product is manufactured without additional
manipulations other than aliquoting, the DP (see Table-1 in the Appendix of this
guidance for a list of suggested test parameters for DS and DP).
E. 	Drug Product
The drug product is the final formulated product used for patient administration. In the
case of an MVGT product, this would include, for example, the vialed microbial vector.
Drug product release criteria testing should be performed on each lot of product
manufactured. We recommend that you use a table format to provide all of your
proposed specifications (tests for safety, purity, potency, and identity as described in
section III.C of this guidance).
F. 	Stability Testing
As provided in § 312.23(a)(7)(ii), you must conduct stability testing in all phases of the
IND to demonstrate that the product is within acceptable chemical and physical limits for
the planned duration of the proposed clinical investigation. Stability testing is performed
during early phases of the clinical trial to establish that the product is sufficiently stable
for the time period required by the study. Data supporting a final formulation and dating
period will be necessary for licensure.
You should submit a stability protocol and data for both in-process material and the final
MVGT product. A proposed stability protocol should include a measure of product
identity, purity, quality, and potency. For each test conducted you should describe the
test method, sampling time points (there should be a zero-time point), testing
temperature, and other appropriate information, including your justification of the assays
used to indicate product stability, measuring these parameters for the duration of storage
required by the clinical protocol. For further information, refer to International
Conference on Harmonisation (ICH) Q5C Guideline for Industry: Quality of
Biotechnological Products: Stability Testing of Biotechnological/Biological Products
(Ref. 6).

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1. 	In-process Stability Testing
If the MVGT is cryopreserved or lyophilized, your stability protocol
should be designed to ensure that the product is stable during the period of
cryopreservation/lyophilization, and to measure the parameters described
above, as appropriate. In order to assess the biological function of the
MVGT, we recommend that evaluation of the duration of lag phase for
post-storage revival be conducted in rich medium under physiological
conditions.
Testing for live and dead bacteria should be part of your stability protocol.
We expect that the appropriate acceptance criteria will be different for
each bacterial species and we recommend that you establish specifications
based on manufacturing and testing experience with your MVGT product.
2. 	Final Drug Product Stability Testing
You should include any data that demonstrate that the product is stable
between the time of product formulation and patient infusion to aid in
establishing an expiration-dating period. We recommend that you conduct
the testing at the appropriate temperatures and at time points consistent
with predicted storage times. If the product is shipped from the
manufacturing site to the clinical site, describe the time and shipping
conditions (i.e., packaging, temperature). Your stability protocol should
be adequate to demonstrate that product integrity, purity, recoverable
sample volume, product aggregation profile, and potency are maintained
under the proposed storage and shipping conditions. We recommend that
you initiate and complete validation studies using conditions that stress the
system by Phase 3.
G. 	Additional Testing
1. 	Antibiotic Sensitivity Tests
Antibiotic sensitivity testing should be performed on the seed stock, MCB
and DP, and the results included in the IND, when available. Antibiotic
sensitivity tests should contain at least two first line therapy and two
second line therapy antibiotics normally used to treat infections with the
microbial agent(s) for which the MVGT was derived.
2. 	Container/Closure
You should describe the types of containers and closures used and ensure
that the containers and closures are compatible with the product (Ref. 7).

