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) 

Additional copies of this guidance are available from the Office of Communication, Training, 

and Manufacturers Assistance (HFM-40), 1401 Rockville Pike, Suite 200N, Rockville, MD 

20852-1448, or by calling 1-800-835-4709 or 301-827-1800, or from the Internet at 

http://www.fda.gov/cber/guidelines.htm. 

For questions on the content of this guidance, contact the Office of Cellular, Tissue, and Gene 

Therapies at 301-827-5102. 

U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Biologics Evaluation and Research 

April 2008
Contains Nonbinding Recommendations 

Table of Contents 

I. INTRODUCTION............................................................................................................. 1

A. How will FDA Reviewers and Sponsors Use this Guidance?............................ 2

B. How is this Guidance Organized? ....................................................................... 2

II. ADMINISTRATIVE INFORMATION TO BE DOCUMENTED BY FDA 

REVIEWERS .................................................................................................................... 3

III. PRODUCT MANUFACTURING AND CHARACTERIZATION INFORMATION 

TO BE SUBMITTED BY SPONSORS AND DOCUMENTED BY FDA 

REVIEWERS .................................................................................................................... 4

A. Product Manufacturing – Components and Materials..................................... 4

1. Vector.......................................................................................................... 4 

2. Cells............................................................................................................ 5 

3. Reagents...................................................................................................... 9 

4. Excipients.................................................................................................. 12 

5. Additional Considerations ........................................................................ 12 

B. Product Manufacturing – Procedures .............................................................. 14

1. Vector Production/Purification .................................................................. 14 

2. Preparation of ex Vivo Gene-Modified Autologous or Allogeneic Cells... 15 

3. Process Timing and Intermediate Storage ................................................ 16 

4. Final Formulation ..................................................................................... 16 

IV. PRODUCT TESTING.................................................................................................... 16

A. Microbiological Testing...................................................................................... 17

1. Sterility Testing (Bacterial and Fungal Testing) ...................................... 17 

2. Mycoplasma.............................................................................................. 19 

3. Adventitious Agent Testing...................................................................... 19 

B. Identity................................................................................................................. 22

C. Purity.................................................................................................................... 22

1. Residual Contaminants............................................................................. 22 

2. Pyrogenicity/Endotoxin ............................................................................ 23 

D. Potency................................................................................................................. 23

E. Other .................................................................................................................... 24

1. General Safety Testing.............................................................................. 24 

2. Viability .................................................................................................... 24 

3. Cell Number/Dose .................................................................................... 24 

V. FINAL PRODUCT RELEASE CRITERIA TESTING.............................................. 24

VI. PRODUCT STABILITY................................................................................................ 25

VII. OTHER ISSUES ............................................................................................................. 26

A. Product Tracking................................................................................................ 26

B. Labeling ............................................................................................................... 26

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C. Container/Closure............................................................................................... 27

D. Environmental Impact ....................................................................................... 27

E. Qualification of the Manufacturing Process .................................................... 27

F. Biostatistics.......................................................................................................... 28

VIII. PRECLINICAL STUDIES TO BE DOCUMENTED BY FDA REVIEWERS........ 29

A. Summary of Concept Studies............................................................................. 29

B. Gonadal Distribution.......................................................................................... 29

IX. CLINICAL STUDIES TO BE DOCUMENTED BY FDA REVIEWERS................ 29

A. Protocol Title ....................................................................................................... 29

B. Subject Population.............................................................................................. 29

C. Route of Administration..................................................................................... 30

D. Dose ...................................................................................................................... 30

E. Frequency ............................................................................................................ 30

F. Genetic, Biochemical, and Immunological Testing.......................................... 30

G. Informed Consent ............................................................................................... 30

H. Recombinant DNA Advisory Committee (RAC) Review................................ 30

X. RECOMMENDATION TO BE DETERMINDED BY FDA REVIEWERS ............ 30

XI. COMMENTS TO SPONSOR GENERATED BY FDA REVIEWERS .................... 31

A. Clinical Hold........................................................................................................ 31

B. Non-Clinical Hold ............................................................................................... 31

XII. REFERENCES................................................................................................................ 32

