Early Clinical Trials With Live Biotherapeutic Products: Chemistry, Manufacturing, and Control Information: Guidance for Industry

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

Early Clinical Trials with Live
Biotherapeutic Products: Chemistry,
Manufacturing, and Control
Information
Guidance for Industry
This guidance is for immediate implementation
FDA is issuing this guidance for immediate implementation in accordance with
21 CFR 10.115(g)(3) without seeking additional comments after determining that prior public
participation is not reasonable or appropriate (see 21 CFR 10.115(g)(2)). FDA notes that we
already sought comments on the issues addressed by the revisions in this guidance in the Federal
Register notice of March 31, 2015 (80 FR 17050) entitled “Early Clinical Trials with Live
Biotherapeutic Products: Chemistry, Manufacturing, and Control Information; Guidance for
Industry: Request for Comments,” under Docket No. FDA-2010-D-0500. Further delay in
implementing these revisions could impede the progress of certain investigations that are of low
risk and may be of benefit to the public health.
FDA invites comments on this guidance. Submit one set of either electronic or written
comments on this guidance at anytime. Submit electronic comments to
http://www.regulations.gov. Submit written comments to the Division of Dockets Management
(HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, Rockville, MD
20852. You should identify all comments with docket number FDA-2010-D-0500.
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
February 2012
Updated June 2016

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TABLE OF CONTENTS
I. 	INTRODUCTION............................................................................................................. 1
A. 	Purpose and Scope ................................................................................................ 1
B. 	Definitions .............................................................................................................. 2
II. 	BACKGROUND ............................................................................................................... 3
III. 	ADMINISTRATIVE AND REGULATORY PROCEDURES ..................................... 5
A. 	Pre-IND Advice and Meetings ............................................................................. 5
B. 	Address for Submission of INDs.......................................................................... 5
C. 	Drug Master Files ................................................................................................. 6
IV. 	CHEMISTRY, MANUFACTURING, AND CONTROL (CMC) INFORMATION . 6
A. 	Regulatory Considerations................................................................................... 6
B. 	Drug Substance ..................................................................................................... 7
1. 	Description .................................................................................................. 7
2. 	Characterization .......................................................................................... 8
3. 	Manufacturer ............................................................................................... 9
4. 	Method of Manufacture .............................................................................. 9
5. 	Drug Substance Specifications ................................................................. 11
C. 	Drug Product ....................................................................................................... 12
1. 	Composition .............................................................................................. 12
2. 	Manufacture .............................................................................................. 13
3. 	Drug Product Specification ....................................................................... 13
4. 	Stability ..................................................................................................... 14
5. 	Placebo ...................................................................................................... 14
6. 	Environmental Assessment ....................................................................... 14
D. 	IND Studies Utilizing Commercially Available Live Biotherapeutic Products
(LBPs)................................................................................................................... 14
V. 	NON-CLINICAL INFORMATION.............................................................................. 15
VI. 	CLINICAL INFORMATION ........................................................................................ 15
A. 	Previous Human Experience.............................................................................. 15
B. 	Proposed Initial Studies...................................................................................... 16
VII. 	REFERENCES ................................................................................................................ 17

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Early Clinical Trials with Live Biotherapeutic Products:
Chemistry, Manufacturing, and Control Information
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
A. 	Purpose and Scope
We, FDA (or Agency), are providing you, Investigational New Drug Application (IND)
sponsors, with recommendations regarding IND submissions for early clinical trials with
live biotherapeutic products (LBPs) in the United States (U.S.), including LBPs lawfully
marketed as foods (such as conventional foods and dietary supplements) in the U.S. and
proposed for clinical uses regulated under section 351 of the Public Health Service (PHS)
Act (42 U.S.C. 262).1
This guidance focuses on the chemistry, manufacturing, and control (CMC) information
that you should submit in an IND for an LBP. This guidance is applicable to all INDs of
LBPs, whether clinical trials are conducted commercially, in an academic setting, or
otherwise under Title 21 of the Code of Federal Regulations Part 312 (21 CFR Part 312).2
1The term “live biotherapeutic product” is defined in the “definitions” section (section I.B.) of this document.
2 This guidance is not intended to address when an IND is required prior to conducting clinical trials. For further
discussion of that subject see “Guidance for Clinical Investigators, Sponsors, and IRBs: Investigational New Drug
Applications (INDs) – Determining Whether Human Research Studies Can Be Conducted Without an IND” dated
September 2013, available at
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM229175.pdf. A
stay is in effect for parts of this guidance document. See “Investigational New Drug Applications – Determining
Whether Human Research Studies Can Be Conducted Without an Investigational New Drug; Guidance for Clinical
Investigators, Sponsors, and IRBs; Partial Stay and Republication of Guidance” (80 FR 66907, October 30, 2015).