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3. 	Tests for Residual Moisture Content
If using lyophilized/dried bacterial products, please refer to FDA’s
previous recommendations regarding tests for residual moisture content
(Ref. 8).
4. 	Environmental Assessment
During clinical development of an MVGT product under an IND, you
must submit either a claim for categorical exclusion under 21 CFR 25.31,
or an environmental assessment under 21 CFR 25.40
(§ 312.23(a)(7)(iv)(e)). Please refer to FDA’s guidance for industry
entitled, “Determining the Need for and Content of Environmental
Assessments for Gene Therapies, Vectored Vaccines, and Related
Recombinant Viral or Microbial Products” dated March 2015, for
additional information (Ref. 9).
5. 	Qualification of the Manufacturing Process
The manufacturing process for MVGT products entails the use of reagents
and source materials of differing complexity, variability, and risk for
introduction of adventitious agents. You should use the appropriate
methods, facilities, and manufacturing controls to ensure that the MVGT
manufactured for Phase 1 clinical studies meets appropriate standards of
safety, identity, quality, and purity. MVGT products manufactured for
Phase 2 and Phase 3 clinical studies, and for marketing under a BLA, must
comply with 21 CFR Parts 210 and 211 requirements. FDA has
previously issued additional guidance regarding cGMP recommendations
for Phase 1 clinical trials (Ref. 10).
IV. 	PRECLINICAL STUDIES
This section of the guidance describes the considerations for evaluating the activity, safety, and
biodistribution of MVGT products in preclinical studies. Preclinical studies are conducted to
support the scientific rationale for the use of a particular MVGT product in a target patient
population. Adequate information about pharmacological and toxicological studies of the
MVGT on the basis of which the sponsor has concluded that it is reasonably safe to conduct the
proposed clinical trial, must be submitted in the IND (§ 312.23(a)(8)). FDA has previously
issued a guidance document entitled, “Preclinical Assessment of Investigational Cellular and
Gene Therapy Products” dated November 2013 (Preclinical Assessment Guidance) that provides
comprehensive recommendations regarding the selection of appropriate animal species and
animal models of disease, as well as the overall design of preclinical proof-of-concept and
toxicology studies for investigational products, including MVGT products (Ref. 11). Some
additional considerations of preclinical study design for MVGTs are highlighted below.

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A. 	Animal Species and Models
Considerations for selection of biologically relevant animal species and models include
the permissiveness/susceptibility of the species to the MVGT vector; the pharmacological
response to the MVGT and to the expressed transgene; and the comparative
physiology/pathophysiology of the animal species/model to the targeted clinical
population. When feasible, we recommend the use of animal models of disease to
characterize a dose response/biological activity relationship of the MVGT product.
These animal models should also be considered when designing studies to evaluate the
toxicology and biodistribution profile of the MVGT product, as disease status may affect
the safety data generated from these studies, as well as provide a more relevant estimate
of the risk-benefit assessment of the MVGT product. Refer to sections III.A and V.B of
the Preclinical Assessment Guidance (Ref. 11) for a more detailed discussion of this
topic.
B. 	Safety Evaluation
The overall objectives of the preclinical safety studies are to identify, characterize, and
quantify any potential local, systemic, acute, or chronic toxicity of the MVGT product.
The data generated from these studies will help guide the selection of the starting dose
level, dose-escalation scheme, dosing schedule, monitoring parameters, and other
elements of the clinical trial. General recommendations for preclinical study designs
applicable to MVGT products are provided in sections III and V of the Preclinical
Assessment Guidance (Ref. 11). Specific to this product class, parameters to assess
include: the potential induction of proinflammatory cytokines and chemokines; and
innate and adaptive immune responses mediated by the MVGT or the expressed
transgene. A possible association of an immune response with any off-target toxicities
resulting from administration of the MVGT product should be explored as this
information could affect clinical trial design.
C. 	Attenuation Evaluation
The majority of the clinical population receiving an MVGT may be immuno-
compromised or immunosuppressed to some extent. Therefore, the degree of product
attenuation and the stability of the attenuated phenotype should be assessed. For
example, if MVGT product replication is dependent on special nutrient requirement(s) or
a specific microenvironment, then in vitro and/or in vivo data demonstrating the
rigorousness of this dependence and the lack of product replication in the absence of
these factors should be submitted in the IND application. As applicable, the effect of
abrogation of virulence factors or the introduction of new virulence factors in the MVGT
product on the host cells/tissues should also be evaluated. For MVGT products capable
of forming spores, the conditions under which the spores form or replicate, including the
target site(s), should be well-characterized.