APPENDIX A – PRODUCT REVIEW TEMPLATE (GENE THERAPY).......................... 35

APPENDIX B – CONSIDERATIONS FOR DEVELOPMENT OF FINAL PRODUCT 

RELEASE CRITERIA SPECIFICATIONS AND STABILITY PROTOCOLS ..... 40

A. Development of Release Acceptance Criteria .................................................. 40

B. Development of Acceptance Criteria Analytical Procedures ......................... 40

C. Development of Stability Protocols................................................................... 41

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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) 

This guidance represents the Food and Drug Administration’s (FDA’s) current thinking on this 

topic. It does not create or confer any rights for or on any person and does not operate to bind 

FDA or the public. You can use an alternative approach if the approach satisfies the 

requirements of the applicable statutes and regulations. If you want to discuss an alternative 

approach, contact the appropriate FDA staff. If you cannot identify the appropriate FDA staff, 

call the appropriate number listed on the title page of this guidance. 

I. INTRODUCTION 

This guidance document provides to you, sponsors of a human gene therapy investigational new 

drug application (IND), recommendations on the chemistry, manufacturing, and control (CMC) 

information to include in an original IND. This guidance also applies to combination products 

that contain a human gene therapy biological product in combination with a drug or device as 

part of the final product. Also, this guidance instructs FDA CMC reviewers about the 

information to record and assess as part of an IND review, taking into consideration the various 

manufacturing challenges for these products. 

In order to deliver a safe and effective product, human gene therapies present many 

manufacturing challenges. Some of these challenges include the variability and complexity 

inherent in the components used to generate the final product, such as the source of cells (i.e., 

autologous or allogeneic), the potential for adventitious agent contamination, the need for aseptic 

processing, and in the case of ex vivo genetically modified cell therapies the inability to 

“sterilize” the final product because it contains living cells. Distribution of these products can 

also be a challenge due to stability issues and the frequently short dating period of many ex vivo 

genetically modified cell products, which may necessitate release of the final product for 

administration to a patient before certain test results are available. 

This guidance finalizes the draft guidance entitled, “Guidance for FDA Review Staff and 

Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information 

for Human Gene Therapy Investigational New Drug Applications (INDs)” dated November 2004 

(69 FR 64958; November 9, 2004).

FDA’s guidance documents, including this guidance, do not establish legally enforceable 

responsibilities. Instead, guidances describe the FDA’s current thinking on a topic and should be 

viewed only as recommendations, unless specific regulatory or statutory requirements are cited. 

The use of the word should in FDA’s guidances means that something is suggested or 

recommended, but not required. 

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A. How will FDA Reviewers and Sponsors Use this Guidance? 

FDA’s primary objectives in the review of INDs are to help ensure the safety and rights 

of human subjects in all phases of an investigation and, in Phases 2 and 3, to help ensure 

that the quality of the scientific evaluation of the investigational product is adequate to 

permit an evaluation of its safety and effectiveness (21 CFR 312.22(a)). This guidance 

will help sponsors and FDA reviewers to assess, given the phase of the investigation, 

whether sufficient information is provided to assure the proper identification (identity 

testing), quality, purity, and strength (one aspect of potency) of the investigational 

product (21 CFR 312.23(a)(7)(i)). These principles apply to investigational biological 

products and drugs; however, specific terms, such as safety, identity, purity, and potency, 

are generally understood to be applicable to biological products and are used throughout 

this document. 

If you are a FDA reviewer, you will use this guidance as you assess the safety, identity, 

purity, and potency of an investigational product and you will use the format of the 

human gene therapy CMC review template (Appendix A) in preparing your reviews. 

Because of the wide variability of the contents of IND amendments, you are only 

expected to use the attached template during review of original IND submissions. 

However, you should consult this document for guidance throughout the investigational 

new drug development process. 

The human gene therapies CMC review instructions and template described in this 

guidance are tools to assist FDA in the review of human gene therapy INDs. They are 

designed to serve as a guide to help ensure that all applicable regulatory requirements are 

reviewed at the appropriate stage of product development. In addition to the CMC 

review instructions and template, some general considerations are discussed in Appendix 

B that should be helpful in assessing proposed release criteria testing and specifications. 

Section 10.70 (21 CFR 10.70) provides further instruction to FDA reviewers regarding 

documentation of review decisions. 