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This guidance is not applicable to LBPs intended as gene therapy vectors, to oncolytic
bacteria, or to oncolytic viruses.3
This guidance updates the guidance of the same title dated February 2012 (February 2012
guidance) by including the new section IV.D to address when the label on commercially
available product(s) would be considered adequate to satisfy the purpose of the CMC
information requirements under 21 CFR 312.23(a)(7). In addition, this guidance updates
the February 2012 guidance by removing the General Safety Test (21 CFR 610.11) as an
example of a test method in section IV.C and by updating certain footnotes, references,
and addresses.4
FDA’s guidance documents, including this guidance, do not establish legally enforceable
responsibilities. Instead, guidances describe 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.
B. 	Definitions
For purposes of this guidance:
A drug includes, but is not limited to, articles intended for use in the diagnosis, cure,
mitigation, treatment, or prevention of disease in man or other animals. (see section
201(g)(1) of the Federal Food, Drug, and Cosmetic Act (FD&C Act) (21 U.S.C.
321(g)(1)) for the full definition).
A biological product means a virus, therapeutic serum, toxin, antitoxin, vaccine, blood,
blood component or derivative, allergenic product, protein (except any chemically
synthesized polypeptide), or analogous product, or arsphenamine or derivative of
arsphenamine (or any other trivalent organic arsenic compound) applicable to the
prevention, treatment, or cure of a disease or condition of human beings (see section
351(i) of the Public Health Service (PHS) Act (42 U.S.C. 262(i)). The FD&C Act
applies to a biological product subject to regulation under 42 U.S.C. 262, except that,
under 42 U.S.C. 262(j), a product for which a license has been approved under subsection
(a) of 42 U.S.C. 262 shall not be required to have an approved application under section
505 of the FD&C Act (21 U.S.C. 355).
3As a general matter, the Office of Vaccines Research and Review in the Center for Biologics Evaluation and
Research (CBER) reviews the clinical studies for the LBPs described in this guidance. CBER’s Office of Cellular,
Tissue and Gene Therapies (OCTGT) reviews the clinical studies for certain microbial products, such as microbial
vectors for gene therapy (MVGT), oncolytic bacteria and oncolytic viruses. OCTGT encourages potential sponsors
of INDs for such products to contact its office for additional guidance prior to submission of their IND.
4 We are removing reference to the General Safety Test because the underlying regulations were revoked in 2015.
See “Revocation of General Safety Test Regulations That Are Duplicative of Requirements in Biologics License
Applications” (80 FR 37971, July 2, 2015).

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A virus as used in the PHS Act, is understood to be a product containing the minute
living cause of an infectious disease and includes but is not limited to filterable viruses,
bacteria, rickettsia, fungi, and protozoa (21 CFR 600.3(h)(1)).
The sponsor of an IND is a person who takes responsibility for and initiates a clinical
investigation. The sponsor may be an individual or pharmaceutical company,
governmental agency, academic institution, private organization, or other organization
(21 CFR 312.3(b)).
An LBP, for the purposes of this guidance document, is a biological product that:
1) contains live organisms, such as bacteria; 2) is applicable to the prevention, treatment,
or cure of a disease or condition of human beings; and 3) is not a vaccine. For the
purposes of this document, LBPs are not filterable viruses, oncolytic bacteria, or products
intended as gene therapy agents and, as a general matter, are not administered by
injection. An example of an LBP, for the purposes of this document, would be one or
more strains of lactobacilli administered orally to treat patients with ulcerative colitis, or
administered vaginally to prevent bacterial vaginosis.
A recombinant LBP is a live biotherapeutic product composed of microorganisms that
have been genetically modified through the purposeful addition, deletion, or modification
of genetic material. A recombinant LBP which is subject to this guidance is likely to
raise additional considerations and thus would require additional information to be
submitted in an IND. Potential sponsors of an IND for a recombinant LBP are
encouraged to contact FDA to obtain additional guidance prior to submission of their
IND.
The drug substance is the unformulated active substance that may be subsequently
formulated with excipients to produce the drug product (Refs. 1 and 5). The
microorganisms contained in an LBP are typically cellular microbes such as bacteria and
yeast. Thus, the drug substance for an LBP is typically the unformulated live cells.
The drug product is the finished dosage form of the product (Refs. 1 and 5).
A clinical investigation means any experiment in which a drug is administered or
dispensed to, or used involving, one or more human subjects (21 CFR 312.3(b)).
An experiment is any use of a drug except for the use of a marketed drug in the course of
medical practice (21 CFR 312.3(b)).
II. 	BACKGROUND
Proposed clinical trials to evaluate LBPs vary in design, depending on the product, proposed use,
and intended population for enrollment. For example, live microorganisms contained in LBPs
may be isolated from human hosts, genetically modified for use as vectors, and/or engineered to
exhibit enhanced functions or new characteristics. Dosage forms and routes of administration