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D. 	Biodistribution/ Shedding
In many clinical trials, administration of the MVGT product is not via its natural route(s)
of infection or transmission. Thus, the biodistribution/shedding profile of the MVGT and
the expressed transgene (if applicable) following administration via the planned clinical
route of administration in animals is an important safety endpoint. We recommend that
you collect biodistribution/shedding data from target and non-target tissues/biological
fluids at multiple time points following administration of the MVGT product. The
tissues/samples should be analyzed using well-characterized methods that use appropriate
positive and negative controls, and the number of live and dead microorganisms should
be determined. FDA has previously provided recommendations for general principles
regarding study design (Ref. 2). You may also refer to the FDA guidance entitled,
“Design and Analysis of Shedding Studies for Virus or Bacteria-Based Gene Therapy and
Oncolytic Products” dated August 2015 (Shedding Studies Guidance) (Ref. 12), for
additional information on the design and analysis of shedding studies.
E. 	Antibiotic Use
We recommend evaluating in vivo biological activity, safety, and
biodistribution/persistence profiles of the MVGT product in the presence and absence of
various antibiotics (see section III.G.1 of this guidance for recommendations on the types
of antibiotics to include). Data obtained from these studies can help guide the selection
of effective antibiotics, as well as appropriate timing of antibiotic administration in the
clinical trial.
V. 	CLINICAL STUDIES
This section of the guidance focuses on clinical development and early clinical trial design issues
that are unique to the study of MVGT products. General trial issues are discussed in the ICH
guideline E8 General Considerations for Clinical Trials (Ref. 13). Clinical considerations that
are particularly relevant to early-phase clinical trials of MVGT products are described below.
A. 	General Considerations
Administration of MVGT products in clinical trials may present substantial safety
concerns. Such concerns derive from the infectious nature of the product, the risks of
necessary concomitant medications, and the risks of the study procedures. The purpose
of this section is to discuss general elements of risk mitigation that may guide the design
and conduct of an early-phase trial of an MVGT product.
Specific MVGT products (e.g., live bacterial product) and specific study populations
(e.g., immunocompromised patients) entail a risk of clinically significant sepsis and/or
disease. Prophylactic, concomitant, or post treatment antibiotics may be indicated to
decrease this risk of infection. Antibiotic sensitivity assays (see section III.G.1) and
preclinical studies with antibiotics (see section IV.E of this guidance) may support the

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choice of first- and second-line antibiotics for the clinical trial. Relevant clinical or
preclinical data should also support the dosing schedules and duration of administration
of these antibiotics.
In vivo survival of an MVGT product may depend on concomitant administration of
immunosuppressive drugs. However, such immunosuppressive drugs may substantially
increase the risk of serious infection for the study subjects. Therefore, the administration
of immunosuppressive drugs must be justified and incorporated into the study with
appropriate precautions (e.g., careful dose selection and monitoring for infectious adverse
events).
MVGT products may be studied in combination with other products (e.g., antibiotics or
immunosuppressive drugs, as described above), administered through invasive
procedures (e.g., surgery), or delivered systemically. In such cases, the rationale and data
to justify the starting dose, dosing schedule, and dose escalation should consider the
expected risks from both the product(s) and the study procedures. The study protocol
should include a plan to monitor for these expected risks.
In early-phase clinical trials, a pre-specified plan for management of risks reduces the
variability and severity of the risk outcomes due to differences in interventions. Thus, a
pre-specified risk management plan may improve the interpretability of the safety data.
Risk assessments and managements for clinical trials of MVGT products will continue to
evolve as new data emerge.
B. 	Prior Human Experience
The specific investigational MVGT product, or a related product, may have been
previously administered to humans. If so, then relevant clinical safety and activity data
may be available to inform the design of the proposed study. To assess the relevance of
the available data to the specific investigational MVGT product, adequate
characterization of the previously administered MVGT product(s) is necessary (please
refer to section III of this guidance). Thus, the characterization of these previously
administered MVGT products determines the extent to which the available data can
inform the design of a trial for a proposed MVGT product.
In situations where previous human data exist, and depending on the relevance of those
data, the conduct of additional preclinical studies may not be necessary to support the
starting dose and/or the dose-escalation scheme. FDA recommends that you contact
CBER to discuss these dosing issues prior to conducting additional preclinical studies.
You should provide comprehensive activity and safety data from the previous human
experience to support the safety of the proposed dosing of the MVGT product.

