Chemistry, Manufacturing, and 
Control (CMC) Information for 
Human Gene Therapy Investigational 
New Drug Applications (INDs) 
 
 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
Additional copies of this guidance are available from the Office of Communication, Outreach 
and Development (OCOD), 10903 New Hampshire Ave., Bldg. 71, Rm. 3128, Silver Spring, 
MD 20993-0002, or by calling 1-800-835-4709 or 240-402-8010, or email ocod@fda.hhs.gov, or 
from the Internet at https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-
regulatory-information-biologics/biologics-guidances. 
 
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 
January 2020  Contains Nonbinding Recommendations 
 
Table of Contents 
 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  BACKGROUND ............................................................................................................... 2 
III.  ADMINISTRATIVE INFORMATION (MODULE 1 OF THE CTD) ........................ 3 
A.  Administrative Documents ................................................................................... 3 
B.  Labels ..................................................................................................................... 4 
C.  Environmental Analysis ....................................................................................... 4 
D.  Previously Submitted Information ...................................................................... 4 
IV.  SUMMARY OF QUALITY INFORMATION (MODULE 2 OF THE CTD) ............ 5 
A.  General Information ............................................................................................. 5 
B.  Drug Substance and Drug Product ..................................................................... 6 
C.  Combination Products .......................................................................................... 7 
D.  Product Handling at the Clinical Site ................................................................. 7 
V.  MANUFACTURING PROCESS AND CONTROL INFORMATION (MODULE 3 
OF THE CTD) ................................................................................................................... 8 
A.  Drug Substance (3.2.S) ......................................................................................... 8 
1.  General Information (3.2.S.1) ........................................................................... 8 
2.  Drug Substance Manufacture (3.2.S.2) ........................................................... 10 
3.  Drug Substance Characterization (3.2.S.3) ..................................................... 28 
4.  Control of Drug Substance (3.2.S.4) ............................................................... 31 
5.  Reference Standards or Materials (3.2.S.5) .................................................... 37 
6.  Container Closure System (3.2.S.6) ................................................................ 37 
7.  Stability (3.2.S.7) ............................................................................................ 38 
B.  Drug Product (3.2.P) ........................................................................................... 39 
1.  Drug Product Description and Composition (3.2.P.1) .................................... 39 
2.  Pharmaceutical Development (3.2.P.2) .......................................................... 39 
3.  Manufacture (3.2.P.3) ..................................................................................... 42 
4.  Control of Excipients (3.2.P.4) ....................................................................... 44 
5.  Control of Drug Product (3.2.P.5) .................................................................. 45 
6.  Reference Standards or Materials (3.2.P.6) .................................................... 50 
7.  Container Closure System (3.2.P.7) ................................................................ 50 
8.  Stability (3.2.P.8) ............................................................................................ 51 
C.  Appendices (3.2.A) .............................................................................................. 51 
1.  Facilities and Equipment (3.2.A.1) ................................................................. 51 
2.  Adventitious Agents Safety Evaluation (3.2.A.2) .......................................... 52 
D.   Regional Information (3.2.R) ............................................................................. 52 
VI.  REFERENCES ................................................................................................................ 53 
 
i  Contains Nonbinding Recommendations 
 
Chemistry, Manufacturing, and Control (CMC) Information for 
Human Gene Therapy Investigational New Drug Applications 
(INDs) 
 
 
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 
 
Human gene therapy seeks to modify or manipulate the expression of a gene or to alter the 
biological properties of living cells for therapeutic use.  We, FDA, are providing you, sponsors 
of human gene therapy Investigational New Drug Applications (INDs), recommendations 
regarding chemistry, manufacturing, and control (CMC) information submitted in an IND.  The 
purpose of this guidance is to inform sponsors how to provide sufficient CMC information 
required to assure product safety, identity, quality, purity, and strength (including potency) of the 
investigational product (21 Code of Federal Regulations (CFR) 312.23(a)(7)(i)).  This guidance 
applies to human gene therapy products and to combination products1 that contain a human gene 
therapy in combination with a drug or device.2 
 
This guidance finalizes the draft guidance of the same title dated July 2018 and supersedes the 
document entitled Guidance for FDA Reviewers and Sponsors:  Content and Review of 
Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy 
Investigational New Drug Applications (INDs), dated April 2008 (April 2008 guidance).  The 
field of gene therapy has progressed rapidly since we issued the April 2008 guidance.  Therefore, 
we are updating that guidance to provide you with current FDA recommendations regarding the 
CMC content of a gene therapy IND.  This guidance is organized to follow the structure of the 
FDA guidance on the Common Technical Document (CTD).  Information on the CTD can be 
found in FDAs Guidance for Industry:  M4Q:  The CTD  Quality, (Ref. 1).  For information 
on the submission of an electronic CTD (eCTD), please see the FDA website 
https://www.fda.gov/drugs/electronic-regulatory-submission-and-review/electronic-common-
technical-document-ectd. 
                                                 
1 Combination products are comprised of any combination of a drug and a device; a device and a biological product; 
a biological product and a drug; or a drug, a device, and a biological product; see 21 CFR 3.2(e) for the complete 
definition of combination product.  Combination products are assigned to a lead center for review; see 21 CFR 3.4. 
2 This guidance does not apply to vaccines for infectious disease indications, bacteriophage products, live 
biotherapeutic products, fecal microbiota for transplantation (FMT) products and allergenic products.   
1  Contains Nonbinding Recommendations 
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe the FDAs current thinking on a topic and should be 
viewed only as recommendations, unless specific regulatory or statutory requirements are cited.  
The use of the word should in FDAs guidance means that something is suggested or 
recommended, but not required. 
 
 
II.  BACKGROUND 
 
FDA generally considers human gene therapy products to include all products that mediate their 
effects by transcription or translation of transferred genetic material or by specifically altering 
host (human) genetic sequences.  Some examples of gene therapy products include nucleic acids 
(e.g., plasmids, in vitro transcribed ribonucleic acid (RNA)), genetically modified 
microorganisms (e.g., viruses, bacteria, fungi), engineered site-specific nucleases used for human 
genome editing (Ref. 2),  and ex vivo genetically modified human cells.  Gene therapy products 
meet the definition of biological product in section 351(i) of the Public Health Service (PHS) 
Act (42 U.S.C. 262(i)) when such products are applicable to the prevention, treatment, or cure of 
a disease or condition of human beings.3 
 
FDA requires all sponsors of investigational new drug products (DPs), including investigational 
gene therapy products, to describe the CMC information for the drug substance (DS) (21 CFR 
312.23(a)(7)(iv)(a)) and the DP (21 CFR 312.23(a)(7)(iv)(b)).  FDA may place the IND on 
clinical hold if the IND does not contain sufficient CMC information to assess the risks to 
subjects in the proposed studies (21 CFR 312.42(b)(1)(iv)).  
 
The CMC information submitted in an IND describes the sponsors commitment to perform 
manufacturing and testing of the investigational product as stated.  We acknowledge that 
manufacturing changes may be necessary as product development proceeds, and you should 
submit information amendments to supplement the initial information submitted for the CMC 
processes (21 CFR 312.31(a)(1)).  The CMC information submitted in the original IND for an 
early phase study may be limited, and therefore, the effect of manufacturing changes, even minor 
changes, on product safety and quality may not be sufficiently understood.  Thus, if a 
manufacturing change could affect product safety, identity, quality, purity, potency, or stability, 
you should submit the manufacturing change for FDA review prior to implementation. 
 
We published a guidance document entitled Providing Regulatory Submissions in Electronic 
Format  Certain Human Pharmaceutical Product Applications and Related Submissions Using 
the eCTD Specifications; Guidance for Industry, addressing the electronic submission of certain 
applications in the CTD format (Ref. 3).  As of May 5, 2017, all New Drug Applications 
(NDAs), Abbreviated New Drug Applications (ANDAs), and Biologics License Applications 
                                                 
3 See Federal Register Notice:  Application of Current Statutory Authorities to Human Somatic Cell Therapy 
Products and Gene Therapy Products (58 FR 53248, October 14, 1993), 
https://www.fda.gov/media/76647/download. 
 
2  Contains Nonbinding Recommendations 
 
(BLAs) must be submitted in eCTD, and commercial IND submissions and Master Files4 must 
be submitted in eCTD beginning May 5, 2018 (Ref. 3).5  Products that are not intended to be 
distributed commercially, such as in investigator-sponsored and expanded access INDs (e.g., 
emergency use INDs and treatment INDs), are excluded from the eCTD requirement.  Also 
excluded from the eCTD requirement are INDs for devices under section 351 of the PHS Act, 
such as in vitro diagnostic devices.  However, devices used in gene therapy product delivery or 
combination products (e.g., scaffolds) are not excluded, and should follow the recommendations 
in this guidance.  In preparation for meeting the eCTD requirements, we recommend that 
sponsors begin to organize and categorize their CMC information according to the CTD format.   
 
You are not required to complete all CTD sections in your original IND submission.  The 
amount of CMC information to be submitted in your IND depends on the phase of investigation 
and the scope of the clinical investigation proposed (21 CFR 312.23(a)(7)).  The emphasis for 
CMC review in all phases of development is product safety and manufacturing controls.  We 
expect that sponsors may need to make modifications and additions to previously submitted 
information as clinical development proceeds and additional product knowledge and 
manufacturing experience is collected (21 CFR 312.31).   
 
We are providing detailed recommendations for submitting CMC information in Module 3 of the 
CTD.  We are also providing general recommendations regarding administrative and quality 
summary information for Modules 1 and 2, respectively, of the CTD submissions.  
 
 
III.  ADMINISTRATIVE INFORMATION (MODULE 1 OF THE CTD) 
 
A.  Administrative Documents 
 
Administrative documents (e.g., application forms, such as Form FDA 1571, cover 
letters, reviewer guides, and cross-reference authorization letters), claims of categorical 
exclusion, and labeling information should be included in Module 1 of the CTD 
submissions.  The cover letter for your submission should include a brief explanation of 
your submission and its contents.  When amendments are submitted to the IND for 
manufacturing changes, your cover letter should clearly describe the purpose of the 
amendment and highlight proposed changes.  For amendments containing numerous or 
significant changes (e.g., manufacturing process, assays for critical quality attributes 
(CQAs), new manufacturing site or manufacturer, etc.), we recommend that you include a 
Reviewers Guide, as described in FDAs eCTD Technical Conformance Guide:  
Technical Specifications Document, (Ref. 4) or a document with all changes tracked, 
and that you allow sufficient lead time (e.g., 30 days) for FDA review before release of a 
new lot of clinical trial material. 
 
   
                                                 
4 This includes Master Files other than Type III Drug Master Files (DMFs).  Beginning May 5, 2020, Type III 
DMFs must be submitted in eCTD. (Ref. 3). 
5 See also section 745A(a) of the Federal Food, Drug, and Cosmetic Act (FD&C Act).  
3  Contains Nonbinding Recommendations 
 
B.   Labels 
 
Your IND must contain a copy of all labels and labeling to be provided to each 
investigator in the clinical study (21 CFR 312.23(a)(7)(iv)(d)).  We recommend that you 
include sample or mock-up labels in Module 1 of the CTD.  Please note that IND 
products must bear a label with the statement, Caution:  New DrugLimited by Federal 
(or United States) law to investigational use (21 CFR 312.6).  For products derived from 
autologous donors and other situations described in 21 CFR 1271.90(a) for which a donor 
eligibility determination is not required, you must include the required labeling in 21 
CFR 1271.90(c), as applicable.  For example, for cells intended for autologous use, you 
must label the product FOR AUTOLOGOUS USE ONLY (21 CFR 1271.90(c)(1)) and 
NOT EVALUATED FOR INFECTIOUS SUBSTANCES if donor testing and 
screening is not performed (21 CFR 1271.90(c)(2)). 
 
C.  Environmental Analysis 
 
Your IND must contain either an environmental analysis or a claim for categorical 
exclusion (21 CFR 312.23(a)(7)(iv)(e)).  Please note that, under ordinary circumstances, 
most INDs are eligible for categorical exclusion under 21 CFR 25.31(e) (Ref. 5).  This 
information can be submitted in Module 1 of the CTD. 
 
D.  Previously Submitted Information 
 
For INDs, you generally are not required to resubmit information that you have 
previously submitted to the Agency, and you may incorporate such information by 
reference.  You may submit a written statement in your IND that appropriately identifies 
information you have previously submitted (21 CFR 312.23(b)), usually by providing a 
submission tracking number (STN), for the IND or biologics Master File.  We 
recommend that you describe the information you are referencing and identify where that 
information is located in the previously submitted file.   
 
You may also reference information previously submitted by another individual if proper 
authorization has been granted.  Proper authorization may be granted with a Letter of 
Authorization (LOA) from the individual who submitted the information (21 CFR 
312.23(b)).  We recommend that the LOA include a description of the information being 
cross-referenced (e.g., reagent, container, vector manufacturing process) and identify 
where that information is located (e.g., file name, reference number, volume, page 
number).  Please note that this LOA only allows you to cross-reference the information 
outlined in the LOA and submitted by the author of the LOA.  The LOA does not provide 
you permission to cross-reference information that was submitted by another individual 
and cross-referenced by the author of the LOA.  In other words, you may not cross-
reference information that is cross-referenced by the author of the LOA.  You are 
required to submit a LOA for all information submitted by another individual (21 CFR 
312.23(b)). 
 
4  Contains Nonbinding Recommendations 
 
In addition to including LOAs in Module 1 of the CTD, you should list these files in the 
IND cover sheet (i.e., Form FDA 1571) of each IND submission.  If the LOA is absent or 
inadequate or the information in the cross-referenced file is inadequate for the purpose 
cited, we intend to notify you that the information in the cross-referenced file is not 
sufficient to support your IND.  In the event a cross-referenced IND is placed on clinical 
hold or is withdrawn, your IND may also be placed on clinical hold if critical cross-
referenced information is no longer available or adequate. 
 
 
IV.  SUMMARY OF QUALITY INFORMATION (MODULE 2 OF THE CTD) 
 
A.  General Information 
 
Your IND should contain a general introduction to the gene therapy product under 
investigation, including a description of its active ingredient(s), mode of action, and 
proposed clinical use.  We recommend that you provide this summary in the Quality 
Overall Summary (QOS), located in Module 2 of the CTD (Ref. 1).  The information in 
the QOS is a condensed summary of the quality-related data and information provided in 
Module 3 of the CTD.  It can also serve to help reviewers more effectively assess 
applications, understand the quality information presented, and relate this information to 
potential patient risks.  This summary should include an overview of the manufacturing 
process, controls in place to ensure product quality, and general information regarding the 
qualification of components and starting materials.  You should describe the composition 
of the DS and DP, whether the gene therapy is a combination product, and how your gene 
therapy will be handled at the clinical site prior to administration, as described in the 
sections below.   
 