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can vary from ready-mixed oral preparations with the appearance of a traditional food or drink to
pre-filled vaginal applicators. The proposed mechanisms of action are generally to interfere with
the growth of a pathogenic or potentially pathogenic microorganism in the body or to stimulate
other potentially beneficial cellular processes as a result of transient persistence and/or long-term
colonization with the microorganisms contained in the LBP. Investigational objectives vary with
respect to prevention versus treatment, and stand-alone therapy versus adjunct therapy to
antimicrobial, or other therapy. For example, potential indications include treatment of bacterial
vaginosis in combination with antibiotics, prevention of necrotizing enterocolitis, prevention of
allergic rhinitis, and maintenance of remission of acute pouchitis. Intended study populations
vary from premature neonates to older adults, and from healthy individuals who may be at risk
for specific diseases to individuals severely afflicted with particular diseases or conditions.
Regulations applicable to investigational biological products that meet the FD&C Act’s
definition of “drug” can be found at 21 CFR Part 312. Except as provided under the limited
exemptions set out at 21 CFR 312.2(b), these requirements apply to all clinical investigations of
products that are subject to section 505 of the FD&C Act or to section 351 of the PHS Act (see
21 CFR 312.2(a)). The intended use of a product plays a central role in how it is regulated. For
example, products that contain live microorganisms may be regulated as dietary supplements,
conventional foods, or drugs under the FD&C Act, depending on the product’s intended use and
other factors relevant to the statutory definition of the product category.5 The intended use of a
product in a clinical investigation determines whether IND requirements for clinical
investigation of the product are applicable.6 Note that under section 201(ff)(3)(B)(ii) of the
FD&C Act (21 U.S.C. 321(ff)(3)(B)(ii)), if an LBP that meets the other parts of the dietary
supplement definition in section 201(ff) is authorized for investigation under an IND, and was
marketed as a dietary supplement or conventional food before the IND was authorized, it may
continue to be marketed as a dietary supplement despite the existence of the IND.7 Under
section 301(ll) of the FD&C Act (21 U.S.C. 331(ll)), if substantial clinical investigations of an
LBP as a drug or biologic were instituted before the LBP was marketed in food, a food
5 See “Draft Guidance for Industry: Complementary and Alternative Medicine Products and Their Regulation by
the Food and Drug Administration” dated December 2006 at page 4, issued for public comment and available at
http://www.fda.gov/RegulatoryInformation/Guidances/ucm144657.htm.
6 See also “Guidance for Clinical Investigators, Sponsors, and IRBs: Investigational New Drug Applications (INDs)
– Determining Whether Human Research Studies Can Be Conducted Without an IND” dated September 2013,
available at
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM229175.pdf. A
stay is in effect for parts of this guidance document. See “Investigational New Drug Applications (INDs) –
Determining Whether Human Research Studies Can Be Conducted Without an IND; Guidance for Clinical
Investigators, Sponsors, and IRBs; Partial Stay and Republication of Guidance” (80 FR 66907, October 30, 2015).
7 If an LBP was not marketed as a dietary supplement or conventional food before the IND was authorized, the LBP
may only be marketed as a dietary supplement if one or more of the following is true: (1) substantial clinical
investigations of the LBP have not been instituted; (2) the existence of substantial clinical investigations of the LBP
(if such clinical investigations were instituted) has not been made public; or (3) the Secretary has issued a regulation,
after notice and comment, finding that the article would be lawful under the FD&C Act
(21 U.S.C. 321(ff)(3)(B)(ii)). As of the date of issuance of this guidance, no such regulations have been issued.

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containing the LBP may continue to be marketed only if the existence of such investigations has
not been made public, or if one of the exceptions in section 301(ll)(2)-(3) of the FD&C Act
applies.8
FDA’s primary goals in reviewing an IND are, in all phases of the investigation (phases 1, 2, and
3), to assure the safety and rights of subjects, and in Phases 2 and 3, to help assure that the
quality of the scientific evaluation of drugs is adequate to permit an evaluation of the drug’s
effectiveness and safety (21 CFR 312.22(a)). The general principles of the IND submission and
IND content and format are contained in 21 CFR 312.22 and 312.23, respectively. This
guidance describes information that should be provided in an IND in order to meet the
requirements under 21 CFR 312.23 for early clinical trials evaluating LBPs.
III. 	ADMINISTRATIVE AND REGULATORY PROCEDURES
A. 	Pre-IND Advice and Meetings
Formal communication with FDA representatives before submission of an IND may be
helpful, especially for sponsors that have not previously interacted with CBER, for
sponsors who have limited experience with IND submissions, or for sponsors who have
new products, technology, or assays under development. The purpose of a “pre-IND
meeting” is generally to discuss information required under an IND, such as product
characterization, final and in-process testing of the product, any animal data, and the
proposed clinical protocol (see 21 CFR 312.23). For information on how to request,
schedule, conduct, and document pre-IND meetings, please refer to the FDA document
entitled “Guidance for Industry: Formal Meetings Between the FDA and Sponsors or
Applicants” dated May 2009 (Ref. 2).
B. 	Address for Submission of INDs
An original IND submission, as well as IND amendments, must be submitted in triplicate
(21 CFR 312.23(d)). Each submission should be accompanied by a cover letter and must
include Form FDA-1571 (21 CFR 312.23(a)(1)). You should sequentially number all
pages of the entire submission, including its attachments and appendices. The mailing
address for submission of INDs to CBER is (21 CFR 312.140(a)(3)):
Food and Drug Administration
Center for Biologics Evaluation and Research
Document Control Center
10903 New Hampshire Ave.
Bldg. 71, Rm. G112
Silver Spring, MD 20993–0002.
8 See Footnote 6.