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C. 	Patient Population
The clinical development of MVGT products may involve initial testing in a wide
spectrum of medical conditions. These range from relatively benign conditions that are
easily treated to advanced stages of fatal or disabling diseases with only limited treatment
options, and also include rare genetic diseases without available treatments. In general,
the eligibility criteria for early-phase trials should define an appropriate disease setting
that is relevant to the treatment indication being pursued. You should consider selection
of a population with relatively reduced risks from the intrinsic or allergenic properties of
the MVGT product. For example:
• 	Anaerobic bacterial spore-based products may produce spores that could
proliferate in necrotic tissues. To reduce the risk of proliferation of spores,
consider excluding patients with the presence of, or propensity to develop,
necrotic tissue (e.g., patients with brain abscess, diverticulitis, or recent radiation).
• 	MVGT products with organ-specific tropism may have inherent risks to those
organs. Consider selection of subjects to minimize these potential inherent risks.
For example, in a trial of an MVGT product with infectious tropism to the liver,
excluding patients with underlying liver disease may reduce the risk from
hepatotoxicity.
D. 	Starting Dose, Dose Escalation, and Dosing Schedule
The dose selection and dosing schedule in early-phase clinical trials may substantially
impact the risks associated with the MVGT product. These potential risks have been
discussed in the CMC and preclinical sections of this guidance. To minimize the risks to
human subjects, the results of preclinical proof-of-concept and safety studies can help
guide the selection of the initial clinical dose level, dose-escalation scheme, and dosing
schedule. Sponsors are encouraged to actively engage with the FDA early in product
development to discuss the preclinical data needed to support early-phase trials for
MVGT products.
The starting dose, dose-escalation scheme, and dosing schedule may also be supported by
previous clinical data, if the rationale and justification provided for the dose-escalation
plan are adequate. Appropriate definitions for dose-limiting toxicities (DLTs), maximum
tolerated dose (MTD), and recommended Phase 2 dose (RP2D) also minimize risks
associated with dose escalation. Consider including specific MVGT-related risks in these
definitions. For example:
• 	Clinically symptomatic septicemia may be a criterion for study DLTs.
• 	Fever and positive blood culture, even without hospitalization, after
administration of an MVGT product may be defined as serious and unexpected
adverse reactions for expedited reporting to the FDA and incorporated into DLTs
and/or stopping rules (Ref. 14).
• 	MVGT products administered systemically may have expected risks such as fever
or positive blood cultures immediately following administration. Prolonged

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duration or delayed occurrence of these expected events may serve as a DLT
and/or stopping criterion, taking into account factors such as the type of organism,
specific disease to be treated, and the expected or observed clearance.
E. 	Treatment Modifications
Study stopping rules and “off-treatment” criteria for individual subjects enhance the
safety of an early-phase clinical trial. An example of an MVGT-related risk reduction
procedure includes study stopping rules based on excessive rates or severity of expected
infectious risks (e.g., abscess formation in studies of anaerobic MVGT products;
septicemia).
F. 	Monitoring
Safety monitoring plans should consider the potential risks associated with the specific
MVGT product and the transgene product as identified in the previous preclinical and
clinical experience. Examples of specific monitoring plans include the following:
• 	Long-term safety monitoring for MVGT products with the potential to germinate,
re-germinate, or re-seed.
• 	Monitoring plans to perform blood cultures and imaging studies at onset of non-
specific symptoms to mitigate potential risks for abscess formation due to
anaerobic microbial products.
• 	Consider long-term safety monitoring for MVGT products with anticipated or
known delayed adverse events or cumulative toxicities. Safety data, particularly
the nature and timing of adverse events, from prior human experience with related
MVGT products may help determine an appropriate duration for long-term
monitoring. Assessment of the relevance of the prior clinical experience with
related MVGT products should include consideration of the dose level, route of
administration, duration of exposure, and number of subjects exposed and
evaluated.
For early-phase clinical trials, monitoring for shedding of MVGT products is
recommended. Monitoring for shedding should be continued until after adequate
successive measurements do not demonstrate the presence of the MVGT product. The
choice of samples (e.g., bodily secretions and/or excretions) may depend on the specific
MVGT product and the proposed route of administration. Please refer to FDA’s
Shedding Studies Guidance for additional information (Ref. 12).

