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  Contains Nonbinding Recommendations 
 
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 
 
i  Contains Nonbinding Recommendations 
 
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). 
1  Contains Nonbinding Recommendations 
 
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    
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe 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.   
 
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.  CBERs 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). 
2  Contains Nonbinding Recommendations 
 
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 
3  Contains Nonbinding Recommendations 
 
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 Acts 
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 products 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. 
4  Contains Nonbinding Recommendations 
 
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 
 
FDAs 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 drugs 
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 209930002. 
 
                                                 
8 See Footnote 6. 
5  Contains Nonbinding Recommendations 
 
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. 
6  Contains Nonbinding Recommendations 
 
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 LBPs 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 LBPs 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.   
 
7  Contains Nonbinding Recommendations 
 
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 products 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. 
8  Contains Nonbinding Recommendations 
 
 
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 manufacturers 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.   
9  Contains Nonbinding Recommendations 
 
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; 
10  Contains Nonbinding Recommendations 
 
  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.   
 
11  Contains Nonbinding Recommendations 
 
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  
12  Contains Nonbinding Recommendations 
 
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 
13  Contains Nonbinding Recommendations 
 
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. 
14  Contains Nonbinding Recommendations 
 
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 
15  Contains Nonbinding Recommendations 
 
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 investigators 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).   
 
16  Contains Nonbinding Recommendations 
 
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 
 
17  Contains Nonbinding Recommendations 
 
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  
18