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For information on the submission of electronic INDs, please refer to the following FDA
guidance documents: “Guidance for Industry: Providing Regulatory Submissions to
CBER in Electronic Format — Investigational New Drug Applications (INDs)” dated
March 2002 (Ref. 3), “Providing Regulatory Submissions in Electronic Format —
Standardized Study Data; Guidance for Industry” dated December 2014 (Ref. 16),
“Providing Regulatory Submissions in Electronic Format — Submissions Under Section
745A(a) of the Federal Food, Drug, and Cosmetic Act; Guidance for Industry” dated
December 2014 (Ref. 17) and “Providing Regulatory Submissions in Electronic Format
— Certain Human Pharmaceutical Product Applications and Related Submissions Using
the eCTD Specifications; Guidance for Industry” dated May 2015 (Ref. 18).
C. 	Drug Master Files
An IND sponsor who does not manufacture the product proposed for use in a clinical trial
may ask the manufacturer of the product to submit a Drug Master File (DMF). A DMF is
a submission of information to FDA that may be used to provide confidential information
about the methods used in the manufacturing, processing, packaging, or storing of a drug
product. DMFs can be used by a commercial entity to allow an IND sponsor to reference
the information in the DMF in support of the IND, without disclosure of the contents of
the DMF to the IND sponsor. The DMF holder submits to FDA written authorization
permitting the IND sponsor to reference the DMF.9 When submitted in support of an
IND reviewed by CBER, original DMF submissions and updates should be sent to CBER
(to the same address in section III.B of this document) rather than to the Center for Drug
Evaluation and Research. If a DMF will be submitted or substantially updated, the DMF
holder should alert the appropriate review division and relevant IND sponsors.
IV. 	CHEMISTRY, MANUFACTURING, AND CONTROL (CMC) INFORMATION
A. 	Regulatory Considerations
In 2008, FDA issued a guidance entitled, “Guidance for Industry: CGMP for Phase 1
Investigational Drugs” dated July 2008 (Ref. 4). The approach described in this guidance
recognizes that both manufacturing controls and the extent of such manufacturing
controls needed to achieve appropriate product quality differ not only between
investigational and commercial manufacture, but also among the various phases of
clinical trials.
Production of investigational drug and biological products is subject to section
501(a)(2)(B) of the FD&C Act (21 U.S.C. 351(a)(2)(B)) and the IND regulations at
21 CFR Part 312. During Phase 1 studies, emphasis should generally be placed on
elements to assure the safety of subjects (see 21 CFR 312.22). This should include
9 For more information on DMFs, see the FDA guidance entitled “Guideline for Drug Master Files,” dated
September 1989, available at
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm122886.htm.

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identification and control of the raw materials and the drug substance, stability assurance,
and, where appropriate, non-clinical safety assessments (see, e.g., 21 CFR 312.23(a)(8)).
Quality control and quality assurance should be refined as product development proceeds.
Although in each phase of the investigation, sufficient information is required to assure
the proper identification, quality, purity, and strength of the investigational drug, the
amount of information necessary to make that assurance will vary with the phase of the
investigation, proposed duration, dosage form and amount of information otherwise
available (21 CFR 312.23(a)(7)). In preparing your IND, you should refer to the FDA
guidance on the content and format of INDs for Phase 1 studies entitled, “Guidance for
Industry: Content and Format of Investigational New Drug Applications (INDs) for
Phase 1 Studies of Drugs, Including Well-Characterized, Therapeutic, Biotechnology-
derived Products” dated November 1995 (Ref. 5).
Section IV.B (Drug Substance) and section IV.C (Drug Product) of this guidance detail
the information that should be included in the CMC section of an IND in order to support
proceeding to clinical evaluation of an LBP’s safety in human subjects. The terms “drug
substance” and “drug product” are used in this guidance document for consistency with
documentation on traditional drug products and biological products regulated under the
PHS Act (Ref. 1). The CMC information detailed in the drug substance and drug product
sections may be submitted in the IND or in a supporting DMF.
B. 	Drug Substance
1. 	Description
A description of the LBP’s drug substance, including its physical, chemical, or
biological characteristics, must be included in the IND
(21 CFR 312.23(a)(7)(iv)(a)). A description of the drug substance should include
the following:
• 	Biological name and strain designations;
• 	Original source of cells from which the drug substance was derived;
• 	Culture/passage history of the strains;
• 	If cells were obtained from a clinical specimen, a description of the
clinical health of the donor(s), if known (merely noting procurement from
a commercial provider would not permit a conclusion that the product is
adequately safe);
• 	Summary of the phenotype and genotype of the product strains, with
special attention to biological activity or genetic loci that may indicate
activity or potency; and
• 	Documentation and summary of modifications, if any, to the LBP, e.g.,
intentional introduction of foreign genes or mutations, along with details
of the genetic construction.