If you are a sponsor of a human gene therapy IND, you may use this guidance in 

developing an IND submission that will be adequate to permit FDA reviewers to make an 

assessment of the safety, identity, purity, and potency of your investigational product. 

Other regulatory documents that may be relevant are listed in the references (see Section 

XII below). 

B. How is this Guidance Organized? 

This guidance is organized in a format that generally corresponds to the sections in the 

CMC review template provided in Appendix A. In each section, where necessary, we 

(FDA) provide recommendations as to the information you may submit in your original 

IND submission. As necessary throughout this document, we give specific instructions 

to FDA reviewers concerning their documentation and assessment of an IND submission 

during the CMC review. Many of the instructions for FDA reviewers provided in this 

guidance are distinguished by the designation “Note to FDA Reviewers.” 

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II. ADMINISTRATIVE INFORMATION TO BE DOCUMENTED BY FDA 

REVIEWERS 

Note to FDA Reviewers: Document in your review all of the IND information listed below. 

Most of this information should be available on Form FDA 1571, the sponsor’s cover letter, or 

the reviewer assignment notice from the Regulatory Project Manager (RPM) of the application 

division. 

• BB-IND Number (assigned by Center for Biologics Evaluation and Research (CBER) 

after receipt); 

• Date of submission; 

• 30-day review due date; 

• Sponsor – name, address, title, phone, fax; 

• Sponsor point of contact (sponsor’s authorized representative) – name, address, title, 

phone, fax; 

• Title of IND; 

• Proposed use; 

• Product description; 

• Phase of study; 

• Cross-referenced INDs, investigational device exemptions (IDEs), and master files 

(MFs): List all regulatory files (IND, IDE, MF) that the sponsor has obtained permission 

to cross-reference in support of this file. The file under review must contain a letter 

signed by the person who submitted the cross-referenced file (21 CFR 312.23(b)), giving 

FDA permission to cross-reference the file. This letter should identify the nature of the 

information being cross-referenced (e.g., pre-clinical, product manufacturing, and/or 

clinical) and where it is located within the file being cross-referenced. You should verify 

that the cross-referenced information satisfies the IND requirement for which the 

information is cited. If the letter of cross-reference is absent or inadequate, or the crossreferenced information is inadequate for the purpose cited, the RPM or the reviewer 

should notify the sponsor to obtain additional information; 

• Key words: Include three to four words that can be used to identify the product, 

indication, and any materials, components, or devices that may be part of the final 

product or used in the manufacturing process. These key words should be general 

enough to be used in a database search; 

• Introduction/rationale: Summarize relevant information on the development of the 

product if the sponsor provides this information. In addition, document and assess, as 

appropriate, the sponsor’s scientific rationale and justification for using the product for 

the indication under review; and 

• Study objectives. 

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III. PRODUCT MANUFACTURING AND CHARACTERIZATION INFORMATION 

TO BE SUBMITTED BY SPONSORS AND DOCUMENTED BY FDA 

REVIEWERS 

As described in the following sections, you should provide a detailed description of where and 

how the gene therapy product is manufactured. You should include all of the components and 

materials used during the manufacture of the gene therapy product, such as the vector, cells, cell 

bank systems, and any reagents or excipients. In addition, you should describe all procedures 

used during the manufacturing process. Examples of these procedures may include vector 

derivation, purification, preparation of cell banks systems and testing, including final 

formulation of the product. This information will allow us to assess the identity, quality, purity, 

and potency of your product. For further information, refer to the guidance on “Content and 

Format of Investigational New Drug Applications (INDs) for Phase 1 Studies of Drugs, 

Including Well-Characterized, Therapeutic, Biotechnology-Derived Products” (Ref. 1). In 

addition, you may refer to the other documents listed in the references (see Section XII below). 

We also encourage sponsors to use the format and headings described in Appendix A to facilitate 

an efficient review by FDA. 

Note to FDA Reviewers: Document and assess product manufacturing and characterization 

information in your IND reviews. Organize the CMC review using the format and headings 

described in Appendix A and below, as appropriate. 