Your summary should also include a description of potential CQAs that are relevant to 
the safety and biological activity of the product as they are understood at the time of 
submission.  A CQA is a physical, chemical, biological, or microbiological property or 
characteristic that should be within an appropriate limit, range, or distribution to ensure 
the desired product quality (Ref. 6).  We acknowledge that limits may be broader during 
early development when you are still gaining information about your product.  In 
addition, as your product progresses through development the list of potential CQAs may 
be revised as your knowledge of the product increases.  
 
We further acknowledge that understanding and defining product characteristics that are 
relevant to the clinical performance of the gene therapy may be challenging during early 
stages of product development, when product safety and quality may not be sufficiently 
understood.  Therefore, we recommend that you evaluate a number of product 
characteristics during early clinical development to help you identify and understand 
CQAs.  This will also help ensure your ability to assess manufacturing process controls, 
manufacturing consistency, and stability as development advances.  This is especially 
important for sponsors of gene therapy products who are pursuing expedited development 
programs (Ref. 7).   
 
5  Contains Nonbinding Recommendations 
 
CQAs may be used to specify key characteristics of the DS and DP including, but not 
limited to, their specifications for a later phase clinical study or BLA.  It may be crucial 
to establish CQAs as early as possible particularly when you plan to make manufacturing 
changes during product development because well-established CQAs are generally 
necessary for demonstrating product comparability by analytical methods.  For additional 
information regarding establishing CQAs, please see FDAs Guidance for Industry:  
Q8(R2) Pharmaceutical Development (Ref. 6), and Q11 Development and 
Manufacture of Drug Substances (Ref. 8).  Information to support a CQA and results 
from specific studies or published literature may be included in Module 3 of the CTD in 
the Manufacturing Process Development (3.2.S.2.6) section or the Pharmaceutical 
Development (3.2.P.2) section (see Ref. 1) depending on whether the attribute pertains 
to a DS or a DP.  Information may also be linked to the relevant nonclinical or clinical 
sections of the application in the CTD.  
 
 
B.  Drug Substance and Drug Product 
 
Your IND must contain a description of the DS (21 CFR 312.23(a)(7)(iv)(a)) and DP (21 
CFR 312.23(a)(7)(iv)(b)), including the physical, chemical, or biological characteristics, 
manufacturing controls, and testing information, to ensure the DS and DP meet 
acceptable limits for identity, strength (potency), quality, and purity.  A bulk DS means 
the same as active pharmaceutical ingredient (21 CFR 207.3; see also 21 CFR 207.1), 
which is further described as any substance that is intended for incorporation into a 
finished DP and is intended to furnish pharmacological activity or other direct effect in 
the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the 
structure or any function of the human body (21 CFR 207.1).  An active ingredient is 
defined as any component that is intended to furnish pharmacological activity or other 
direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to 
affect the structure or any function in the human body of man or other animals.  The term 
includes those components that may undergo chemical change in the manufacture of the 
DP and be present in the DP in a modified form intended to furnish the specified activity 
or effect (21 CFR 210.3(b)(7)).  For these products, it follows that a vector6 used to 
transduce cells ex vivo and which furnishes a pharmacological activity for the treatment 
of disease is a critical component.  Without the vector, the resulting cell product would 
not have the same pharmacological activity.  
 
Similarly, a vector in its final formulation for administration of the genetic material is 
generally considered a DP.  A DP is defined as a finished dosage form, for example, 
tablet, capsule, solution, etc., that contains an active drug ingredient generally, but not 
necessarily, in association with inactive ingredients.  The term also includes a finished 
dosage form that does not contain an active ingredient but is intended to be used as a 
placebo (21 CFR 210.3(b)(4)).  Additionally, a vector in its final formulation used for 
                                                 
6 Where a vector is defined as a vehicle consisting of, or derived from, biological material that is designed to 
deliver genetic material.  Examples include plasmids, viruses, and bacteria that have been modified to transfer 
genetic material. (Long Term Follow-Up After Administration of Human Gene Therapy Products; Guidance for 
Industry; January 2020, available at https://www.fda.gov/media/113768/download).    
6  Contains Nonbinding Recommendations 
 
administration of the genetic material may also be an active ingredient, depending upon 
the manufacturing process and formulation of the finished dosage form.    
 
We recognize that distinguishing a DS from a DP may be difficult for some gene therapy 
products due to the complex nature of the manufacturing processes.  Some gene therapy 
products may not have a distinct DS.  Others may consist of two or more different DSs 
that are combined to make the DP.  This guidance does not recommend how sponsors 
should distinguish the DS and DP.  However, we do recommend that you provide an 
explanation to support the definition of DS/DP for your product in the summary 
information in Module 2 of CTD submissions and that you submit the required 
information for each DS and DP, as outlined in Module 3 of the CTD (Ref. 1).  A 
separate DS section should be provided for vectors used for ex vivo modification of cells.  
 
When the manufacturing process includes more than one DS, we recommend that you 
provide separate DS sections for each active ingredient of the final product.  The CTD 
DS sections should follow the format and numbering scheme recommended in Module 3 
of FDAs Guidance for Industry:  M4Q:  The CTD  Quality (Ref. 1), and the sections 
should be distinguished from one another by including the DS name and manufacturer in 
the heading (e.g., General Information, 3.2.S.1 [DS name, manufacturer]). 
 
A summary of the available stability data for the DS and the DP, recommended storage 
conditions, and shelf life, if applicable, should also be included in each DS and DP 
section.  Information on stability protocols and stability data should be included in the 
appropriate sections of Module 3 of the CTD (Ref. 1). 
 
C.  Combination Products 
 
For submissions in which the gene therapy is a component of a combination product, as 
defined in 21 CFR 3.2(e), we recommend that you briefly describe the combination 
product in the summary and briefly state the regulatory status of each component.  To 
clearly delineate the different components of a combination product, you should include 
manufacturing and engineering information for the gene therapy and drug or device in 
separate entries of the CTD submission, as described in the FDA eCTD Technical 
Conformance Guide:  Technical Specifications Document (Ref. 4).  
 
D.  Product Handling at the Clinical Site 
 
Proper control of the finished DP is critical to your investigational studies.  Therefore, 
your IND should include a description of how the product will be shipped to, received, 
and handled at the clinical site to ensure safety, product quality, and stability.  Your IND 
should also include information on shipping conditions, storage conditions, expiration 
date/time (if applicable), and chain of custody from the manufacturer to the site of 
administration in the summary information of the CTD.  Your summary in Module 2 
should also include information for product handling at the clinical site prior to 
administration (such as thawing, the addition of diluent or adjuvant, loading into a 
7  Contains Nonbinding Recommendations 
 
delivery device, and transport to the bedside) and summary information on product 
stability prior to and during administration (e.g., in-device hold times and temperatures).  
 
Details regarding product stability after preparation for delivery and delivery device 
compatibility data should be included in Module 3 (sections 3.2.P.8 and 3.2.P.2.6, 
respectively) of the CTD (Ref. 1).  Instructions for drug handing and preparation for 
administration at the clinical site (e.g., Pharmacy Manual, Instructions for Use, 
Investigators Brochure) should be provided in the appropriate section of your IND 
(Module 5 of the CTD) and hyperlinked to the QOS in Module 2.  Detailed information 
about the delivery device may be included in the Regional Information (3.2.R) section 
of the CTD (Ref. 1). 
 
 
V.  MANUFACTURING PROCESS AND CONTROL INFORMATION (MODULE 3 
OF THE CTD) 
 
The headings and text below include CTD section numbers in parentheses for reference (Ref. 1). 
 
A.  Drug Substance (3.2.S) 
 
1.  General Information (3.2.S.1) 
 
a.  Nomenclature (3.2.S.1.1) 
 
You should provide the name of the DS(s).  If the name of the DS has 
changed during clinical development, you should provide the names used 
to identify the DS at all stages of development.  If the United States 
Adopted Name (USAN) Council has given it a nonproprietary name, you 
may provide it here. 
 
b.  Structure (3.2.S.1.2) 
 
You should submit information on the molecular structure (including 
genetic sequence, if applicable) and/or cellular components of the DS.  
The genetic sequence can be represented in a schematic diagram that 
includes a map of relevant regulatory elements (e.g., promoter/enhancer, 
introns, poly(A) signal), restriction enzyme (RE) sites, and functional 
components (e.g., transgene, selection markers).  Please note that you 
should also submit information on your sequence analysis and the 
annotated sequence data in your IND.  We recommend that your sequence 
data, including any sequence data collected to support the genetic stability 
of your vector, be submitted in the Regional Information (3.2.R) section 
of the CTD.  Recommendations for sequence analysis are described in 
section V.A.2.c.iii., Control of Materials (3.2.S.2.3) of this guidance.  
 
8  Contains Nonbinding Recommendations 
 
Some specific examples of structure and structural elements of different 
gene therapy products are outlined below: 
 
  For viral-based gene therapies, you should include a description of 
the composition of the viral capsid and envelope structures, as 
appropriate, and any modifications to these structures (e.g., 
modifications to antibody binding sites or tropism-changing 
elements).  We recommend that you include biophysical 
characteristics (e.g., molecular weight, particle size) and 
biochemical characteristics (e.g., glycosylation sites).  You should 
also describe the nature of the genome of viral vectors, whether 
single-stranded, double-stranded, or self-complementary, 
deoxyribonucleic acid (DNA) or RNA, and the copy number of 
genomes per particle.   
 
  For microbial-based gene therapies, you should include defining 
physical and biochemical properties, growth characteristics, 
genetic markers (e.g., auxotrophic or attenuating mutations, 
antibiotic resistance), and the location (e.g., on plasmid, episome, 
or chromosome) and description of any inserted foreign genes and 
regulatory elements, if applicable.  For additional details on 
microbial vectors, please see FDAs Recommendations for 
Microbial Vectors used for Gene Therapy, Guidance for Industry 
(Ref. 9).  
 
  For ex vivo genetically modified cell-based gene therapies, you 
should describe the expected major and minor cell populations, as 
well as the vector that contains the transgene cassette that is 
transferred into the cell.  For cells that have been genetically 
modified using genome editing, you should describe the gene(s) 
that is altered and how the change(s) was made (i.e., the gene 
editing technology used).  A separate DS section should be 
provided for vectors used for ex vivo modification of cells (see 
section IV.B. of this guidance) and the structure of the vector 
should be included in this section.   
 
c.  General Properties (3.2.S.1.3) 
 
You should provide a section in the IND that describes the composition 
and properties of the DS, including the biological activity and proposed 
mechanisms of action.  
 
   
9  Contains Nonbinding Recommendations 
 
2.  Drug Substance Manufacture (3.2.S.2) 
 
a.  Manufacturer(s) (3.2.S.2.1) 
 
You must provide the name and address of each manufacturer, including 
contract manufacturer(s), involved in the manufacture, testing, and storage 
of the DS (21 CFR 312.23(a)(7)(iv)(a)).  You should indicate the 
responsibility of each manufacturer.  Your IND should contain complete 
information on the DS manufacturer, regardless of whether the process is 
performed by you or by a contract manufacturing organization (CMO).  
As the sponsor of the IND, you are ultimately responsible for the safety of 
subjects in the clinical investigation (21 CFR 312.3); therefore, we 
recommend that you and the CMO understand and document your 
respective responsibilities for ensuring product quality.  Additional 
information on quality agreements can be found in Contract 
Manufacturing Arrangements for Drugs: Quality Agreements; Guidance 
for Industry (Ref. 10).  
 
b.  Description of Manufacturing Process and Process Controls 
(3.2.S.2.2) 
 
Your description of the DS manufacturing process and process controls 
should include the following, as applicable:  cell culture; transduction; cell 
expansion; harvest(s); purification; filling; and storage and shipping 
conditions.  Your description should also accurately represent your 
process and process controls (Ref 16).  We acknowledge that information 
on process controls may be limited early in development and recommend 
that sponsors provide additional information and updates as product 
development proceeds.  Changes and updates to the DS manufacturing 
process and process controls information should be submitted as an 
amendment to the IND (21 CFR 312.31(a)(1)) prior to implementation for 
investigational use in clinical studies, as indicated in section II. of this 
guidance. 
 
i.  Batch and Scale 
 
A description of how you define each manufacturing run (i.e., 
batch, lot, other) should be submitted with an explanation of the 
batch (or lot7) numbering system.  You should clearly state 
whether any pooling of harvests or intermediates occurs during 
manufacturing.  If pooling is necessary during production, we 
recommend that you control the storage conditions (e.g., time, 
temperature, container closure system) for each pool and that you 
describe the testing performed prior to pooling to ensure the 
quality of each pool.  
                                                 
7 For purpose of this guidance, batch and lot are used interchangeably. 
10  Contains Nonbinding Recommendations 
 
 
We also recommend that you provide an explanation for how the 
batch scale is defined (e.g., bioreactor volume, cell processing 
capacity) and how the DS is quantified (e.g., vector genomes, 
transducing units, infectious particles, mass, number of gene 
modified cells) to facilitate review and allow a better 
understanding of the manufacturing process.  When known, please 
include the yield expected per batch. 
 
ii.  Manufacturing Process 
 
The description of your manufacturing process should include a 
process flow diagram(s) and a detailed narrative.  Your description 
should clearly identify any process controls and in-process testing 
(e.g., titer, bioburden, viability, impurities) as well as acceptable 
operating parameters (e.g., process times, temperature ranges, cell 
passage number, pH, CO , dissolved O , glucose level).  We 
2 2
acknowledge that this information may be gathered over the course 
of product development and may be submitted in a stage-
appropriate manner.     
 
We recommend that you monitor process performance parameters 
for process consistency.  Process trend analysis and evaluation of 
process parameters and materials will help to determine and 
establish process control strategies.  You should clearly describe 
any controls for cleaning and change over as well as tracking and 
segregation procedures that are in place to prevent cross-
contamination throughout the manufacturing process (see also 
section V.C.1., Appendices  Facilities and Equipment (3.2.A.1), 
of this guidance). 
 
a.  Cell Culture (for Vector Production) 
 
The description of all cell culture conditions should contain 
sufficient detail to make understandable any of the process 
steps that apply, process timing, culture conditions, hold 
times and transfer steps, and materials used (e.g., media 
components, bags/flasks).  You should describe whether 
the cell culture system is open or closed and any aseptic 
processing steps.  If extensive culture times are needed, you 
should outline the in-process controls you have in place to 
monitor cell quality (e.g., viability, bioburden, pH, 
dissolved O ).  Expectations for media components and cell 
2
bank qualification are outlined in section V.A.2.c., Control 
of Materials (3.2.S.2.3), of this guidance.  
 