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2. 	Characterization
Characterization of an LBP must include a description of the acceptable limits and
analytical methods used to assure the identity, strength, quality, and purity of the
drug substance (21 CFR 312.23(a)(7)(iv)(a)). Test results should contain actual
laboratory data in tabulated form rather than summaries. Results for quantitative
assays should be presented as actual data and not simply as “Pass,” “Satisfactory,”
or “Within Specification.” Additionally, you should submit in your IND detailed
descriptions of the methods and assays used to develop and initially characterize
your LBP, such as the following:
• 	You should provide identification of the cells used to establish the Master
Cell Bank (MCB), as described in section IV.B.4.c. of this guidance. This
information should be at the species and strain level. We recommend that
you use at least two complementary methods of identification, e.g.,
biochemical identification and genetic identification.
• 	You should determine minimum inhibitory or minimum bactericidal
concentrations for a panel of antibiotics proposed by the investigator and
include your assessment of whether the product strains are sensitive or
resistant to each of the antibiotics in the panel.
• 	You may need to develop an assay to determine whether or to what extent
antibiotic resistance is transferable from a product strain to relevant
microbial flora.
• 	When an antibiotic resistance gene has been intentionally introduced in the
product strain(s) for selection or maintenance of genetic modifications,
you should discuss the need for such a gene and any possible alternative
approaches.
• 	You should provide the methods you used to attenuate an otherwise
virulent strain, as well as documentation addressing the stability of such
attenuation.
• 	If the ability of the product strain(s) to cross a mucosal barrier is a critical
safety concern, you should use a reproducible assay for translocation,
preferably in an appropriate animal model, such as germ-free mice, for
oral products.
• 	If the product’s mechanism(s) of action(s) is known, you should submit
data in the IND to support the mechanism(s) of action(s). Investigating
the mechanism(s) of action(s) can be valuable for developing quality
assurance criteria. For example, if you are able to identify a limited
number of genes that may be potency-indicating, we recommend that you
investigate the genetic stability of those genes.

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3. 	Manufacturer
The IND submission must contain the name and address of the manufacturer(s) of
the drug substance (21 CFR 312.23(a)(7)(iv)(a)). You should include any other
pertinent organizational information. You should also provide a comprehensive
list of all additional products that are manufactured or manipulated in the same or
adjacent areas used to produce the drug substance. In addition, you should
indicate whether the production of other products will utilize the same product
contact equipment and, if so, how that equipment will be cleaned between
operations for the manufacturing of different products. A floor diagram is
suggested as the most effective presentation to enable visualization of the
production flow and to identify adjacent operations that may create particular
concerns, such as contamination by extraneous microorganisms.
4. 	Method of Manufacture
a. 	Raw Materials
You should provide a list of all materials (e.g., culture media, buffers,
etc.) used in the manufacture of the drug substance, and their tests and
specifications, or reference(s) to official compendia. For purchased
materials, you should provide representative certificates of analysis
from the supplier(s) and/or manufacturer’s acceptance criteria. The
source and quality of materials of ruminant origin are of particular
importance and you should provide the appropriate documentation.10
b. 	Flow Charts
You should provide a flow chart of the manufacturing process for each
drug substance. For drug substances prepared from more than a single
microbial strain, a common flow chart is acceptable, with indications
of where the processing diverges. A flow chart should show the steps
in production, equipment and materials used, the room or area where
the operation is performed, and a complete list of the in-process
controls and tests performed on the product at each step. You should
include in-process holding steps and indicate time and temperature
limits. Your flow chart should also identify manufacturing steps that
10 Many medical products are manufactured with, or otherwise use, ruminant-derived material because this material
can provide necessary nutrients for cell growth. Bovine derived materials are frequently used. If a proposed rule
published in 2007 entitled “Use of Materials Derived from Cattle in Medical Products Intended for Use in Humans
and Drugs Intended for Use in Ruminants” (72 FR 1583, January 12, 2007), is finalized as proposed, drugs for
humans could not be manufactured from, or otherwise contain, prohibited cattle materials without written
permission from FDA.

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are computer controlled. You may reference other sections of your
IND for more detailed process information. You should also
document equipment dedicated to specific areas or products.
c. 	Cell Bank System
The cell bank system generally consists of two tiers: an MCB; and
Working Cell Banks (WCBs) generated from the MCB for product
manufacturing (although a WCB may not have been generated prior to
Phase 1 studies). You should provide a detailed description of the cell
banking procedures you have used including: the banking system; the
size of the cell banks; the methods, reagents, and media used for
preparation of the cell banks; the conditions employed for
cryopreservation and storage; in-process controls; and storage
conditions. You should provide a description of the procedures used
to avoid extraneous microbial contamination. You should also include
a discussion of precautions (e.g., storage of cell banks in multiple
freezers or at different sites) taken to prevent any event that could
render the cell banks unusable.
You should identify the cells comprising the MCB and provide a
complete history and characterization of the MCB (see section IV.B.2
on “Characterization”). The history and characterization should
include: the original source of cells used in the establishment of the
cell banks and the culture/passage history of the cells; the method used
to derive the cell bank; phenotypic and genotypic characterization as a
means for identification; biochemical and/or genetic markers that may
be potency-indicating; purity of culture (screening procedures for
adventitious agents); and a description of all media components.
You should provide the same information for any WCBs that you
establish.
d. 	Cell Growth and Harvesting
This section should generally contain descriptions of:
• 	each step in propagation, from retrieval of the working cell
bank to culture harvest (stages of growth);
• 	the media used at each step (including water quality), with
details of their preparation and sterilization;
• 	the inoculation and growth of initial and sub-cultures,
including volumes, time and temperature of incubation(s);
• 	how transfers are performed;