A. Product Manufacturing – Components and Materials 

Note to FDA Reviewers: Document the source of all materials and components and 

summarize the testing performed on those materials and components, and review the 

specific instructions and recommendations set out below. 

Your IND must include a list of all components used in manufacturing of your product 

(21 CFR 312.23(a)(7)(iv)(b)). The sections below detail the information on 

manufacturing components that we recommend you submit in an IND, and that FDA 

reviewers will document and assess. 

1. Vector 

You should provide the following information about your vector: 

a. Gene Therapy Vector Construct 

A description of the history and detailed derivation of the gene therapy vector 

including: 

• The gene map, with relevant restriction sites, and any vector constructs 

used during generation of the final vector and their sources; 

• The gene insert; 

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• Regulatory elements, such as promoter, enhancer, and poly-adenylation 

signal; and 

• Selection markers. 

b. Vector Diagram 

A diagram of the vector identifying the gene insert and regulatory regions, and any 

other relevant elements, such as pertinent restriction endonuclease sites: 

Note to FDA Reviewers: Document the vector diagram by scanning it into the 

review document. 

c. Sequence Analysis 

Vectors 40 kilobases (kb) or less: We recommend that you fully sequence all 

vectors under 40 kb, perform sequence analysis, and submit an annotated sequence 

of the entire vector. You should provide a summary of the sequence analysis, 

indicating the origin and function of each component of the vector that accounts for 

all nucleotides such as promoters, known coding sequences, polyadenylation 

signals, origins of replication and restriction sites used during construction of the 

vector or for diagnostic tests. 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 recommend viral vectors be 

sequenced from the master viral bank (MVB) when appropriate. Plasmid sequence 

should be obtained from the master cell bank (MCB), and retroviral vector sequence 

should be obtained from the MVB/packaging cell line or from DNA obtained after 

transduction of a stable cell line. 

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. We recommend that you perform sequence 

analysis of the gene insert, flanking regions, and any regions of the vector that are 

modified. 

2. Cells 

a. Allogeneic and/or Autologous Cell Components 

You should describe the following information in your IND: 

• Cell source: tissue and cell type (e.g., colon, hematopoietic, neuronal, Tcells); 

• Mobilization protocol: document whether or not donor cells are mobilized 

or activated in vivo in the donor; 

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• Collection or recovery method: state the procedure used to obtain cells 

(e.g., surgery or leukapheresis indicating the device used if possible), the 

name and location of the collection facility, and transport conditions if 

shipped to a processing facility for further manufacturing; and 

• Donor screening and testing: the donor screening and testing that is 

performed to determine donor eligibility. Requirements for screening and 

testing donors of human cells and tissues are described in 21 CFR Part 

1271 (see final rule, “Eligibility Determination for Donors of Human 

Cells, Tissues and Cellular and Tissue-Based Products (HCT/Ps)”) (Ref. 

2). When appropriate, you should document the donor safety testing that 

is performed. In addition, FDA has published a final “Guidance for 

Industry: Eligibility Determination for Donors of Human Cells, Tissues, 

and Cellular and Tissue-Based Products (HCT/Ps)” (Ref. 3). We 

recommend that you review this guidance to ensure that the donor 

qualification criteria described in your IND are consistent with current 

recommendations. 

1) Autologous 

You are not required to make a donor eligibility determination or to perform 

donor screening for cells and tissues for autologous use (21 CFR 

1271.90(a)(1)). However, you should determine whether your manufacturing 

procedures increase the risk of propagation of pathogenic agents that may be 

present in the donor. If so, you should document whether the donor is 

reactive for specific pathogens. Also, you should describe precautions to 

prevent the spread of viruses or other adventitious agents to persons other 

than the autologous recipient (see Ref. 2). 

2) Allogeneic 

You must perform donor screening and testing as required in 21 CFR Part 

1271 for all allogeneic cells or tissues except those that meet the exceptions 

in 21 CFR 1271.90(a). Donors of all types of cells and tissues must be 

screened and tested for HIV-1, HIV-2, hepatitis B virus (HBV, surface and 

core antigen), hepatitis C virus (HCV), Treponema pallidum (syphilis), and 

CJD (screening only). Donors of viable leukocyte-rich cells or tissues should 

be screened and tested for human T-lymphotropic virus types 1 and 2 

(HTLV-1, HTLV-2) and CMV. In addition, you should document whether 

FDA-licensed, cleared, or approved test kits are used in these detection 

assays and document which tests are used. Include a description of the type 

of serological, diagnostic, and clinical history data obtained from the donor. 