11  Contains Nonbinding Recommendations 
 
b.  Vector Production 
 
For the manufacture of gene therapy vectors (e.g., virus, 
bacteria, plasmids), you should provide a description of all 
production and purification procedures.  Production 
procedures should include the cell culture and expansion 
steps, transfection or infection procedures, harvest steps, 
hold times, vector purification (e.g., density gradient 
centrifugation, column purification), concentration or 
buffer exchange steps, and the reagents/components used 
during these processes.  You should outline any in-process 
testing to ensure vector quality as appropriate (e.g., titer, 
impurities).  
 
You should describe whether the vector DS will be 
formulated into the DP for administration of the genetic 
material (section V.B.3.b., Batch Formula (3.2.P.3.2), of 
this guidance) or whether it will be formulated as a bulk DS 
for ex vivo genetic modification of cells (as outlined in 
section IV.B., Drug Substance and Drug Product, of this 
guidance).  As an active pharmaceutical ingredient, an 
appropriate level of control should be applied to each DS, 
and each DS should be manufactured under current good 
manufacturing practice (CGMP) conditions that are 
appropriate for the stage of development (Ref. 11).  
Information on Quality Unit is outlined in section V.C.1., 
Appendices  Facilities and Equipment (3.2.A.1), of this 
guidance.  
 
c.  Genetically Modified Cell Production 
 
If your gene therapy product consists of genetically 
modified cells, your cell processing description should 
contain sufficient detail to make understandable any of the 
following process steps that apply: source material (e.g., 
autologous or allogeneic cells; donor eligibility when 
applicable); collection of cellular source material (e.g., 
leukapheresis, biopsy); storage at the collection site; 
shipping to and handling at the manufacturing facility; cell 
selection, isolation, or enrichment steps (including 
methods, devices, reagents); cell expansion conditions; 
hold times and transfer steps;  cell harvest and purification, 
if any, and materials used.  
 
You should also provide a complete description of all 
procedures used for gene modification (such as 
12  Contains Nonbinding Recommendations 
 
transfection, infection or electroporation of vectors, or 
genome editing components) and any additional culture, 
cell selection, or treatments after modification.  The vector 
used should be described in detail as indicated above. 
 
d.  Irradiated Cells 
 
If your product contains or is processed with irradiated 
cells, you should provide information on the irradiator 
source, documentation for the calibration of the irradiator 
source and provide supporting data to demonstrate that the 
irradiated cells are rendered replication-incompetent, while 
still maintaining their desired characteristics. 
 
e.  Filling, Storage, and Transportation (Shipping) 
 
You should provide a detailed description and identify any 
associated process controls for formulation, filling, storage, 
and shipping of the DS, if applicable.  You should also 
describe the container closure system used for storage and 
shipping of the DS.  Information on container closure 
systems is outlined in section V.A.6., Container Closure 
System (3.2.S.6), of this guidance.  We recommend that 
you describe procedures that are in place to ensure 
appropriate storage and transport (as needed). 
 
c.  Control of Materials (3.2.S.2.3) 
 
You must provide a list of all materials used in manufacturing and a 
description of the quality or grade of these materials (21 CFR 
312.23(a)(7)(iv)(b)).  This information which may be provided in tabular 
format, includes the identity of the material, the supplier, the quality (e.g., 
clinical-grade, FDA-licensed), the source of material (e.g., animal, human, 
insect), and the stage at which each material is used in the manufacturing 
process (e.g., culture media, vector purification).  This includes 
information on components, such as cells, cell and viral banking systems, 
and reagents, as described in more detail below; it also includes raw 
materials and equipment that come into contact with the product, such as 
culture bags, culture flasks, chromatography matrices, and tubing.  
 
You should establish a qualification program and provide documentation 
that the materials used for manufacturing meet standards appropriate for 
their intended use (e.g., test results, certificates of analysis (COAs), 
package inserts).  COAs for materials may consist of sample or 
representative COAs and can be provided in the Appendices -
Adventitious Agents Safety Evaluation (3.2.A.2) section of the CTD and 
13  Contains Nonbinding Recommendations 
 
hyperlinked to relevant sections of your IND.  We recommend that you 
use FDA-licensed, approved, or cleared materials, or other clinical-grade 
materials, when they are available.  If the material is not FDA-licensed, 
approved, or cleared (or in the absence of recognized standards), 
additional information on the manufacturing and/or testing may be needed 
to evaluate the safety and quality of the material.  As outlined below, the 
extent of recommended testing will depend on the specific material and 
the manner in which it is used in the manufacturing process. 
 
i.  Reagents 
 
For purpose of this guidance, reagents (or ancillary materials) are 
those materials used for manufacturing (e.g., cell growth, 
differentiation, selection, purification, or other critical 
manufacturing steps) that are not intended to be part of the final 
product.  Examples include fetal bovine serum, digestive enzymes 
(e.g., trypsin, collagenase, DNase/RNase, restriction 
endonucleases), growth factors, cytokines, monoclonal antibodies, 
antibody-coated beads, antibiotics, media, media components, and 
detergents.  These reagents can affect the safety, potency, and 
purity of the final product, especially by introducing adventitious 
agents or other impurities.   
 
For biologically sourced reagents, including human, bovine, and 
porcine-derived materials, we recommend that you also refer to 
FDAs Guidance for Industry:  Characterization and Qualification 
of Cell Substrates and Other Biological Materials Used in the 
Production of Viral Vaccines for Infectious Disease Indications 
(Ref. 12).  Animal-derived materials increase the risk of 
introducing adventitious agents.  Certain animal-derived materials, 
such as sera, are complex mixtures that are difficult to standardize, 
and such materials may have significant batch-to-batch variations 
that may affect the reproducibility of your manufacturing process 
or the quality of your final product.  We recommend that you 
consider using non-animal-derived reagents if possible (e.g., 
serum-free tissue culture media and recombinant proteases). 
 
a.  Bovine 
 
We recommend that you provide information on any 
bovine material used in manufacturing, including the 
source of the material; information on the location where 
the herd was born, raised, and slaughtered; and any other 
information relevant to the risk of transmissible spongiform 
encephalopathy (TSE).  If serum is used, we recommend 
that it be -irradiated to reduce the risk of adventitious 
14  Contains Nonbinding Recommendations 
 
agents.  This information may be included on the COA and 
Certificate of Origin (COO) provided from the supplier. 
 
Bovine materials used in production of reagents, which are, 
in turn, used in manufacturing a product, should also be 
identified, and the source and qualification of bovine 
material should be documented.   
 
You should provide sample COAs and COOs (in section 
3.2.A.2 of the CTD) for all bovine material lots used in the 
manufacture and establishment of cell and virus banks 
consistent with 9 CFR 113.53. 
 
b.  Porcine 
 
You should provide sample COAs (in section 3.2.A.2 of the 
CTD) for all porcine material lots used in manufacture and 
establishment of cell and virus banks consistent with 9 CFR 
113.53.  In addition, we recommend porcine reagents be 
tested for porcine circovirus 1 and 2, porcine parvovirus 
and other zoonotic viruses, as appropriate. 
 
c.  Murine or Monoclonal Antibodies 
 
Monoclonal antibodies used in manufacturing that have 
product contact should be tested as per the 
recommendations described in FDAs Points to Consider 
in the Manufacture and Testing of Monoclonal Antibody 
Products for Human Use (Ref. 13).  Alternatively, as 
described in section III.D. of this guidance, we recommend 
you provide a LOA to cross-reference this information in a 
different regulatory submission (IND or MF).  You should 
also consider that many monoclonal antibodies and 
recombinant proteins (such as cytokines) used during the 
manufacture of gene therapy products may be purified by 
affinity chromatography using antibodies generated from 
mouse hybridomas.  This may introduce the risk of 
contamination with adventitious agents from rodents, 
which should be controlled for by the supplier. 
 
d.  Human Source 
 
If human albumin is used, you should use FDA-licensed 
products and have procedures in place to ensure that no 
recalled lots were used during manufacture or preparation 
of the product.   
15  Contains Nonbinding Recommendations 
 
 
If human AB serum is used (e.g., for ex vivo genetically 
modified cells), you should ensure the serum is processed 
from blood or plasma collected at FDA licensed facilities.  
Source Plasma, which is often used to make human AB 
serum, must be collected as described in 21 CFR Part 640, 
Subpart G.  Source Plasma is not tested as extensively as 
blood products intended for infusion, and we recommend 
that you ensure the AB serum used in your manufacturing 
does not have the potential to transmit infectious disease.  
For example, if your serum is derived from Source Plasma, 
which may be pooled from a large number of donors, you 
may reduce the risk of infectious disease by conducting 
additional testing for relevant transfusion-transmitted 
infections.  Alternatively, it may be acceptable to include 
viral inactivation or clearance steps in the production of AB 
serum from Source Plasma.  
 
For all other reagents that are human-derived, you should 
identify whether the reagent is a licensed product (e.g., 
HSA, IL-2) or is clinical or research grade and provide a 
COA or information regarding testing of the donor or 
reagent. 
 
ii.  Cells - Autologous and Allogeneic Cells or Tissue 
 
For autologous or allogeneic cells or tissue, you should provide a 
description of the cell source, the collection procedure, and any 
related handling, culturing, processing, storage, shipping, and 
testing that is performed prior to use in manufacture.  Your 
description should include the following information: 
 
  materials used for collection (including devices, reagents, 
tubing, and containers);  
 
  method of cell collection (i.e., standard blood draw, bone 
marrow aspiration, or apheresis); 
 
  enrichment steps, cryopreservation, if performed;  
 
  labeling and tracking of collected samples;  
 
  hold times; and  
 
  transportation conditions to the manufacturing facility.   
 
16  Contains Nonbinding Recommendations 
 
As an example, for cells collected by leukapheresis:  you should 
provide a summary description of the collection device(s); 
operating parameters; volumes or number of cells to be collected; 
and how the collected material is labeled, stored, tracked, and 
transported to the manufacturing facility.  Establishing well-
designed process controls and standard operating procedures 
(SOPs) for manipulating and handling starting and in-process 
materials can help reduce variability in the manufacturing process 
and ultimately in the DS and DP.  
 
This is especially important for multi-center clinical trials, where 
establishing standardized procedures for cell collection and 
handling across all collection sites is critical to assuring the quality 
and safety of the final product as well as ensuring control of the 
manufacturing process.  In your IND, you should include a list of 
collection sites, their FDA Establishment Identifier, and any 
accreditations for compliance with established standards (e.g., 
Foundation for the Accreditation of Cellular Therapy (FACT)), if 
applicable.  
 
a.  Autologous Cells 
 
You are not required to make a donor eligibility 
determination or to perform donor screening on autologous 
cells or tissues (21 CFR 1271.90(a)(1)).  However, you 
should determine based on donor information whether your 
manufacturing procedures increase the risk to the patient by 
further propagation of pathogenic agents that may be 
present in the donor, as applicable.  You should also 
describe precautions to prevent the spread of viruses or 
other adventitious agents to persons other than the 
autologous recipient (Ref. 14). 
 
b.  Allogeneic Cells 
 
For allogeneic cells or tissues, you must perform donor 
screening and testing, as required in 21 CFR Part 1271, 
Subpart C, except for those cells and tissues that meet the 
exceptions in 21 CFR 1271.90(a).  Donors of all types of 
cells and tissues must be screened for risk factors and 
clinical evidence of relevant communicable disease agents 
and diseases, including:  human immunodeficiency virus 
(HIV); hepatitis B virus (HBV); hepatitis C virus (HCV); 
human TSE, including Creutzfeldt-Jakob disease (CJD) and 
variant CJD (vCJD); and Treponema pallidum (syphilis) 
(21 CFR 1271.75).  In addition, donors of viable leukocyte-
17  Contains Nonbinding Recommendations 
 
rich cells or tissues should be screened for human T-
lymphotropic virus (HTLV).  You must also test a 
specimen of donor cells or tissue for evidence of infection 
due to relevant communicable disease agents, including:  
HIV-1; HIV-2; HBV; HCV; syphilis; and if the material is 
leukocyte-rich cells or tissue, HTLV-1, HTLV-2, and 
cytomegalovirus (21 CFR 1271.85).  For donor eligibility 
testing, you must use appropriate FDA-licensed, approved, 
or cleared donor screening tests (21 CFR 1271.80(c)).  
Moreover, the required testing must be performed by a 
laboratory that either is certified to perform such testing on 
human specimens under the Clinical Laboratory 
Improvement Amendments of 1988 (42 U.S.C. 263a) and 
42 CFR Part 493 or has met equivalent requirements as 
determined by the Centers for Medicare and Medicaid 
Services (21 CFR 1271.80(c)).  You should also refer to 
recent Center for Biologics Evaluation and Research 
guidance documents on donor eligibility for additional 
information on testing for emerging relevant communicable 
disease agents and diseases (e.g., West Nile virus (WNV), 
Zika virus).  If cord blood or other maternally-derived 
tissue is used, you must perform screening and testing on 
the birth mothers, as described in 21 CFR 1271.80(a).  
 
Allogeneic cells from a single donor or source tissue may 
sometimes be expanded and stored for greater consistency 
and control in manufacturing.  In these situations, we 
generally recommend that you qualify allogeneic master 
and working cell banks in the same way as cell banks used 
for production of viral vectors (see section V.A.2.c.iii., 
Banking Systems, below), provided that you have 
sufficient material for this testing.  In these situations, we 
are concerned about the introduction of adventitious agents 
(e.g., viruses, bacteria, mycoplasma) during the bank 
manufacturing process, especially from human, bovine or 
porcine materials, animal feeder cells, other animal-derived 
reagents, or human AB serum, if used.  If your allogeneic 
cell bank is small, please consult with the Quality Reviewer 
of your file for more information on appropriate 
qualification of small scale allogeneic cell banks. 
 
iii.  Banking Systems (Starting Materials) 
 
A banking system improves control and consistency in the 
manufacturing of many biologics.  Banking assures an adequate 
supply of equivalent, well-characterized material for production 
18  Contains Nonbinding Recommendations 
 
over the expected lifetime of production.  For these reasons, 
banked materials are a common starting point for many routine 
production applications.  We outline our current thinking for the 
qualification of different banking systems below, including banks 
of cell substrates for production of viral vectors, banks of 
bacterial/microbial cells, allogeneic donor cell banks, and banks of 
viral vectors.  We recommend that you provide a summary of the 
testing in this section, and COAs in section 3.2.A.2 of the CTD.  
Information on bank qualification and adventitious agent testing 
should also be included in your comprehensive Adventitious 
Agents Safety Evaluation (3.2.A.2) section of the CTD. 
 
a.  Master Cell Banks Used as Substrates for Production of 
Viral Vectors 
 
Prior to selecting a cell line for viral vector manufacturing, 
you should carefully consider characteristics of the cells 
that may impact the safety of the final product (such as 
presence of tumorigenic sequences).  This is especially 
important when the viral vector co-packages non-vector 
sequences, such as adeno-associated virus (AAV) (see 
section V.A.3.b., Impurities (3.2.S.3.2), of this guidance).  
We also recommend that you consider cell attributes that 
can affect production capacity (e.g., growth characteristics, 
vector production capacity), prior to generation of a cell 
bank.  
 