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• 	precautions taken and in-process testing conducted to control
contamination;
• 	the nature of the main culture system, including operating
conditions and control parameters, (e.g., temperature of
incubation, static vs. agitated, aerobic vs. anaerobic, culture
vessels vs. fermenter, volume of fermenter, or number and
volume of culture vessels);
• 	the use of antibiotics in the medium and rationale, if
applicable; and
• 	the methods and the criteria used for harvesting and
determining yields, and the criteria for pooling more than one
harvest, if applicable.
e. 	Purification and Downstream Processing
You should provide a description of the methods and materials by
which you separate and/or concentrate intermediate forms and the final
bulk of whole cells. The description of each step in downstream
processing should also include the accompanying analytical tests
developed or adopted by the manufacturer to show identity, purity, and
concentration, and the levels of product related and non-product
related impurities.
f. 	In-Process Testing
For all in-process testing indicated in the flow charts discussed above,
you should provide a brief description of the sampling procedures and
the test methods used. Acceptance limits for the in-process testing
may be necessary to ensure that the product can be manufactured
reproducibly. For testing performed at significant phases of
production, you should specify the criteria for accepting or rejecting an
in-process batch.
5. 	Drug Substance Specifications
You should provide the preliminary specifications and tests for each drug
substance. These should include, but not be limited to, assays for: identity;
purity; microbial bioburden/contamination; potency; and/or biochemical or
physicochemical measurements thought to predict potency, and where applicable,
measures of stability. We recommend that you include upper and lower estimates
of variability, as well as justification for the choice of such limits. Specifications
are likely to be tighter once experience is gained in the manufacturing process and
prior to use of the product in pivotal studies supporting licensure.

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We recommend that the drug specifications section include the following:
• 	The identity of each microbial strain present in the drug substance should
be determined using a specific and reproducible assay. Testing may be
based upon biochemical methods such as fermentation profile or
genotypic methods, including such as ribotyping, restriction fragment
length polymorphism (RFLP), or both. In addition, if one or more genetic
loci, either naturally occurring or engineered, have been identified as
critical for biological activity, we recommend that you develop a specific
identity assay.
• 	Potency of live microbial products is generally a measure of viable cells
per unit or dose, i.e., colony-forming units (CFUs). Additional measures
of product potency may be applicable, depending on the specific product
strain(s) and knowledge of the mechanism(s) of action.
• 	Purity tests of a LBP may include assessment of endotoxin content,
residual antibiotics, and/or the quantification of residual toxic components
or contaminants introduced during manufacture.
• 	Depending on the clinical setting and route of administration, LBPs may
need to be devoid of any extraneous organisms, or alternatively should
have a low level of extraneous organisms. Tests for microbial bioburden
should be in accordance with the US Pharmacopeia (39 USP <61>)
Microbiological Examination of Nonsterile Products: Microbial
Enumeration Tests (Ref. 6). Depending on the nature of other products
campaigned at the manufacturing facility and the proposed use of the
product, the inclusion of tests specific for other organisms may be
necessary.
C. 	Drug Product
The drug product contains the drug substance(s) formulated with other necessary
ingredients in the finished dosage. Other necessary ingredients, active or inactive, may
include adjuvants, stabilizers, and/or excipients. The other ingredients may also be held
separately from the drug product, such as diluents for reconstitution.
1. 	Composition
Composition includes a list of all components in the drug product, including drug
substance(s) and other ingredients (see generally 21 CFR 312.23(a)(7)(iv)(b)).
You should specify quantitative composition of a unit dose and batch. This
section should also contain a description of the tests and preliminary
specifications for all of the drug product ingredients, including the acceptable
upper and lower limits of CFUs for the product strain. You should submit
certificates of analysis for all ingredients in the drug product, if not included in

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the drug substance section. If the drug product is encapsulated or is packaged
within a delivery device11, you should also submit detailed information on the
source, quality, and testing of such encapsulation or device.
2. 	Manufacture
You must submit the name(s) and address(es) of the manufacturer(s) of the drug
product (21 CFR 312.23(a)(7)(iv)(b)). A list of all other products (research and
development, clinical, or approved) manufactured in the same rooms also should
be provided. In addition, you should indicate whether the production of other
products will utilize the same product contact equipment and, if so, how that
equipment will be cleaned between operations for the manufacturing of different
products.
You should submit a description of the manufacturing process flow of the
formulated bulk and finished drug product, including the processing procedures,
lyophilization, and packaging. You should provide records documenting
production of the drug product, as well as manufacturing instructions for
formulation, filling, labeling, and packaging. You must provide the drug product
labeling (see 21 CFR 312.23(a)(7)(iv)(d)).
3. 	Drug Product Specification
Information within this section should include, but not be limited to, sampling
procedures and a description of all test methods not already described in the Drug
Substance section (section IV.B of this document).
Most INDs for LBPs should contain preliminary specifications for the drug
product release/acceptability testing, such as:
• 	Identity (Drug Substance: section IV.B)
• 	Potency (Drug Substance: section IV.B)
• 	Potency Assay: During early clinical development, the potency assay may
be an assessment of CFU. As development proceeds, an alternate or
additional potency assay may be used. Whenever possible, evidence that
the selected potency assay correlates with activity or efficacy observed in
clinical trials should be provided.
• 	Purity (Drug Substance: section IV.B);
• 	Microbial bioburden/contamination (Drug Substance: section IV.B);
• 	Appearance/visual inspection; and
• 	Where applicable, additional tests or modifications thereof, such as
11 Drug products packaged within delivery devices such as pre-filled syringes may meet the definition of a
combination product (21 CFR 3.2(e)). See
http://www.fda.gov/CombinationProducts/AboutCombinationProducts/ucm118332.htm