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VI. 	REFERENCES
1. 	Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry,
Manufacturing, and Control (CMC) Information for Human Gene Therapy
Investigational New Drug Applications (INDs), April 2008.
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformatio
n/Guidances/CellularandGeneTherapy/ucm072587.htm.
2. 	Guidance for Industry: Gene Therapy Clinical Trials - Observing Subjects for Delayed
Adverse Events, November 2006.
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformatio
n/Guidances/CellularandGeneTherapy/ucm072957.htm.
3. 	Centers for Disease Control and Prevention list of invasive bacterial pathogens.
http://www.cdc.gov/abcs/overview/background.html.
4. 	Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products, January
2011.
http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulator
yInformation/Guidances/CellularandGeneTherapy/UCM243392.pdf.
5. 	G. Nebe-von-Caron., et al., “Analysis of Bacterial Function by Multi-Color Fluorescence
Flow Cytometry and Single Cell Sorting.” J. Microb. Methods. 2000. 42:97-114.
6. 	International Conference on Harmonisation Q5C: Guideline for Industry: Quality of
Biotechnological Products: Stability Testing of Biotechnological/Biological Products,
July 1996.
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm0
65005.htm.
7. 	Guidance for Industry: Container Closure Systems for Packaging Human Drugs and
Biologics, May 1999.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guid
ances/UCM070551.pdf.
8. 	Guideline for the Determination of Residual Moisture in Dried Biological Products,
January 1990.
http://www.fda.gov/ohrms/dockets/dockets/05d0047/05d-0047-bkg0001-Tab-11.pdf.
9. 	Determining the Need for and Content of Environmental Assessments for Gene
Therapies, Vectored Vaccines, and Related Recombinant Viral or Microbial Products,
Guidance for Industry, March 2015.
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformatio
n/Guidances/CellularandGeneTherapy/ucm401869.htm.

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10. 	Guidance for Industry: CGMP for Phase 1 Investigational Drugs, July 2008.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guid
ances/ucm070273.pdf.
11. 	Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene
Therapy Products, November 2013.
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformatio
n/Guidances/CellularandGeneTherapy/ucm376136.htm#III.
12. 	Guidance for Industry: Design and Analysis of Shedding Studies for Virus or Bacteria-
Based Gene Therapy and Oncolytic Products, August 2015.
http://www.fda.gov/biologicsbloodvaccines/guidancecomplianceregulatoryinformation/g
uidances/cellularandgenetherapy/ucm404050.htm.
13. 	International Conference on Harmonisation Guidance: E8 General Considerations for
Clinical Trials, 1997 (62 FR 66113, December 17, 1997).
www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/
UCM073132.pdf.
14. 	Guidance for Industry and Investigators: Safety Reporting Requirements for INDs and
BA/BE Studies, December 2012.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guid
ances/UCM227351.pdf.

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APPENDIX
Examples of suggested testing for MVGT products (Table-1)
+ Recommended testing.
(+) May be needed, depending on the manufacturing process.
* IND should include information confirming any attenuating point
mutations/insertions/deletions by sequencing of relevant gene/regions in the vector.
** For lyophilized or dried final product.
*** For killed MVGT final product.
# For live MVGT DP.
Seed
Stock
Master
Cell Bank
Working
Cell Bank
In Process
Testing
Drug
Substance
Drug
Product
Identity 	+ 	+ 	+ 	+
Purity 	+ 	+ 	+ 	(+) 	+
Monoculture Test 	+ 	+ 	+ 	(+) 	+#
Sterility 	+***
Tests for Plasmid(s) 	+ 	+ 	(+) 	(+) 	+ 	(+)
Antibiotic Sensitivity
Profile
+ 	+ 	(+) 	(+) 	+
Host Genetic Makeup 	+ 	+ 	(+) 	+
Potency 	+ 	(+) 	+
Gene Expression 	+ 	(+) 	+
Confirmation of
Attenuation*
(+) 	+ 	(+) 	(+)
Viability 	+ 	+ 	+ 	+ 	+
Cell Number 	+ 	+ 	+ 	+
Live: Dead Cell Ratio 	+
Residual Moisture** 	+
Residual Antibiotics 	(+) 	+
Residual Growth
Medium Components
(+) 	+
Presence of
Aggregates
(+) 	+
Stability Testing 	+ 	+ 	+ 	+