In your IND, you should provide a description of the 
history and detailed derivation of the source material for 
the cell bank.  Your description should include information 
on cell source (including species of origin); how the bank 
was generated (e.g., from a single colony isolate or through 
limiting dilution); testing performed to characterize the 
bank; and if applicable, materials used to genetically 
modify the source material (e.g., packaging cell line).  
 
When a cell substrate has been genetically modified (e.g., 
to provide viral proteins to allow virus replication or 
packaging), you should provide a description of the 
materials used for the genetic modification, including 
information on the quality of the materials (e.g., plasmids, 
viruses, gene editing components) used to introduce the 
genetic changes.  Materials used to generate cell substrates 
for production of viral vectors should be sufficiently 
characterized to ensure safety and purity of the final gene 
therapy product  
19  Contains Nonbinding Recommendations 
 
 
In addition, we recommend that you provide information 
on how the cell banks are stored and maintained as well as 
detailed information on qualification to adequately 
establish the safety, identity, purity, and stability of the 
cells used in your manufacturing process.  Additional 
sources of information regarding qualification of cell 
substrates can be found in FDAs Guidance Q5D Quality 
of Biotechnological/Biological Products: Derivation and 
Characterization of Cell Substrates Used for Production of 
Biotechnological/Biological Products (Ref. 15) and FDAs 
Guidance for Industry:  Characterization and Qualification 
of Cell Substrates and Other Biological Materials Used in 
the Production of Viral Vaccines for Infectious Disease 
Indications. (Ref. 11). 
 
Cell bank qualification should include tests to: 
 
  Ensure absence of microbial contamination, 
including sterility, mycoplasma (and spiroplasma 
for insect cells), and adventitious viral agents.  For 
cell lines used for production of viral vectors, we 
recommend that you test for retroviral 
contamination, using reverse transcriptase assays 
and transmission electron microscopic analysis.  
  The presence of an adventitious viral agent in your 
bank should be vigorously investigated, and re-
derivation of the bank should be considered.  In 
some instances, robust viral clearance studies may 
be necessary to remove and inactivate adventitious 
agents.  
 
-  For additional information on analytical 
methods used for cell bank qualification, 
please see section V.A.4.b., Analytical 
Procedures (3.2.S.4.2), of this guidance. 
 
-  For cell lines that have been exposed to 
bovine or porcine components (e.g., serum, 
serum components, trypsin), appropriate 
testing should include testing for bovine or 
porcine adventitious agents.  See further 
discussion on bovine (section V.A.2.c.i.a.) 
and porcine (section V.A.2.i.b.) reagents, 
above. 
 
20  Contains Nonbinding Recommendations 
 
  Ensure absence of species-specific pathogens. 
 
-  For human cells, this may include testing for 
cytomegalovirus (CMV), HIV-1 & 2, 
HTLV-1 &-2, human herpesvirus-6, -7 and -
8 (HHV-6, -7 & -8), JC virus, BK virus, 
Epstein-Barr virus (EBV), human 
parvovirus B19, HBV, human 
papillomavirus (HPV), and HCV, as 
appropriate. 
 
-  For other animal or insect cells, we 
recommend tests for species-specific 
viruses, as appropriate.  For instance, for 
Vero cells, we recommend testing for simian 
polyomavirus SV40 and simian retrovirus. 
 
-  For insect cells, you should evaluate the 
presence of arboviruses in a susceptible cell 
line, such as baby hamster kidney (BHK21) 
cells.  Insect cell lines with known viral 
contamination should be avoided. 
 
  Identify cells.  You should identify your cells 
through tests that distinguish them from other cell 
lines used in your facility.  For cell lines that you 
have purchased from a type collection, vendor, or 
received from another investigator, we recommend 
master cell bank (MCB) testing to confirm the 
purity of the cells by genetic analysis (i.e., short 
tandem repeat analysis or other profiling analysis) 
(Ref. 16). 
 
  Establish stability of the cell bank.  Stability can be 
assessed by measuring viability of cells over time 
after cryopreservation.  We also recommend a one-
time test of end of production cells (EOP) or mock 
production cells of similar passage history, to be 
tested for their suitability to produce your vector.  
For stable retroviral vector producer cells, we 
recommend that you test the genetic stability of the 
gene insert in the EOP cells. 
 
  Assess the ability of new cell lines to form tumors.  
We recommend that you perform tumorigenicity 
tests for cell lines that have not been previously 
21  Contains Nonbinding Recommendations 
 
characterized for their potential to form tumors.  
This test would not be necessary for cells known to 
form tumors; please see additional information on 
testing for process-related impurities under section 
V.A.3.b.i., Drug Substance Characterization 
(3.2.S.3), of this guidance. 
 
b.  Working Cell Banks 
 
A Working Cell Bank (WCB) may be derived from one or 
more vials of the MCB.  The information to document 
qualification and characterization for a WCB is generally 
less extensive than that for the MCB.  WCB testing should 
include, but is not limited to, sterility, mycoplasma, 
identity, and in vitro adventitious agent tests.  For 
additional information on analytical methods used for 
WCB qualification, please see section V.A.4.b., Analytical 
Procedures (3.2.S.4.2), of this guidance. 
 
c.  Bacterial or Microbial Master Cell Banks 
 
Bacterial MCBs are frequently used as the starting material 
to generate plasmid DNA, which can be used as a gene 
therapy DS or used as a manufacturing intermediate to 
generate a DS for other gene therapy products, such as 
AAV or lentiviral vectors.  Bacterial MCBs also may be 
used to generate a microbial vector for gene therapy (Ref. 
9).  
 
We generally recommend the establishment of a bacterial 
MCB, as it can provide a consistent starting material for the 
manufacture of plasmids or microbial vectors.  However, 
MCBs may not be necessary for all manufacturing 
situations if the plasmid intermediate is appropriately 
qualified (e.g., for early phase studies when the plasmid is 
used to make a vector for ex vivo modification of cells).  
We recognize the diversity of uses for bacterial MCBs, and 
recommend that you appropriately qualify the bank, and 
submit sufficient detailed information for the qualification 
of the banked material regardless of use.    
 
You should provide a description of the history and 
derivation of the materials used to generate the cell bank, 
including information on how plasmid vectors were 
designed and constructed.  For the bank material, itself, you 
should describe the genotype and source of the microbial 
22  Contains Nonbinding Recommendations 
 
cells, provide information on how the material was 
generated, and how the bank is stored and maintained as 
well as information on the qualification of the bank 
(including cell bank COAs) to adequately establish the 
safety, identity, purity, and stability of the microbial cell 
preparation used in the manufacturing process.  
 
For bacterial cell banks used to manufacture a DNA 
plasmid, we recommend the testing include: 
 
  Bacterial host strain identity; 
 
  Plasmid presence, confirmed by bacterial growth on 
selective medium, restriction digest, or DNA 
sequencing; 
 
  Bacterial cell count; 
 
  Bacterial host strain purity (no inappropriate 
organisms, negative for bacteriophage); 
 
  Plasmid identity by RE analysis; 
 
  Full plasmid sequencing.  We recommend that you 
fully sequence plasmids and submit an annotated 
sequence for the vector, as described in more detail 
in the section below on viral vector banks; and 
 
  Transgene expression and/or activity, as applicable. 
 
For microbial cell banks used to manufacture microbial 
vectors, our recommendations for MCB testing are outlined 
in FDAs Recommendations for Microbial Vectors used 
for Gene Therapy, Guidance for Industry (Ref. 9).  
 
d.  Master Viral Banks 
 
Viral banks may be expanded to manufacture a viral vector 
DS (e.g., herpesvirus-based vectors, adenovirus-based 
vectors), or they may be used to generate helper viruses for 
manufacturing non-replicating vectors (e.g., AAV, gutless 
adenovirus).  We acknowledge the diversity of uses of viral 
banks, and we recommend the same level of detail be 
submitted for the qualification of banked material, 
regardless of use.  You should provide a detailed 
description of the history and derivation of the source or 
23  Contains Nonbinding Recommendations 
 
seed materials for these banks.  You should describe how 
the seed stock was generated and what cells and animal-
derived materials were used in the derivation process.  
 
A gene map of the final vector and vector intermediates is 
useful when describing the history and derivation of 
recombinant viral vectors.  We recommend that you state 
whether the seed material was plaque-purified, purified by 
limiting dilution, or rescued from DNA or RNA clones and 
how many times it was passaged, during expansion.  
 
For the banked material, you should describe the 
manufacturing process and the conditions under which the 
banked material was generated, for example, in a research 
laboratory or a good manufacturing practice facility.  You 
should provide a list of animal-derived materials used in 
the generation of the bank and state whether the master 
virus bank (MVB) is expected to represent a single clone or 
a distribution of viral variants or sequences.  
 
We also recommend that you provide information on how 
the bank is stored and maintained as well as detailed 
information on the qualification of the bank to adequately 
establish the safety, identity, purity, and stability of the 
virus preparation used in the manufacturing process.  If a 
COA is available, it should be submitted to the IND 
(section 3.2.A.2 of the CTD).  For additional information 
on analytical methods used for MVB qualification, please 
see Analytical Procedures (3.2.S.4.2) section V.A.4.b. of 
this guidance. 
 
We recommend viral vector bank qualification includes 
tests to: 
 
  Ensure absence of contamination, including 
sterility, mycoplasma, and in vivo and in vitro 
testing for adventitious viral agents. 
 
  Ensure absence of specific pathogens that may 
originate from the cell substrate, such as human 
viruses if the cell line used to produce the MVB is 
of human origin, or pathogens specific to the origin 
of the production cell line (e.g., murine, non-human 
primate, avian, insect). 
 
24  Contains Nonbinding Recommendations 
 
  Ensure absence of replication competent virus in 
replication incompetent vectors. 
 
  Ensure viral titer or concentration. 
 
  Ensure sensitivity to anti-viral drugs, as applicable, 
for example, herpes simplex virus (HSV) sensitivity 
to ganciclovir. 
 
  Ensure transgene activity, if appropriate. 
 
  Identify the viral vector and therapeutic transgene 
(e.g., Southern blot or restriction endonuclease 
analysis), as needed. 
 
  Ensure the correct genetic sequence.  We 
recommend that you fully sequence all vectors that 
are 40 kb or smaller, analyze the sequence, and 
submit an annotated sequence of the entire vector.  
You should provide an evaluation of the 
significance of all discrepancies between the 
expected sequence and the experimentally 
determined sequence and an evaluation of the 
significance of any unexpected sequence elements, 
including open reading frames.  We have the 
following recommendations, regarding sequence 
analysis: 
 
-  We recommend that viral vectors be 
sequenced from the MVB, when possible. 
 
-  For integrating viral vectors, we recommend 
that you perform DNA sequencing on the 
integrated vector.  The material for 
sequencing can be collected from the 
producer cell line or, in the case of vectors 
generated by transient transfection, from 
material collected from cells that you have 
transduced after isolation of a vector lot. 
 
-  For other situations in which no MVB 
exists, sequencing should be performed from 
the DS or DP.  For example, AAV vectors 
are typically made by plasmid transfection, 
and the AAV vector is harvested directly 
from transfected cells to produce a DS.  In 
25  Contains Nonbinding Recommendations 
 
this situation, we recommend that you 
sequence one or more lots (either material 
from DS or DP) to confirm that the vector 
sequence is stable, during manufacturing.  
 
-  For viral vectors greater than 40 kb, you 
should summarize the extent and results of 
sequence analysis that you have performed, 
including any testing performed by 
restriction endonuclease analysis.  You 
should perform sequence analysis of the 
gene insert, flanking regions, and any 
regions of the vector that are modified or 
deleted or that could be susceptible to 
recombination.  The entire vector sequence 
will be necessary to confirm identity for 
licensure. 
 
e.  Working Viral Banks 
 
A working viral bank (WVB) may be derived from one or 
more vials of the MVB, and the information to document 
qualification and characterization of the WVB is less 
extensive than that needed for the MVB.  You should 
describe the process used to generate the WVB and 
whether animal-derived materials were used.  Testing for 
WVB should include, but is not limited to, sterility, 
mycoplasma, identity, and in vitro adventitious agent tests.  
For additional information on the analytical methods used 
for WCB qualification, please see section V.A.4.b., 
Analytical Procedures (3.2.S.4.2), of this guidance. 
 
d.  Control of Critical Steps and Intermediates (3.2.S.2.4) 
 
You should describe the control of critical steps and intermediates in the 
manufacturing process.  Critical control steps include those outlined in 
section V.A.2.b., Description of Manufacturing Process and Process 
Controls (3.2.S.2.2), of this guidance.  We recommend that you also 
consider any steps in which in-process tests with acceptance criteria are 
performed as critical control steps.  The Agency acknowledges that this 
information may be limited in the early phases of development and 
recommends that sponsors provide additional information and updates as 
product development proceeds.   
 
You should define manufacturing intermediates and provide information 
on the quality and control of intermediates.  Intermediates may include 
26  Contains Nonbinding Recommendations 
 
material from collection or hold steps, such as temporary storage of bulk 
harvest, concentration steps, or purification intermediates (e.g., column 
fractions or eluate).  The duration of production steps and hold times 
should be controlled and recorded to facilitate the establishment of process 
limits and to allow for future validation of each step and hold time within 
the proposed limits in support of a license application. 
 