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percent viable cells, a determination of particulate matter (39 USP <788>)
(Ref. 7), the rabbit pyrogenicity test (39 USP <151>) (Ref. 8), pH testing,
and/or residual moisture (21 CFR 610.13(a)(1)).
4. 	Stability
In an IND submission, you must provide stability data to demonstrate that the new
drug substance and drug product are within acceptable chemical and physical
limits for the planned duration of the clinical investigation
(21 CFR 312.23(a)(7)(ii), 312.23(a)(7)(iv)(a)-(b)). For lyophilized products, you
should develop and initiate an additional stability protocol that examines the
shelf-life after reconstitution. If the product is frozen, you should develop data
supporting product stability through a stated number of freeze-thaw cycles.
The stability study protocol should include, but not be limited to: testing for
potency; viable cell determination; microbial contamination; pH; and residual
moisture, if applicable.
5. 	Placebo
You must include a brief general description of the composition, manufacture,
and control of any placebo used in a controlled clinical trial in your IND
submission (21 CFR 312.23(a)(7)(iv)(c)).
6. 	Environmental Assessment
You must include a claim for a categorical exclusion with the basis for the
exclusion or an environmental assessment (EA), as outlined in
21 CFR 312.23(a)(7)(iv)(e).12 An EA is likely to be needed for use of virulent
organisms, organisms that are ecologically more fit than their wild-type
counterparts, or organisms wherein eradication is problematic or difficult to
document.
D. 	IND Studies Utilizing Commercially Available Live Biotherapeutic Products
(LBPs)
For certain investigations of commercially marketed conventional foods and dietary
supplements, we anticipate that the label on a commercially available LBP will be
sufficient to satisfy the purpose of the CMC information under
21 CFR 312.23(a)(7)(iv)(a)-(b) and that noncompliance with
21 CFR 312.23(a)(7)(iv)(a)-(b) would not pose a significant and unreasonable risk to
human subjects of the investigation (see 21 CFR 312.10(b)). Specifically, FDA generally
12 For more information on EAs, see the FDA guidance entitled “Guidance for Industry: Environmental Assessment
of Human Drug and Biologics Applications” dated July 1998, available at
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070561.htm.

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intends to grant a waiver provided that: 1) the LBP that is proposed for investigational
use is lawfully marketed as a conventional food or dietary supplement; 2) the
investigation does not involve a route of administration, dose, patient population, or other
factor that significantly increases the risk (or decreases the acceptability of risk)
associated with the use of the food or dietary supplement; 3) the investigation is not
intended to support a marketing application of the LBP as a drug for human use or a
biological product for human use; and 4) the investigation is otherwise conducted in
compliance with the requirements for INDs (21 CFR Part 312). For FDA to grant a
waiver from the requirements under 21 CFR 312.23(a)(7)(iv)(a)-(b), sponsors must
request a waiver from FDA under 21 CFR 312.10(a). We recommend that sponsors
submit the following information to the IND to meet the requirements for a waiver
request under
21 CFR 312.10(a): (1) information showing the above; (2) a copy of the label of the
commercially available food or dietary supplement; and (3) a commitment to record the
lot(s) number and date of expiry in the case report form.
V. 	NON-CLINICAL INFORMATION
You must submit adequate information about pharmacological and toxicological studies of the
LBP in laboratory animals, or in vitro, to support a proposed clinical trial evaluating the
investigational LBP (21 CFR 312.23(a)(8)). We recommend that you summarize available
information and that you include studies you conduct as well as those reported in the relevant
literature. Conclusions based on data obtained from animal studies and/or in vitro studies
evaluating the clinical formulation or individual ingredients in the clinical formulation, as
opposed to similar or unknown formulations, are generally most supportive of a proposed
clinical trial. Depending on the product and as appropriate for the proposed clinical study, we
recommend that you address: general toxicity; target organs or systems of toxicity; teratogenic,
carcinogenic, or mutagenic potential of any ingredient in the product; and relationship of dosage
and duration to toxic response and pharmacological activity. Additional FDA guidance
documents, prepared under the auspices of the International Conference on Harmonization (ICH)
are available at http://www.fda.gov/RegulatoryInformation/Guidances/ucm122049.htm (Refs. 9
and 10).
As discussed above, we encourage you to consult the applicable CBER review division,
preferably in a pre-IND meeting, regarding the extent and type of non-clinical data necessary to
support the proposed clinical investigation (Ref. 2).
VI. 	CLINICAL INFORMATION
A. 	Previous Human Experience
Previous human experience varies from prospectively designed, randomized, controlled
clinical trials to case reports. Similarly, varying degrees of product characterization may
be reported for the products used in the respective study or studies. Drawing conclusions

