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  Contains Nonbinding Recommendations 
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 
i  Contains Nonbinding Recommendations 
E.  Treatment Modifications .................................................................................... 21 
F.  Monitoring ........................................................................................................... 21 
VI.  REFERENCES ................................................................................................................ 22 
APPENDIX .................................................................................................................................. 24 
ii  Contains Nonbinding Recommendations 
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). 
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe the FDAs 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 FDAs 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 
1  Contains Nonbinding Recommendations 
genetic modifications that may alter an MVGTs 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 FDAs 
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.   
2  Contains Nonbinding Recommendations 
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 MVGTs 
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 
MVGTs 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, 
3  Contains Nonbinding Recommendations 
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).  
4  Contains Nonbinding Recommendations 
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. 
5  Contains Nonbinding Recommendations 
  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 
 
6  Contains Nonbinding Recommendations 
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. 
 
7  Contains Nonbinding Recommendations 
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 
8  Contains Nonbinding Recommendations 
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  
9  Contains Nonbinding Recommendations 
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: 
10  Contains Nonbinding Recommendations 
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. 
11  Contains Nonbinding Recommendations 
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. 
12  Contains Nonbinding Recommendations 
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.  
13  Contains Nonbinding Recommendations 
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).  
 
14  Contains Nonbinding Recommendations 
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). 
 
15  Contains Nonbinding Recommendations 
3.  Tests for Residual Moisture Content   
 
If using lyophilized/dried bacterial products, please refer to FDAs 
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 FDAs 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. 
 
16  Contains Nonbinding Recommendations 
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. 
 
17  Contains Nonbinding Recommendations 
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 
18  Contains Nonbinding Recommendations 
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. 
 
19  Contains Nonbinding Recommendations 
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 
20  Contains Nonbinding Recommendations 
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 FDAs 
Shedding Studies Guidance for additional information (Ref. 12). 
 
21  Contains Nonbinding Recommendations 
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. 
 
22  Contains Nonbinding Recommendations 
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. 
 
23  Contains Nonbinding Recommendations 
APPENDIX   
 
Examples of suggested testing for MVGT products (Table-1) 
  Seed  Master  Working  In Process  Drug  Drug 
Stock  Cell Bank  Cell Bank  Testing  Substance  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    +  +  +    + 
 
+ 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.   
24