Intermediates in gene therapy manufacturing may also include DNA 
plasmids that are used in the manufacture of other gene therapy products, 
such as AAV or lentiviral vectors.  We recommend that DNA plasmid 
intermediates be derived from qualified banks, as described in more detail 
above and in section V.A.2.c., Control of Materials (3.2.S.2.3), of this 
guidance.  In addition, we recommend that you provide information on the 
plasmid manufacturing procedures, reagents, and plasmid specifications 
for use, regardless of whether they were made by the IND sponsor or a 
contract manufacturer.  In general, we recommend that this testing include 
assays to ensure the identity, purity, potency, and safety of the final 
product.  For a DNA plasmid, this may include sterility, endotoxin, purity 
(including percent of supercoiled form and residual cell DNA, RNA, and 
protein levels), and identity testing (restriction digest and sequencing if 
sequencing was not performed on the bacterial bank).  A COA 
documenting plasmid quality testing should be included in the IND 
(section 3.2.A.2 of the CTD). 
 
e.  Process Validation and/or Evaluation (3.2.S.2.5) 
 
Process validation studies are generally or typically not required for early 
stage manufacturing, and thus, most original IND submissions will not 
include process performance qualification.  We recommend that you use 
early stage manufacturing experience to evaluate the need for process 
improvements and to support process validation studies in the future. 
Additional information on process validation can be found in FDAs 
Guidance for Industry: Process Validation:  General Principles and 
Practices (Ref. 17). 
 
In general, we recommend that INDs at all stages of development should 
have established written SOPs to ensure proper manufacturing control and 
oversight.  Manufacturing oversight is usually performed by a dedicated 
Quality Unit, the duties of which include implementing procedures to 
prevent microbial contamination, cross-contamination, and product mix-
ups.  Your Quality Unit should have procedures in place to investigate lot 
failures, out-of-specification results, and ways to implement corrective 
actions.  We recommend that your IND include a description or summary 
of your Quality Unit, including the manner in which quality control testing 
and oversight are separated from the manufacturing unit.  For more 
27  Contains Nonbinding Recommendations 
 
information see section V.C.1., Appendices  Facilities and Equipment 
(3.2.A.1), of this guidance. 
 
f.  Manufacturing Process Development (3.2.S.2.6) 
 
You should provide a description and discussion of the developmental 
history of the manufacturing process as described in the Description of 
Manufacturing Process and Process Controls (3.2.S.2.2) section of the 
CTD.   
 
For early stage INDs, there may be differences between the manufacturing 
and testing of the toxicology lots and the material you plan to use in the 
clinical studies.  For later stage INDs, there may be changes to the 
manufacturing process as part of process development, optimization, or 
under certain conditions there may be reprocessing step(s).  In both 
situations, we recommend that you describe how manufacturing 
differences are expected to impact product safety and activity and to 
provide batch analysis information in the Batch Analysis (3.2.S.4.4) 
section of the CTD.   
 
If you make significant manufacturing changes, then comparability studies 
may be necessary to determine the impact of these changes on the identity, 
purity, potency, and safety of the product.  The extent of comparability 
testing would depend on the manufacturing change, the ability of 
analytical methods to detect changes in the product, and the stage of 
clinical development.  For first-in-human studies, any differences between 
toxicology lots and clinical lots should be assessed for their impact on 
product safety.  For later phase studies, especially those designed to 
measure product efficacy, differences in clinical lots should be assessed 
for their impact on product safety and activity.  
 
Please note that it is important to retain samples of the DS and 
manufacturing intermediates, when possible, in the event that 
comparability studies are requested during future product development or 
after licensure of the investigational DP. 
 
3.  Drug Substance Characterization (3.2.S.3) 
 
a.  Elucidation of Structure and Other Characteristics (3.2.S.3.1) 
 
We recommend that you include annotated sequence analysis for your 
vector in the original IND submission and any additional sequence 
information gathered during the course of product development in 
subsequent submissions.  Raw sequence data may be included in the 
Regional Information (3.2.R) section of the CTD.  In addition, we 
recommend that you provide any further information confirming the 
28  Contains Nonbinding Recommendations 
 
primary, secondary, or higher order structure; post-translational 
modifications; and/or distribution of cell types for the DS if it has not 
already been described in the Structure (3.2.S.1.2) section of the CTD. 
 
b.  Impurities (3.2.S.3.2) 
 
We recommend that your manufacturing process be designed to remove 
process- and product-related impurities and that you have tests in place to 
measure levels of residual impurities.  You should describe your test 
procedures in the IND and set appropriate limits.  Your initial 
specification, including acceptance limits, for impurities may be refined 
with additional manufacturing experience.  We recommend that you 
measure impurities throughout product development, as this will help 
ensure product safety, contribute to your understanding of the 
manufacturing process, and provide a baseline for comparing product 
quality after manufacturing changes, if needed. 
 
i.  Process-Related Impurities 
 
We recommend testing for process-related impurities.  These 
include, but are not limited to, residual cell substrate proteins, 
extraneous nucleic acid sequences, helper virus contaminants (i.e., 
infectious virus, viral DNA, viral proteins), and reagents used 
during manufacture, such as cytokines, growth factors, antibodies, 
selection beads, serum, and solvents. 
 
A common process-related impurity for many viral preparations is 
residual nucleic acid, such as cell substrate DNA, which can co-
purify with the vector.  Some vectors, including AAV, can also 
package (i.e., inside the viral capsid) a large amount of plasmid 
DNA sequences (used during transfection) as well as cellular 
DNA.  The presence of these impurities may have adverse effects 
on product quality and safety.  We recommend that you optimize 
your manufacturing process to reduce non-vector DNA 
contamination in your product.  Additionally, you should monitor 
and control the amount of extraneous nucleic acid sequences in 
your product.   
 
Since some cell substrates also harbor tumorigenic genetic 
sequences or retroviral sequences that may be capable of 
transmitting infection, we recommend that you take steps to 
minimize the biological activity of any residual DNA associated 
with your viral preparation.  This can be accomplished by reducing 
the size of the DNA to below the size of a functional gene and by 
decreasing the amount of residual DNA.  We recommend that you 
limit the amount of residual DNA for continuous non-tumorigenic 
29  Contains Nonbinding Recommendations 
 
cells to less than 10 ng/dose and the DNA size to below 
approximately 200 base pairs (Ref. 12).   
 
If you are using cells that are tumor-derived (e.g., Hela) or have 
tumorigenic phenotypes (e.g., HEK293, HEK293T) or other 
characteristics that may give rise to special concerns, the limitation 
of specific residual DNA quantities may be needed to assure 
product safety.  In addition to controlling host cell DNA content 
and size, as described above, you should also control the level of 
relevant transforming sequences in your product with acceptance 
criteria that limit patient exposure.  For example, products made in 
293T cells should be tested for adenovirus E1 and SV40 Large T 
antigen sequences, similarly products made in Hela cells should be 
tested for E6/E7 genes.  Your tests should be appropriately 
controlled and of sufficient sensitivity and specificity to determine 
the level of these sequences in your product.   
 
Some vectors, including AAV, can package a large amount of non-
vector DNA (e.g., plasmid DNA, helper virus sequences, cellular 
DNA), and it may not be possible to remove or reduce this DNA 
from the product to a level to assure safety based on current 
guidance (Ref. 12).   Therefore, we strongly recommend that the 
cell lines and helper sequences used to make viral vectors that 
package non-vector DNA, such as AAV, be carefully chosen to 
reduce the risks of the product.  Sponsors should provide necessary 
quality data, risk assessments, and/or details of their process and 
product control strategy to address and mitigate potential risks 
posed by the manufacturing systems used. 
 
ii.  Product-Related Impurities 
 
For viral vectors, typical product-related impurities may include 
defective interfering particles, non-infectious particles, empty 
capsid particles, or replicating recombinant virus contaminants.  
These impurities should be measured and may be reported as a 
ratio, for example, full:empty particles or virus particles:infectious 
units.  
 
For ex vivo genetically modified cells, product-related impurities 
include non-target cells, which may be present after selection or 
enrichment, and unmodified target cells, which may be present 
after the ex vivo modification step.  We recommend that you 
evaluate the nature and number of non-target cells and measure the 
percentage of cells that have been genetically modified.  As you 
develop a greater understanding of the cellular phenotypes present 
in your product during clinical development, you may also 
30  Contains Nonbinding Recommendations 
 
consider adding impurity tests for specific cell populations in order 
to establish greater manufacturing control.   
 
4.  Control of Drug Substance (3.2.S.4) 
 
a.  Specification (3.2.S.4.1) 
 
You should list DS specifications in your original IND submission.  
Specifications are defined as a list of tests, references to analytical 
procedures, and appropriate acceptance criteria used to assess quality.  
Acceptance criteria should be established and justified, based on data 
obtained from lots used in preclinical and/or clinical studies, data from lots 
used for demonstration of manufacturing consistency, data from stability 
studies, and relevant development data. 
 
For products in the early stages of clinical development, very few 
specifications are finalized, and some tests may still be under 
development.  However, the testing plan submitted in your IND should be 
adequate to describe the physical, chemical, or biological characteristics of 
the DS necessary to ensure that the DS meets acceptable limits for 
identity, strength (potency), quality, and purity (21 CFR 
312.23(a)(7)(iv)(a)).  
 
Your IND should include specifications with established acceptance 
criteria for safety testing at Phase 1.  Safety testing includes tests to ensure 
freedom from extraneous material, adventitious agents, microbial 
contamination, and replication competent virus.  Information on some 
common safety test methods is provided in more detail in section V.A.4.b., 
Analytical Procedures (3.2.S.4.2), of this guidance.  To maximize the 
sensitivity of safety testing, it is important that you perform each test at the 
stage of production at which contamination is most likely to be detected.  
For example, tests for mycoplasma or adventitious viruses (in vivo or in 
vitro) should be performed on cell culture harvest material (cells and 
supernatant) prior to further processing, e.g., prior to clarification, 
filtration, purification, and inactivation.8 
 
Your IND should also include specifications for measuring an appropriate 
dose level (i.e., strength or potency) at Phase 1.  To ensure consistent 
dosing in your clinical investigations, assays used to determine dose (e.g., 
vector genome titer by quantitative polymerase chain reaction (qPCR), 
transducing units, plaque-forming units, flow cytometry for transduced 
cells) should be qualified as suitable for use prior to initiating clinical 
studies.  Information on how to qualify your dose determining assay is 
                                                 
8 For additional information regarding the use of control-cell cultures, refer to FDAs Guidance for Industry: 
Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of 
Viral Vaccines for Infectious Disease Indications (Ref. 12).  
31  Contains Nonbinding Recommendations 
 
provided in section V.A.4.c., Validation of Analytical Procedures 
(3.2.S.4.3), of this guidance.  
 
Additional testing will depend on the type of gene therapy product and the 
phase of clinical development.  These tests may include assays to assess 
product characteristics, such as identity, purity (including endotoxin and 
contaminants, such as residual host cell DNA, bovine serum albumin 
(BSA), DNase), and potency/strength.  For additional information on 
potency tests, please refer to FDAs Guidance for Industry:  Potency 
Tests for Cellular and Gene Therapy Products (Ref. 18).  
 
Please note that the same types of tests listed in this section of the 
guidance may not be necessary for the release of both the DS and DP.  In 
certain situations, the DS and DP may not be readily distinguishable due 
to the design of manufacturing process and control.  In such cases, the 
sponsor may inquire with FDA on how to define DS and DP and meet 
release requirements.  In some cases, repeat testing may be good practice; 
however, redundant testing may not always be feasible or provide 
additional information.  In this case, we recommend that you provide a 
rationale to support the selection of testing performed for release of either 
the DS or DP. 
 
We provide some additional comments regarding tests for product 
characterization and impurities under section V.B.5.a., Specifications 
(3.2.P.5.1), of this guidance.   
 
b.  Analytical Procedures (3.2.S.4.2) 
 
You should provide a description of the analytical procedures used during 
manufacturing to assess your manufacturing process and product quality.  
In your original IND submission, your descriptions should have sufficient 
detail, including a description of system suitability control, so that we can 
understand and evaluate the adequacy of your procedures.  We 
recommend that you develop detailed SOPs for how your analytical 
procedures are conducted at early stages of product development as a part 
of your quality system.  We acknowledge that, during product 
development, analytical methods may be modified to improve control and 
suitability.  However, assay control is critical during all phases of clinical 
development to ensure product quality and safety and to allow for 
comparability studies, following manufacturing changes.  When possible, 
we recommend trending assay performance to gain additional 
understanding of the method and method improvements during product 
development.  
 