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or making assumptions as to the relevance of previous studies may be difficult or
impossible without adequate available CMC information.
In general, the relevance of previous human experience to support a proposed study is
based upon the similarity of the product(s) under study, as well as study design,
objectives and endpoints, the number of individuals exposed, the level and duration of
exposure, the type and duration of active monitoring and passive surveillance, and the
integrity of study conduct, data collection and subsequent analyses. If you wish to
reference a study submitted to the Agency by a person other than you (the sponsor), you
must obtain and include in your submission, a signed letter of cross-reference stating that
FDA has permission to access this information (21 CFR 312.23(b)). Also, an accounting
of the final disposition of all randomized or enrolled study subjects is important in order
to provide a context for the study data, since a large number of dropouts, withdrawals, or
protocol violations make data analysis and conclusions less convincing. Multiple
statistical analyses without appropriate correction and post hoc analyses, including
analyses of population subsets, can be useful to generate hypotheses in designing
subsequent studies; however, conclusions based solely upon multiple statistical analyses
without appropriate correction may be misleading. Finally, safety data based on active
adverse event monitoring provides a more meaningful safety profile of a product than
data based on passive monitoring and/or reporting of only those events that an individual
investigator deems “related” based upon an individual investigator’s judgment.
B. 	Proposed Initial Studies
Safety data obtained from the administration of an investigational product to healthy
volunteers can be important in identifying common product-associated adverse events
before proceeding to studies in more vulnerable populations, e.g., children or those with
the disease of interest. Proposed studies evaluating treatment effects should include
disease definitions and criteria for worsening, improvement, relapse, etc., applicable to
the disease and population studied. For useful sources of information on clinical
investigations generally, and clinical studies in pediatric or geriatric populations
specifically, you should refer to the appropriate FDA guidance documents, including
those prepared under the auspices of ICH at
http://www.fda.gov/RegulatoryInformation/Guidances/ucm122049.htm (Refs. 11 through
15).

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VII. 	REFERENCES
1. 	Guidance for Industry: Content and Format of Chemistry, Manufacturing and Controls
Information and Establishment Description Information for a Vaccine or Related Product,
January 1999.
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/
Guidances/Vaccines/ucm076612.htm
2. 	Guidance for Industry: Formal Meetings Between the FDA and Sponsors or Applicants,
May 2009.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/
Guidances/UCM079744.htm (Note: The document entitled “Formal Meetings Between the
FDA and Sponsors or Applicants of PDUFA Products; Guidance for Industry,” dated March
2015, when finalized will supersede this May 2009 guidance.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidance
s/UCM437431.pdf.)
3. 	Guidance for Industry: Providing Regulatory Submissions to CBER in Electronic Format —
Investigational New Drug Applications (INDs), March 2002.
http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInf
ormation/Guidances/General/UCM150028.htm
4. 	Guidance for Industry: CGMP for Phase 1 Investigational Drugs, July 2008.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/
Guidances/UCM070273.htm
5. 	Guidance for Industry: Content and Format of Investigational New Drug Applications
(INDs) for Phase 1 Studies of Drugs Including Well-Characterized, Therapeutic,
Biotechnology-derived Products, November 1995.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/
Guidances/UCM071597.htm
6. 	US Pharmacopeia (39 USP <61>) Microbiological Examination of Nonsterile Products:
Microbial Enumeration Tests
7. 	US Pharmacopeia (39 USP <788>) Particulate Matter in Injections
8. 	US Pharmacopeia (39 USP <151>) Pyrogen Test
9. 	Guidance for Industry: S6 Preclinical Safety Evaluation of Biotechnology-Derived
Pharmaceuticals, July 1997, ICH.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidance
s/UCM074957.pdf

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10. Guidance for Industry: M3(R2) Non-Clinical Safety Studies for the Conduct of Human
Clinical Trials and Marketing Authorization for Pharmaceuticals, January 2010, ICH.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidance
s/UCM073246.pdf
11. Guidance for Industry: E6 Good Clinical Practice: Consolidated Guidance, April 1996,
ICH.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidance
s/UCM073122.pdf
12. Guidance for Industry: E11 Clinical Investigation of Medicinal Products in the Pediatric
Population, December 2000, ICH.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidance
s/UCM073143.pdf
13. Guideline for Industry: Studies in Support of Special Populations: Geriatrics, August 1994,
ICH-E7.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidance
s/UCM073131.pdf
14. E8 General Considerations for Clinical Trials, December 1997, ICH.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidance
s/UCM073132.pdf
15. Guidance for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers
Enrolled in Preventive Vaccine Clinical Trials, September 2007.
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/
Guidances/Vaccines/ucm074775.htm
16. Providing Regulatory Submissions in Electronic Format — Standardized Study Data;
Guidance for Industry, December 2014.
http://www.fda.gov/downloads/Drugs/.../Guidances/UCM292334.pdf
17. Providing Regulatory Submissions in Electronic Format — Submissions Under Section
745A(a) of the Federal Food, Drug, and Cosmetic Act; Guidance for Industry, December
2014.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidance
s/UCM384686.pdf
18. Providing Regulatory Submissions in Electronic Format — Certain Human Pharmaceutical
Product Applications and Related Submissions Using the eCTD Specifications; Guidance for
Industry, May 2015.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidance
s/UCM333969.pdf