You should consider other issues such as typing for polymorphisms and 

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human leukocyte antigen (HLA) matching, where appropriate. If cord blood 

or other maternally derived tissue is used, you should describe testing and 

screening performed on birth mothers. 

Note to FDA Reviewers: Communicate with the clinical reviewer regarding any 

issues or concerns relating to the screening or testing of the donor cells. 

b. Cell Bank System 

You should describe pertinent information, as described in Sections 1) through 3) 

below, relating to the cell bank system (i.e., master cell bank (MCB), and working 

cell bank (WCB)) used in product manufacture. In addition, you should describe 

the history, source, derivation, characterization of each cell bank (both MCB and 

WCB), and the frequency at which testing is performed. For further information, 

refer to the document on “Points to Consider in the Characterization of Cell Lines 

Used to Produce Biologicals” (Ref. 4). See also ICH document Q5D, “Derivation 

and Characterization of Cell Substrates Used for Production of 

Biotechnological/Biological Products” (Ref. 5), and, where applicable, “Guidance 

for Industry: Source Animal, Product, Preclinical, and Clinical Issues Concerning 

the Use of Xenotransplantation Products in Humans” (Ref. 6) and the “PHS 

Guideline on Infectious Disease Issues in Xenotransplantation” (Ref. 7).1 

Note to FDA Reviewers: Document and assess the testing that is performed on 

each cell bank. Determine if the most relevant and critical testing for the 

particular gene therapy product has been performed. Appropriate tests should be 

performed depending on the species of origin used to derive the cell bank. 

1) Master Cell Bank (MCB)/Packaging Cell Line2

You should include in the IND information regarding MCB history, source, 

derivation and characterization, including testing to adequately establish the 

safety, identity, purity, and stability of the cells. This section will likely 

address: 

• Product microbiologic characteristics, including sterility, mycoplasma, in 

vivo and in vitro testing for adventitious viral agents, as appropriate (see 

Section IV below); 

• Freedom from the presence of specific pathogens, including, for human 

 

1

 If a feeder cell line of animal origin is used to propagate human cells (i.e., human and non-human animal cells are 

co-cultivated), the final product falls within the definition of a xenotransplantation product (see both Refs. 9 and 

10). 

2

 If an ecotropic cell line was used during the generation of a retroviral producer cell line, we recommend that 

sponsors test for ecotropic retrovirus (see “Product testing”). Reviewers would assess and document the testing that 

was performed. 

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cells, testing for CMV, HIV-1 & 2, HTLV-1 & 2, EBV, B19, HBV, and 

HCV, as appropriate. For cell lines that are exposed to bovine or porcine 

components (e.g., serum, serum components, trypsin), appropriate testing 

would include testing for bovine and/or porcine adventitious agents. See 

further discussion of bovine components and reagents in Section III.A.2.a; 

• Identity of the cells, including tests to distinguish the specified cells 

through physical or chemical characteristics of the cell line (i.e., 

phenotype, genotype, or other markers); 

• Purity of banked cells, including identification and quantification of any 

contaminating cells; 

• Testing for activity of cells (e.g., activated lymphocytes, dopamine 

secretion, insulin secretion) and cell maturation (e.g., dendritic cells). 

This should be performed if activity is relevant to the therapeutic nature of 

the product; 

• End of production cells (EOP). This should be tested on a one time basis 

to assess genetic stability of the MCB/packaging cell line; and 

• Processes critical to product safety, as applicable, including: 

o Culture conditions used, including documentation of all media, and 

reagents/components used during production, with copies of 

relevant certificates of analysis (COA);

o Method of introduction of vector (transfection, transduction, 

infection) into MCB/parental cells to establish vector producer 

cell; 

o Analysis and selection of producer cell clone; and

o Cryopreservation, storage, and recovery of the MCB, including 

information pertaining to cell density, number of vials frozen, 

storage temperature, and cell bank location; and 

o Genetic and phenotypic stability of the MCB after multiple 

passages as well as viability of cells after cryopreservation. We 

recommend that, while the IND is in effect, you perform a stability 

assessment EOP as a one-time test. This testing is usually 

performed later in product development and would be included as 

part of the biologics license application (BLA). 