Documentation submitted in support of your analytical procedures should 
describe in detail how a procedure is performed and should specify any 
32  Contains Nonbinding Recommendations 
 
reference standards, equipment, and controls to be used.  Submission of 
information, such as individual SOPs or batch records, will generally not 
be necessary, provided descriptions of your analytical procedures are 
sufficiently detailed in your IND.  Contractor test reports are acceptable, 
provided there is adequate description of the analytical procedure, test 
sensitivity, specificity, and controls. 
 
i.  Safety Testing 
 
Safety testing on the DS should include microbiological testing, 
such as bioburden (or sterility, as appropriate), mycoplasma, and 
adventitious viral agent testing, to ensure product quality.  
Guidelines and/or procedures for many safety tests have been 
described in detail, elsewhere (e.g., bioburden,9 sterility,10 
mycoplasma (Ref. 19), adventitious agent testing, and tests for 
specific pathogens (Ref. 12)).  Analytical procedures different than 
those outlined in the United States Pharmacopeia (USP), FDA 
guidance, or CFR may be acceptable under an IND if you provide 
adequate information about your test method, including specificity, 
sensitivity, and robustness.  Examples of alternative methods, 
which may be needed for live cells, include rapid sterility tests, 
rapid mycoplasma tests (including PCR-based tests), and rapid 
endotoxin tests.  For these non-compendial tests we recommend 
that you qualify/validate them to ensure they are fit for their 
intended use.  We provide some additional comments regarding 
these tests under section V.B.5.a., Specifications (3.2.P.5.1), of 
this guidance, as well as comments regarding replication 
competent virus and wild-type oncolytic virus testing, below.   
 
ii.  Replication Competent Virus 
 
For non-replicating gene therapy viral vectors, we recommend 
specific testing, due to the potential for these vectors to recombine 
or revert to a parental or wild-type (WT) phenotype at a low 
frequency.  Tests for replication-competent, parental, or wild-type 
viruses that may be generated during production (e.g., replication-
competent adenovirus (RCA) and replication-competent retrovirus 
(RCR)) should be performed on material collected at the 
appropriate stage of the manufacturing process.  For example, we 
recommend testing banked material for the presence of replication-
                                                 
9 USP<61> describes membrane filtration, plate count, and most probable number methods that can be done to 
quantitatively determine the bioburden of non-sterile DPs.  Although 21 CFR 211.110(a)(6) does not specify a test 
method, it requires that bioburden in-process testing be conducted pursuant to written procedures during the 
manufacturing process of DPs.   
10 Sterility testing may be performed on the DS when it cannot be performed on the DP, as outlined in the final rule: 
Amendments to Sterility Test Requirements for Biological Products (May 3, 2012; 77 FR 26162 at 26165).  Sterility 
tests are described in 21 CFR 610.12 and USP<71> Sterility Tests. 
33  Contains Nonbinding Recommendations 
 
competent viruses and as a specification for in-process or release 
testing of the DS or DP, as appropriate (please see further details 
and examples, below, within this section). 
 
a.  Replication-Competent Retrovirus (RCR) Testing 
 
Most retroviral-based products (including lentivirus and 
foamy virus-based products) used for gene therapy 
applications are designed to be replication defective.  To 
ensure the absence of RCR, you should perform testing for 
RCR at multiple points during production of a retroviral 
vector.  For further information on retroviral testing, refer 
to Testing of Retroviral Vector-Based Human Gene 
Therapy Products for Replication Competent Retrovirus 
During Product Manufacture and Patient Follow-up; 
Guidance for Industry (Ref. 20). 
 
b.  Replication-Competent Adenovirus (RCA) Testing 
 
Most adenoviral-based products used for gene therapy 
applications are designed to be replication defective.  
Notable exceptions include oncolytic adenoviruses (see 
section V.A.4.b.ii.D., Wild-Type Oncolytic Virus 
Testing, of this guidance).  RCA may be generated at a 
low frequency as a result of homologous recombination 
between viral vector sequences and viral sequences present 
in the cell substrate during manufacturing.  Therefore, for 
adenoviral-based gene therapy products, we recommend 
that you qualify your MVB for RCA and test either the DS 
or DP of each production lot for RCA.  We recommend a 
maximum level of 1 RCA in 31010 viral particles.  
 
c.  Replication-Competent AAV (rcAAV) Testing 
 
Preparations of AAV vectors can be contaminated with 
helper virus-dependent rcAAV, also referred to as wild-
type AAV or pseudo wild-type AAV.  These rcAAV are 
generated through homologous or non-homologous 
recombination events between AAV elements present on 
the vector and AAV rep and cap sequences that are present 
during manufacture.  While wild-type AAV has no known 
associated pathology and cannot replicate without helper 
virus, expression of cap or rep genes in infected cells can 
result in unintended immune responses, which can reduce 
effectiveness and may have unintended safety risks.  
 
34  Contains Nonbinding Recommendations 
 
Therefore, we recommend that you test for rcAAV, which 
could potentially replicate in the presence of helper virus, 
and report these results in the IND.  A number of methods 
have been published for evaluating the level of rcAAV, 
including amplification of AAV in the presence of helper 
virus, followed by PCR for rep/inverted terminal repeats 
(ITR) junctions, and PCR for rep and cap sequences, 
following DNase digestion of the vector preparation.  We 
do not recommend a specific method for determining 
rcAAV in this guidance.  You should describe your test 
method and assay sensitivity in the IND. 
 
d.  Wild-Type Oncolytic Virus Testing 
 
Most oncolytic viruses used in gene therapy applications 
not only carry transgenes but also have been attenuated or 
adapted from a parental virus strain to grow selectively in 
cancer cells.  It may be possible for these attenuated or 
adapted viruses to either recombine or revert to a parental 
(or WT) genotype during manufacture.  Therefore, we 
recommend that you conduct tests to determine whether the 
parental virus sequences are present in your product.  In 
addition, we recommend that you select production cells 
that do not contain viral sequences that may allow 
homologous recombination with the product.  For example, 
we do not recommend 293 cell substrates for the 
manufacture of E1-modified oncolytic adenoviruses due to 
the potential for homologous recombination with E1 
sequences in the 293 cells. 
 
c.  Validation of Analytical Procedures (3.2.S.4.3) 
 
Validation of analytical procedures is usually not required for original 
IND submissions for Phase 1 studies; however, you should demonstrate 
that test methods are appropriately controlled.  In general, scientifically 
sound principles for assay performance should be applied (i.e., tests 
should be specific, sensitive, and reproducible and include appropriate 
controls or standards).  We recommend that you use compendial methods 
when appropriate and qualify safety-related tests prior to initiation of 
clinical trials.  
 
To ensure safety of gene therapy products, you should also qualify the 
assays used to determine dose (e.g., vector genome titer by qPCR, 
transducing units, plaque forming units, transduced cells) prior to 
initiating clinical studies.  In your original IND submission, you should 
provide a detailed description of the qualification protocol (e.g., samples; 
35  Contains Nonbinding Recommendations 
 
standards; positive/negative controls; reference lots; and controls 
evaluated, such as operators, reagents, equipment, dates) and data 
supporting the accuracy, reproducibility, sensitivity, and specificity of the 
method.  Also, critical to ensuring safety is the ability to compare the dose 
used for preclinical evaluations to the dose to be used for clinical studies.  
One way to compare doses is to use the same qualified method to 
quantitate preclinical and clinical lots.  If it is not possible to use the same 
qualified method, we recommend that you retain sufficient quantities of 
preclinical material to enable side by side testing with the clinical material, 
using the same qualified method.  In addition, you should validate tests 
used to determine dose prior to initiating clinical studies to support 
licensure. 
 
Assays used to measure RCR and RCA should also meet our current 
recommendations for sensitivity at an early stage of development (see 
descriptions of RCR Testing in section V.A.4.b.ii.a. of this guidance, and 
RCA Testing in section V.A.4.b.ii.b. of this guidance).  We recommend 
that you include relevant positive and negative controls when conducting 
these tests and include positive controls spiked in the test article to assess 
whether there are any inhibitory effects of the test article on detection.   
 
For all analytical procedures, we recommend that you evaluate assay 
performance throughout product development, have a validation plan in 
place prior to initiating later phase clinical studies, and complete 
validation before BLA submission.  For more information on validation of 
analytical methods, please see FDAs Guidance for Industry:  Q2B 
Validation of Analytical Procedures:  Methodology (Ref. 21).  
 
d.  Batch Analysis (3.2.S.4.4) 
 
You should include a table with test results for batches (or lots) of the DS 
that you have manufactured.  For early stage INDs, this may include 
toxicology lots, developmental batches, engineering runs, or a single 
manufacturing run for clinical grade material.  We recommend that you 
gain adequate experience with a new clinical manufacturing processes 
prior to making clinical material.  This is especially critical following 
technology transfer to a new manufacturing facility, when manufacturing 
changes occur during development, and when multiple manufacturing 
facilities will be utilized.  Please note that batches manufactured in 
different ways should be clearly identified in the submission.  Information 
regarding process development of these materials should be outlined in the 
Manufacturing Process Development (3.2.S.2.6) section of the CTD.  
We recommend that you annually update this section of your IND as new 
batches are produced.  You should indicate any batches that fail to meet 
release specifications and any action taken to investigate the failure 
according to your Quality Unit procedures (for Quality Unit information 
36  Contains Nonbinding Recommendations 
 
please see section V.C.1., Appendices  Facilities and Equipment 
(3.2.A.1), of this guidance).  We recommend that you retain samples of 
production lots for use in future assay development, validation, or 
comparability studies. 
 
e.  Justification of Specification (3.2.S.4.5) 
 
You should provide justification for the DS specifications in your IND.  
We recognize that acceptance criteria may be adjusted throughout the 
product development stages, based on both manufacturing and clinical 
experience.  For early stage clinical studies, assays used to characterize 
production lots may be more variable than those used in later phase 
investigations. 
 
For later stage investigational studies in which the primary objective is to 
gather meaningful data about product efficacy, we recommend that 
acceptance criteria be tightened to ensure batches are well-defined and 
consistently manufactured. 
 
5.  Reference Standards or Materials (3.2.S.5) 
 
You should provide information on the reference standards or reference materials 
used for testing the DS in your original IND submission.  We recommend that 
you provide the source and lot number; expiration date; COAs, when available; 
and/or internally or externally generated evidence of identity and purity for each 
reference standard.  
 
Three types of reference standards are generally used:  1) certified reference 
standards (e.g., USP compendial standards); 2) commercially supplied reference 
standards obtained from a reputable commercial source; and/or 3) other materials 
of documented purity, custom-synthesized by an analytical laboratory or other 
noncommercial establishment.  In some cases, the reference material for an assay 
will be a well-characterized lot of the gene therapy product, itself.  In this case, 
we recommend that you reserve and maintain a sufficient amount of material 
(e.g., part of a production lot) to serve as a reference material. 
 
You should have a protocol to evaluate the stability of the reference standard and 
plan well in advance for production of a new reference material.  It is also critical 
to retain sufficient material to bridge to a new reference material as needed during 
product development. 
 
6.  Container Closure System (3.2.S.6) 
 
You should describe the type(s) of container and closure used for the DS in your 
original IND submission, including the identity of materials used in the 
construction of the container closure system.  We recommend that you determine 
37  Contains Nonbinding Recommendations 
 
whether the containers and closures are compatible with the DS.  For an original 
IND submission, compatibility with a gene therapy product may be evaluated 
during stability studies or may be based on historical data and experience, using 
similar products.  You should indicate whether the container is an approved or 
cleared device and/or the information is cross-referenced to a master file, as 
described in section III., Administrative Information, of this guidance. 
 
7.  Stability (3.2.S.7) 
 
a.  Stability Summary and Conclusions (3.2.S.7.1) 
 
We recommend that you describe in your original IND submission the 
types of stability studies (either conducted or planned) to demonstrate that 
the DS is within acceptable limits.  The protocol should describe the 
storage container, formulation, storage conditions, testing frequency, and 
specifications (i.e., test methodologies and acceptance criteria).  Please 
note that stability studies may evolve with product development, and if the 
DS is immediately processed into a DP, long term DS stability data may 
not be needed.  
 
Your stability analysis may include measures of product sterility (or 
container integrity), identity, purity, quality, and activity or potency.  We 
recommend that you provide justification for the test methods and 
acceptance criteria used in the stability analysis.  It is often helpful to 
demonstrate that at least one or more of the test methods in your stability 
analysis are stability-indicating.  You may demonstrate a test is stability-
indicating using forced degradation studies, accelerated stability studies, 
or another type of experimental system that demonstrates product 
deterioration.  Information to help you design your stability studies may be 
found in the following guidance documents:  FDAs Guideline for 
Industry:  Quality of Biotechnological Products:  Stability Testing of 
Biotechnological/Biological Products (Ref. 22); FDAs Guidance for 
Industry:  Q1A(R2) Stability Testing of New Drug Substances and 
Products (Ref. 23); and FDAs Guidance for Industry:  Q1E Evaluation 
of Stability Data (Ref. 24).  
 
b.  Post-Approval Stability Protocol and Stability Commitment 
(3.2.S.7.2)  
 
We do not recommend that you provide a post-approval stability protocol 
and stability commitment in the IND.  However, as you progress with 
product development, you may want to consider which stability studies 
would be required to determine an expiry date.  We recommend the 
discussion of these items at your late phase IND meetings. 
 
   
38  Contains Nonbinding Recommendations 
 
c.  Stability Data (3.2.S.7.3) 
 
We recommend that you provide the results of your stability studies in 
your IND and update this information on a regular basis (e.g., annual 
reports).  Information on the qualification of analytical procedures used to 
generate stability data should be included in your original IND 
submission.  For additional information see section V.A.4.C., e.g. 
Validation of Analytical Procedures (3.2.S.4.3), of this guidance. 
 
B.  Drug Product (3.2.P) 
 
1.  Drug Product Description and Composition (3.2.P.1) 
 
You should provide a description of the DP and its composition (21 CFR 
312.23(a)(7)(iv)(b)).  This includes a description of the dosage form and a list of 
all of its components (active and inactive), the amount on a per unit basis, the 
function, and a reference to quality standards for each component (e.g., 
compendial monograph or manufacturers specifications).   
 
If a drug or device will be used with your gene therapy as a combination product, 
we recommend that quality information for the drug or device be included in 
section 3.2.P of the CTD with appropriate hyperlinks to section 3.2.R of the CTD, 
as described in FDAs eCTD Technical Conformance Guide:  Technical 
Specifications Document (Ref. 4).  If a placebo treatment is used in the clinical 
trial, a separate DP section should be provided for the placebo.  In addition, you 
should provide a description of any accompanying reconstitution diluents and a 
description of the container and closure used for the dosage form and 
accompanying reconstitution diluent in a separate DP section, if applicable. 
 
2.  Pharmaceutical Development (3.2.P.2) 
 
The Pharmaceutical Development section should contain information on the 
development studies conducted to establish that product formulation, 
manufacturing process, container closure system, microbiological attributes, and 
instructions for use are appropriate for the stage of clinical development.  The 
studies described here are distinguished from routine control tests conducted, 
according to specifications.  Additionally, this section should identify and 
describe the formulation and process attributes (critical parameters) that can 
influence batch reproducibility, product performance, and DP quality.  The 
Agency acknowledges that this information may be limited in the early phases of 
development and recommends that sponsors provide additional information and 
updates as product development proceeds.  Supportive data and results from 
specific studies or published literature can be included within or attached to the 
Pharmaceutical Development section.  Additional supportive data can be 
referenced to the relevant nonclinical or clinical sections of the application. 
 
39  Contains Nonbinding Recommendations 
 
a.  Components of the Drug Product (3.2.P.2.1) 
 
i.  Drug Substance (3.2.P.2.1.1) 
 
You should describe the compatibility of the DS with the 
components listed in the Description and Composition of the 
Drug Product (3.2.P.1) section of the CTD and the key 
characteristics of the DS (e.g., concentration, viability, aggregation 
state, viral infectivity) that can influence the performance of the 
DP. 
 
ii.  Excipients (3.2.P.2.1.2) 
 
You should describe in your original IND submission the choice of 
excipients and inactive components of the DP listed in the 
Description and Composition of the Drug Product (3.2.P.1) 
section of the CTD, their concentration, and the characteristics of 
these excipients that can influence DP performance. 
 
b.  Drug Product (3.2.P.2.2) 
 
i.  Formulation Development (3.2.P.2.2.1) 
 
You should briefly describe the development of the DP 
formulation, taking into consideration the proposed route of 
administration and usage in your IND.  
 
We recommend that you describe any other formulations that have 
been used in clinical or preclinical studies and provide a reference 
to such studies, if applicable.  If formulation changes were needed 
for stability, device compatibility, or safety concerns, this 
information can be reported here. 
 
ii.  Overages (3.2.P.2.2.2) 
 
In your IND, you should describe whether gene therapy product in 
excess of your label claim is added during formulation to 
compensate for degradation during manufacture or a products 
shelf life or to extend shelf life.  We generally do not recommend 
the use of overages as this can result in inconsistent dosing, and we 
recommend that you provide justification for an overage, as 
described in FDAs Guidance for Industry:  Q8(R2) 
Pharmaceutical Development (Ref. 6). 
 
   
40  Contains Nonbinding Recommendations 
 
iii.  Physicochemical and Biologic Properties (3.2.P.2.2.3) 
 
In your IND you should describe the parameters relevant to the 
performance of the DP, (or reference relevant DS sections, if 
appropriate).  These parameters include physicochemical or 
biological properties of the product (e.g., dosing units, genotypic 
or phenotypic variation, particle number and size, aggregation 
state, infectivity, specific activity (ratio of infectious to non-
infectious particles or full to empty particles), biological activity or 
potency, and/or immunological activity).  Understanding these 
CQAs and how they affect product performance usually occurs 
over the course of product development (see section IV.A., 
General Information, of this guidance) and may not be fully 
understood at the time the IND is submitted.  More information on 
the principles of pharmaceutical development and consideration in 
establishing CQAs during the clinical research phase can be found 
in FDAs Guidance for Industry:  Q8(R2) Pharmaceutical 
Development (Ref. 6).  
 
You should update this section on the physiochemical and 
biological properties of your product as you gain a better 
understanding of the CQAs, during development. 
 
c.  Manufacturing Process Development (3.2.P.2.3) 
 
You should describe the selection and optimization of the DP 
manufacturing process (described in the Description of Manufacturing 
Process and Process Controls (3.2.P.3.3) section of the CTD) if 
development studies have been performed.  The Agency acknowledges 
that this information may be limited in the early phases of development 
and recommends that sponsors provide additional information and updates 
as product development proceeds.   
 
d.  Container Closure System (3.2.P.2.4) 
 
You should describe the suitability of the container closure system, which 
you have described in the Container Closure System (3.2.P.7) section of 
the CTD, for the storage, transportation (shipping), and use of the DP. 
 
We recommend that you consider choice of materials, protection from 
moisture, gases, and light, compatibility with the formulation (including 
adsorption to the container and leaching), safety of materials, and 
performance.  For more information on container closure systems, refer to 
FDAs Guidance for Industry:  Container Closure Systems for Packaging 
Human Drugs and Biologics (Ref. 25). 
 
41  Contains Nonbinding Recommendations 
 
In the selection of your container closure system, we also recommend that 
you consider how lots of your product will be tested for final product 
release.  For gene therapy products that are manufactured in small lot sizes 
(e.g., autologous cell products or products vialed at very high dose levels), 
it may be challenging or not possible to dedicate a final container or 
multiple vials for lot release testing.  In this case, we recommend that you 
consider a final container that can be sampled for release testing or that 
you consider alternatives to final container testing. 
 
e.  Microbiological Attributes (3.2.P.2.5) 
 
We recommend, for products intended to be sterile, that you provide 
details on measures taken to ensure aseptic processing, describe the final 
product microbial testing, and address how the integrity of the container 
closure system to prevent microbial contamination will be assessed.  
 
f.  Compatibility (3.2.P.2.6) 
 
You should discuss the compatibility of the DP with the diluent used for 
reconstitution or the delivery device, as appropriate. 
 
We recommend that compatibility studies include measures of both 
product quantity and product activity (e.g., for viral vectors, a measure of 
physical particles and infectivity (or potency) to assess both adsorption 
and inactivation).  These in-use and in-device stability data should support 
recommended hold times and conditions outlined in the clinical protocol 
for patient administration.  The absence of an understanding of in-use and 
in-device stability, and the potential impacts on product performance, may 
not justify risks associated with clinical study treatment(s).  Therefore, we 
recommend that you carefully control and assess DP compatibility and the 
final steps of product preparation and administration. 
 
3.  Manufacture (3.2.P.3) 
 
a.  Manufacturers (3.2.P.3.1) 
 
You should provide the name, address, and responsibility of each 
manufacturer, including contractor manufacturer(s), involved in the 
manufacture and testing of the DP.  
 
For gene therapy-device combination products, we recommend that you 
list the manufacturing facilities for the device components and describe 
the assembly and testing processes taking place at each site, as described 
in FDAs eCTD Technical Conformance Guide: Technical Specifications 
Document (Ref. 4).  You should also identify whether facilities follow 
the combination product streamlined manufacturing approach (as 
42  Contains Nonbinding Recommendations 
 
described in FDAs Guidance for Industry and FDA Staff:  Current Good 
Manufacturing Practice Requirements for Combination Products (Ref. 
26)) and identify the specific set of regulations (i.e., 21 CFR Part 211 or 
Part 820). 
 
b.  Batch Formula (3.2.P.3.2) 
 
You should provide a batch formula that includes a list of all components 
of the dosage form, their amounts on a per-batch basis, and a reference to 
their quality standards. 
 
c.  Description of Manufacturing Process and Process Controls 
(3.2.P.3.3) 
 
You should provide a detailed description of the DP manufacturing 
process and identify process controls and intermediate tests.  Your 
description should include both flow diagram(s) and narrative 
description(s) as well as packaging, product contact materials, and 
equipment used.  This process can include manufacturing steps, such as 
final formulation, filtration, filling and freezing, and process controls.  
Under certain conditions, repeating sterile filtration (if applicable) may be 
critical.  We recommend that you clearly describe the conditions and 
justifications for each reprocessing procedure (21 CFR 211.115) and 
demonstrate product consistency between reprocessed lots and normal 
production lots.   
 
For ex vivo genetically modified cells that are administered immediately 
after manufacturing, we recommend a negative test result from an in-
process sterility test (on a sample taken 48 to 72 hours prior to final 
harvest) for release of the DP.  Additional recommendations are made in 
section V.B.2.e., Microbiological Attributes (3.2.P.2.5), of this 
guidance.  
 
d.  Controls of Critical Steps and Intermediates (3.2.P.3.4) 
 
You should describe the control of critical steps and intermediates in the 
manufacturing process.  Critical steps should include those outlined in the 
Description of Manufacturing Process and Process Controls (3.2.P.3.3) 
section of the CTD to ensure control as well as steps in which tests with 
acceptance criteria are performed.  We recommend that you provide 
justification for acceptance criteria or limits set for these tests.  In addition, 
you should provide information on the quality and control of intermediates 
of the manufacturing process.  Manufacturing intermediates are defined by 
the manufacturer and may include material from collection steps or hold 
steps.  The Agency acknowledges that this information may be limited in 
43  Contains Nonbinding Recommendations 
 
the early phases of development and recommends that sponsors provide 
this information at the appropriate stage. 
 
e.  Process Validation and/or Evaluation (3.2.P.3.5) 
 
Process validation is not required for early stage manufacturing, and thus, 
most original IND submissions will not include this information.  
However, we do recommend that early stage INDs have information on 
methods used to prevent contamination, cross-contamination, and product 
mix-ups.  For more information on functions of the Quality Unit under an 
IND, please see section V.A.2.e., Process Validation and/or Evaluation 
(3.2.S.2.5), of this guidance. 
 
4.  Control of Excipients (3.2.P.4) 
 
a.  Specifications (3.2.P.4.1) 
 
You should provide specifications for all excipients listed in Excipients 
(3.2.P.2.1.2) section of the CTD.  For purpose of this guidance, an 
excipient is any component, other than to the active ingredient, that is 
intended to be part of the final product (e.g., human serum albumin, 
Dimethyl Sulfoxide (DMSO)).  If the excipient is compendial, information 
in the Specifications (3.2.P.4.1) section of the CTD may not be required. 
 
b.  Analytical Procedures (3.2.P.4.2) 
 
You should describe your analytical procedures for testing excipients.  If 
you are performing any additional testing or qualification of compendial 
excipients, you should describe that testing here. 
 
c.  Validation of Analytical Procedures (3.2.P.4.3) 
 
Validation of analytical procedures is usually not required for original 
IND submissions.  We recommend that you provide any available 
validation or verification information for the analytical procedures used to 
test excipients. 
 
d.  Justification of Specifications (3.2.P.4.4) 
 
You should provide justification for the proposed excipient specifications, 
as appropriate. 
 
e.  Excipients of Human or Animal Origin (3.2.P.4.5) 
 
For excipients of human or animal origin, you should provide information 
regarding source, specifications, description of testing performed, and 
44  Contains Nonbinding Recommendations 
 
viral safety data.  FDA-licensed excipients (e.g., human serum albumin) 
should be used, when available.  If human serum is used, we recommend 
that you submit information documenting donor suitability as well as 
appropriate infectious disease testing.  You should ensure that collection is 
performed by a licensed blood bank and that testing meets the 
requirements described in 21 CFR Part 640. 
 
f.  Novel Excipients (3.2.P.4.6) 
 
For excipients used for the first time in a DP or used for the first time in a 
route of administration, you should provide full details of manufacture, 
characterization, and controls, with cross-references to supporting safety 
data (nonclinical and/or clinical) in a regulatory file submitted to FDA, if 
available. 
 
5.  Control of Drug Product (3.2.P.5) 
 
a.  Specifications (3.2.P.5.1) 
 
You should list DP specifications in your original IND submission.  Your 
testing plan should be adequate to describe the physical, chemical, or 
biological characteristics of the DP necessary to ensure that the DP meets 
acceptable limits for identity, strength (potency), quality, and purity (21 
CFR 312.23(a)(7)(iv)(b)).  Product lots that fail to meet specifications 
should not be used in your clinical investigation without FDA approval.  
For early phase clinical studies, we recommend that assays be in place to 
assess safety (which includes tests to ensure freedom from extraneous 
material, adventitious agents, and microbial contamination) and dose (e.g., 
vector genomes, vector particles, or genetically modified cells) of the 
product.  Additional information on safety testing and measuring product 
dose is described in section V.A.4.a., Specification (3.2.S.4.1), of this 
guidance.  
 
We recommend that product release assays be performed at the 
manufacturing step at which they are necessary and appropriate.  For 
example, mycoplasma and adventitious agents release testing is 
recommended on cell culture harvest material, as discussed in section 
V.A.4.a., Specification (3.2.S.4.1), of this guidance.  In addition, 
sterility, endotoxin, and identity testing are recommended on the final 
container product to ensure absence of microbial contamination or to 
detect product mix-ups that might have occurred during the final DP 
manufacturing steps (e.g., buffer exchange, dilution, or finish and fill 
steps).  DP specifications should be further refined as a part of product 
development under an IND.  We recommend that sponsors establish or, in 
some cases, tighten acceptance criteria, based on manufacturing 
experience as clinical development proceeds.  Acceptance criteria should 
45  Contains Nonbinding Recommendations 
 
also be established, based on clinical lots shown to be safe and effective, 
when appropriate.  We also recommend that sponsors develop testing to 
assess product potency and have this assay in place prior to initiating 
studies used to support product efficacy for licensure.  For licensure, a 
complete set of specifications to ensure the safety and effectiveness of the 
product must include the general biological products standards, as outlined 
in 21 CFR Part 610. 
 
b.  Analytical Procedures (3.2.P.5.2) 
 
You should describe the analytical procedures used for testing the DP.  If 
the analytical procedures are the same as those for the DS, you do not 
need to repeat this information unless there is a matrix effect from the DP 
on assay performance.  Please reference the appropriate section of your 
IND, where this information can be found (e.g., Drug Substance 
Analytical Procedures (3.2.S.4.2) section of the CTD.  We have the 
following additional comments regarding these tests: 
 
i.  Sterility 
 
We recognize that the compendial sterility tests (USP <71>; 
610.12) may not be suitable for all products (e.g., those with 
limited shelf life).  As mentioned in section V.A.4.b., Analytical 
Procedures (3.2.S.4.2), of this guidance, rapid sterility tests may 
be acceptable for ex vivo genetically modified cells administered 
fresh or with limited hold time between final formulation and 
patient administration.  
 
For ex vivo genetically modified cells that are administered 
immediately after manufacturing, in-process sterility testing on 
sample taken 48 to 72 hours prior to final harvest is recommended 
for product release.  For such products, aside from an in-process 
sterility test, we also recommend that sponsors perform a rapid 
microbial detection test, such as a Gram stain, on the final 
formulated product and a sterility test, compliant with 21 CFR 
610.12, on the final formulated product.  
 
Under this approach, the release criteria for sterility would be 
based on a negative result of the Gram stain and a no-growth result 
from the 48 to 72 hour in-process sterility test.  Although the 
results of the sterility culture performed on the final product will 
not be available for product release, this testing will provide useful 
data.  A negative result will provide assurance that an aseptic 
technique was maintained.  A positive result will provide 
information for the medical management of the subject and trigger 
an investigation of the cause of the sterility failure.  The sterility 
46  Contains Nonbinding Recommendations 
 
test on the final formulated product should be continued for the full 
duration (e.g.14 days for USP <71> method) to obtain the final 
sterility test result, even after the product has been administered to 
the patient. 
 
In all cases where product release is prior to obtaining results from 
the sterility test, the investigational plan should address the actions 
to be taken in the event that the sterility test is determined to be 
positive after the product is administered to a subject.  You should 
report the sterility failure to both the clinical investigator and FDA.  
We recommend that you include results of the investigation of the 
cause of the sterility failure and any corrective actions in an 
information amendment submitted to your IND within 30 calendar 
days after initial receipt of the positive culture test result (21 CFR 
312.31). 
 
In the case of a positive microbial test result, the clinical 
investigator should evaluate the subject for any signs of infection 
that may be attributable to the product sterility failure.  If the 
patient experiences any serious and unexpected adverse drug event 
that could be from administration of the non-sterile gene therapy 
product, then you must report this information to FDA in an IND 
safety report no more than 15 calendar days after your initial 
receipt of the information (21 CFR 312.32(c)).  If you determine 
that an investigational drug presents an unreasonable and 
significant risk to subjects of a positive microbial test result or for 
any other reason, you must discontinue those investigations that 
present the risk and notify FDA, all institutional review boards, 
and all investigators (21 CFR 312.56(d)). 
 
In addition, please be aware that a product may sometimes 
interfere with the results of sterility testing.  For example, a 
product component or manufacturing impurities (e.g., antibiotics) 
may have mycotoxic or anti-bacterial properties.  Therefore, we 
recommend that you assess the validity of the sterility assay using 
the bacteriostasis and fungistasis testing, as described in USP <71> 
Sterility Tests.  
 
If you freeze the DP before use, we recommend that you perform 
sterility testing on a sample of the product prior to 
cryopreservation so that results will be available before the product 
is administered to a patient.  However, if the product undergoes 
manipulation after thawing (e.g., washing, culturing), particularly 
if procedures are performed in an open system, you may need to 
perform additional release testing including sterility and identity 
testing to ensure product quality.  
47  Contains Nonbinding Recommendations 
 
We recommend that you incorporate the results of in-process 
sterility testing into your acceptance criteria for final product 
specifications. 
 
ii.  Identity 
 
We recommend that identity assays uniquely identify a product 
and distinguish it from other products in the same facility.  This 
test is performed on the final labeled product to verify its contents 
(21 CFR 610.14).  Sometimes, a single test is not sufficient to 
distinguish clearly among products, and therefore, it is good 
practice to use different types of test methods (e.g., vector genome 
restriction digest and protein capsid analysis).   
 
If the final product is ex vivo genetically modified cells, we 
recommend that identity testing include an assay to measure the 
presence of vector (i.e., expression assay, restriction digest) or 
genetic change and an assay specific for the cellular composition 
of the final product (e.g., cell surface markers). 
 
iii.  Purity 
 
Product purity is defined as the relative freedom from extraneous 
matter in the finished product, whether or not it is harmful to the 
recipient or deleterious to the product (21 CFR 600.3(r)).  Purity 
testing includes assays for pyrogenicity or endotoxin and residual 
manufacturing impurities, as outlined under section V.A.3.b., 
Impurities (3.2.S.3.2), of this guidance, of the DS, which may 
include, but are not limited to: proteins; DNA; cell debris; 
reagents/components used during manufacture, such as cytokines, 
growth factors, antibodies, and serum; and in the case of ex vivo 
genetically modified cells, any unintended cellular populations.  
The assays used to demonstrate product purity should be phase 
appropriate and may evolve during development as you develop 
greater understanding of the impurities present in your product, or 
as you make manufacturing process changes.  
 
Although the rabbit pyrogen test method is the current required 
method for testing certain licensed biological products for 
pyrogenic substances (21 CFR 610.13(b)), we generally accept 
alternative test methods, such as the Limulus Amebocyte Lysate 
(LAL).).  For any parenteral drug, except those administered 
intrathecally or intraocularly, we recommend that the upper limit 
of acceptance criterion for endotoxin be 5 Endotoxin Unit (EU)/kg 
body weight/hour.  For intrathecally-administered drugs, we 
recommend an upper limit of acceptance be set at 0.2 EU/kg body 
48  Contains Nonbinding Recommendations 
 
weight/hour.  For intraocularly-administered drugs, we recommend 
acceptance criterion of not more than (NMT) 2.0 EU/dose/eye for 
injected or implanted DPs or NMT 0.5 EU/mL for ophthalmic 
irrigation products. 
 
iv.  Potency 
 
You should describe and justify in your IND all assays that you 
will use to measure potency.  A potency assay is not required to 
initiate early phase clinical studies, but we recommend that you 
have a well-qualified assay to determine dose, as described below 
and in section V.A.4.c., Validation of Analytical Procedures 
(3.2.S.4.3), of this guidance.  For additional information on 
potency assays, please see FDAs Guidance for Industry:  Potency 
Tests for Cellular and Gene Therapy Products (Ref. 18).  
 
v.  Viability 
 
You should establish minimum release criteria for viability, where 
appropriate.  For ex vivo genetically modified cells, we 
recommend a minimum acceptable viability of at least 70 percent.  
If this level cannot be achieved, we recommend that you submit 
data in support of a lower viability specification, demonstrating, 
for example, that dead cells and cell debris do not affect the safe 
administration of the product and/or the therapeutic effect. 
 
vi.  Cell Number or Dose 
 
Your dose-determining assay is an important part of your DP 
specifications and should be qualified as suitable for use prior to 
initiating clinical studies.  For additional information on your dose-
determining assay, please see section V.A.4.a., Specification 
(3.2.S.4.1), of this guidance.  If your final product is genetically 
modified cell-based gene therapy, you should have an acceptance 
criterion for the minimum number of genetically modified cells in 
a product lot.  We recommend that the product dose for such 
products be based on the total number of genetically modified 
cells. 
 
c.  Validation of Analytical Procedures (3.2.P.5.3) 
 
Validation of analytical procedures is usually not required for original 
IND submissions, but we do recommend that you qualify certain safety-
related or dose-related assays, even at an early stage of development (see  
49  Contains Nonbinding Recommendations 
 
section V.A.4.c., Validation of Analytical Procedures (3.2.S.4.3), of this 
guidance).  If they are the same as those listed for DS testing, you do not 
need to repeat them but should reference that section of your IND. 
 
d.  Batch Analyses (3.2.P.5.4) 
 
You should provide final product COA(s), if available, or a description of 
the batches generated to date, and the results of any batch analyses for the 
DP used in toxicology studies, engineering runs, or clinical studies. 
Sponsors can also include supportive developmental batches, if 
appropriate. 
 
e.  Characterization of Impurities (3.2.P.5.5) 
 
You should provide information on characterization of impurities if not 
previously provided in the Impurities (3.2.S.3.2) section of the CTD. 
 
f.  Justification of Specifications (3.2.P.5.6) 
 
You should provide justification for the DP specifications.  See section 
V.A.4.e., Justification of Specification (3.2.S.4.5), of this guidance for 
additional details. 
 
6.  Reference Standards or Materials (3.2.P.6) 
 
You should provide information on the reference standards or reference materials 
used in testing the DP if not previously provided in the Reference Standards or 
Materials (3.2.S.5) section of the CTD. 
 
7.  Container Closure System (3.2.P.7) 
 
You should provide a description of the container closure systems, including 
identity of construction materials for each primary packaging component and its 
specification.  You should also provide information on how the container is 
sterilized.  
 
Please see section V.B.5.d., Container Closure System (3.2.P.2.4), of this 
guidance for more information and recommendations, regarding the suitability of 
different final product containers.  
 
If the final container is an FDA-cleared device, we recommend that you reference 
the 510(k) number for the device in your submission.  For combination products 
containing a device constituent part, we recommend that you include a table of 
contents for the combination product (with reference links to other files) in this 
section, as described in FDAs eCTD Technical Conformance Guide: Technical 
Specifications Document (Ref. 4). 
50  Contains Nonbinding Recommendations 
 
 
8.  Stability (3.2.P.8) 
 
a.  Stability Summary and Conclusion (3.2.P.8.1) 
 
You should summarize the types of studies conducted, protocols used, and 
the results of the studies.  Your summary should include, for example, 
conclusions regarding storage conditions and shelf life as well as in-use 
and in-device storage conditions. 
 
If a short-term clinical investigation is proposed, or if a DP manufacturing 
process has limited product hold times, stability data submitted may be 
correspondingly limited.  Early in development, stability data for the gene 
therapy may not be available to support the entire duration of the proposed 
clinical investigation.  Therefore, we recommend that you submit a 
prospective plan to collect stability information and update this 
information to the IND in a timely manner (e.g., in an annual IND update). 
 
b.  Post-Approval Stability Protocol and Stability Commitment 
(3.2.P.8.2) 
 
We do not recommend that you provide a post-approval stability protocol 
and stability commitment in your IND submission.  However, as product 
development continues, we recommend that you consult with your Quality 
Reviewer to determine the type of studies that will be necessary to support 
product expiration dates for commercial manufacturing. 
 
c.  Stability Data (3.2.P.8.3) 
 
You should provide results of the stability studies in your IND in an 
appropriate format (e.g., tabular, graphic, narrative).  Information on the 
analytical procedures used to generate the data should also be included, 
and this may be referenced to other sections of your submission (e.g., 
Analytical Procedures (3.2.P.5.2) section of the CTD). 
 
C.  Appendices (3.2.A) 
 
1.  Facilities and Equipment (3.2.A.1) 
 
For early phase studies we recommend that facility information should include a 
diagram illustrating the manufacturing flow of the manufacturing areas, 
information on all developmental or approved products manipulated in this area, a 
summary of product contact equipment, and information on procedures and 
design features of the facility, to prevent contamination or cross-contamination.  
As noted in section III.D., Previously Submitted Information, of this guidance 
51  Contains Nonbinding Recommendations 
 
this information may be cross-referenced to other regulatory files (e.g. Type V 
biologics master file).  
 
We recommend that you include a description of your Quality Unit whose duties 
should include establishing procedures to qualify reagents and critical materials, 
prevent microbial contamination, cross-contamination, and product mix-ups.  In 
addition, your Quality Unit should have procedures in place to investigate lot 
failures, out-of-specification results, and ways to implement corrective actions as 
product development progresses.  We recommend that your IND include a 
summary of your Quality Unit, including the manner in which quality control 
testing and oversight are separated from the manufacturing unit.  
 
Additional information on quality systems can be found in the following FDAs 
Guidance for Industry: CGMP for Phase 1 Investigational Drugs (Ref. 11); 
Quality Systems Approach to Pharmaceutical CGMP Regulations (Ref. 27); 
and Process Validation: General Principles and Practices (Ref. 17).  Please note 
that the application of CGMP is required under section 501(a)(2)(B) of the FD&C 
Act at all stages of clinical investigation.  However, the CGMP regulations (21 
CFR Part 211) are not required for the manufacture of most investigational new 
drugs under Phase 1 INDs (See 21 CFR 210.2(c); Ref. 11).  
 
2.  Adventitious Agents Safety Evaluation (3.2.A.2) 
 
You should provide information assessing the risk of potential contamination with 
adventitious agents.  For non-viral adventitious agents, we recommend that you 
provide detailed information on the avoidance and control of transmissible 
spongiform encephalopathy agents, bacteria, mycoplasma, and fungi.  This 
information can include certification and/or testing of components and control of 
the production process.  For viral adventitious agents, we recommend that you 
provide information on viral safety studies.  Study reports and data to support 
qualification of your manufacturing components (such as adventitious agents test 
reports for banked materials) may be submitted as a part of this appendix.  These 
studies should demonstrate that the materials used in production are considered 
safe and that the approaches used to test, evaluate, and eliminate potential risks, 
during manufacture, are suitable.  
 
In addition, COAs for all raw materials and reagents described in your IND may 
be put in this section to support control of adventitious agents. Also, data 
collected (i.e., study reports) for adventitious agent testing can be placed in this 
section. 
 
D.   Regional Information (3.2.R) 
 
Information that is specific to a regulatory region can be put in the regional section of 
eCTD.  This would include vector and plasmid sequencing information, delivery device 
information, and other information as requested in this guidance.   
52  Contains Nonbinding Recommendations 
 
VI.  REFERENCES 
 
 
1.  Guidance for Industry:  M4Q:  The CTD  Quality, August 2001. 
https://www.fda.gov/media/71581/download.  
2.  Human Genome Editing:  Science, Ethics, and Governance.  The National Academies 
Press; 2017.  https://www.nap.edu/read/24623/chapter/1#xvii. 
3.  Providing Regulatory Submissions in Electronic Format  Certain Human 
Pharmaceutical Product Applications and Related Submissions Using the eCTD 
Specifications; Guidance for Industry, July 2019. 
https://www.fda.gov/media/128935/download. 
4.  eCTD Technical Conformance Guide:  Technical Specifications Document, December 
2019. https://www.fda.gov/media/93818/download. 
5.  Determining the Need for and Content of Environmental Assessments for Gene 
Therapies, Vectored Vaccines, and Related Recombinant Viral or Microbial Products; 
Guidance for Industry, March 2015. https://www.fda.gov/media/91425/download. 
6.  Guidance for Industry:  Q8(R2) Pharmaceutical Development, November 2009, 
https://www.fda.gov/media/71535/download. 
7.  Guidance for Industry:  Expedited Programs for Serious Conditions  Drugs and 
Biologics, May 2014. https://www.fda.gov/media/86377/download. 
8.  Guidance for Industry:  Q11 Development and Manufacture of Drug Substances, 
November 2012. https://www.fda.gov/media/80909/download. 
9.  Recommendations for Microbial Vectors used for Gene Therapy; Guidance for Industry, 
September 2016. https://www.fda.gov/media/94200/download. 
10.  Contract Manufacturing Arrangements for Drugs:  Quality Agreements; Guidance for 
Industry, November 2016. https://www.fda.gov/media/86193/download. 
11.  Guidance for Industry:  CGMP for Phase 1 Investigational Drugs, July 2008. 
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12.  Guidance for Industry:  Characterization and Qualification of Cell Substrates and Other 
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Indications, February 2010. https://www.fda.gov/media/78428/download. 
13.  Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for 
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14.  Final Rule:  Eligibility Determination for Donors of Human Cells, Tissues, and Cellular 
and Tissue-Based Products (69 FR 29786, May 25, 2004). 
https://www.gpo.gov/fdsys/pkg/FR-2004-05-25/pdf/04-11245.pdf. 
15.  Federal Register Notice:  International Conference on Harmonisation; Guidance on Q5D 
Quality of Biotechnological/Biological Products:  Derivation and Characterization of Cell 
Substrates Used for Production of Biotechnological/Biological Products (63 FR 50244, 
September 21, 1998). https://www.fda.gov/media/71463/download. 
53  Contains Nonbinding Recommendations 
 
16.  Reid Y, D Storts, T Riss, L Minor, Authentication of Human Cell Lines by STR DNA 
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for Advancing Translational Sciences; 2004.  
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2011. https://www.fda.gov/media/79856/download. 
19.  Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals, 
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20.  Testing of Retroviral Vector-Based Human Gene Therapy Products for Replication 
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21.  Guidance for Industry:  Q2B Validation of Analytical Procedures:  Methodology, 
November 1996. https://www.fda.gov/media/71725/download. 
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23.  Guidance for Industry:  Q1A(R2) Stability Testing of New Drug Substances and 
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24.  Guidance for Industry:  Q1E Evaluation of Stability Data, June 2004. 
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25.  Guidance for Industry:  Container Closure Systems for Packaging Human Drugs and 
Biologics, May 1999. https://www.fda.gov/media/70788/download. 
26.  Guidance for Industry and FDA Staff:  Current Good Manufacturing Practice 
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27.  Guidance for Industry:  Quality Systems Approach to Pharmaceutical CGMP 
Regulations, September 2006. https://www.fda.gov/media/71023/download.  
 
 
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