2) Master Viral Bank (MVB) 

You should provide a description of the MVB and the testing that you have 

performed to ensure safety, purity, and identity. We recommend that you 

address: 

• History and derivation of the MVB; 

• Culture conditions used during tissue culture scale up; 

• Testing of media and other reagents used during production, including 

COAs; 

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• Product microbiologic characterization – including sterility, mycoplasma, 

in vivo and in vitro testing for adventitious viral agents, as appropriate; 

• Freedom from the presence of specific pathogens, such as human viruses 

if the cell line is of human origin, or pathogens specific to the origin of the 

production cell line (e.g., murine, non-human primate,; 

• Tests to identify presence of replication competent virus; 

• Identity testing to establish the presence of gene therapy vector and 

therapeutic transgene (e.g., Southern blot); and 

• Information pertaining to the cryopreservation of the MVB, including 

condition and storage location(s). 

3) Working Cell Bank (WCB)/Working Viral Bank (WVB) 

The WCB/WVB may have been derived from one or more vials of the 

MCB/MVB. As discussed in the guidance documents referenced in III.A2a, 

the amount of information needed to document characterization of the 

WCB/WVB (derived from the MCB, and MVB, respectively) is usually less 

extensive than that needed to document characterization of the MCB/MVB. If 

there is a two tiered cell bank system in place (MCB, MVB), we recommend 

that you test the WCB/WVB for: 

• In vitro adventitious viral agent testing; 

• Replication competent virus; 

• Bacterial and fungal sterility; 

• Mycoplasma; and 

• Limited identity testing (e.g., Southern blot, flow cytometry). 

3. Reagents 

You must list in your IND any reagents used in manufacturing the product (21 CFR 

312.23(a)(7)(iv)(b)). For the purpose of this guidance, reagents are those materials 

that are used for cellular growth, differentiation, selection, purification, or other 

critical manufacturing steps but are not intended to be part of the final product. 

Examples include fetal bovine serum, trypsin, digestion enzymes (e.g., collagenase, 

DNAse) growth factors, cytokines, monoclonal antibodies, antibiotics, cell 

separation devices, and media and media components. These reagents can affect the 

safety, potency, and purity of the final product, especially by introducing 

adventitious agents. For monoclonal antibodies, refer to the guidance on “Points to 

Consider in the Manufacture and Testing of Monoclonal Antibody Products for 

Human Use” (Ref. 8) for further information. 

a. Tabulation of Reagents Used in Manufacture 

We recommend that you provide the following information on all reagents used 

during product manufacturing: 

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• concentration of the reagent at the manufacturing step at which it is used; 

• vendor/supplier; 

• source: 

o human: If human albumin is used, you should have procedures in 

place to ensure that no recalled lots were used during manufacture 

or preparation of the product. If using human AB serum, ensure 

serum is obtained from an approved blood bank and meets all 

blood donor criteria. For all other reagents that are human derived 

you should identify whether it is a licensed product, or clinical or 

research grade, and provide a COA or information regarding 

testing of the donor and/or reagent. 

o porcine: If porcine products are used, a COA or other 

documentation that the products are free of porcine parvovirus. 

o bovine: If a reagent is derived from bovine material, you should 

identify the bovine material, the source of the material, information 

on the location where the herd was born, raised, and slaughtered, 

and any other information relevant to the likelihood that the animal 

may have ingested animal feed prohibited under 21 CFR 589.2000. 

It may be that bovine material is introduced at different points in 

production of a reagent; the information described above should be 

provided for all bovine materials used. For more information see, 

“Proposed Rule: Use of Materials Derived from Cattle in Medical 

Products Intended for Use in Humans and Drugs Intended for Use 

in Ruminants,” (72 FR 1581; January 12, 2007); found at 

http://www.fda.gov/cber/rules/catruminant.htm. In addition, you 

should provide a COA to document that bovine materials are 

compliant with the requirements for the ingredients of animal 

origin used for production of biologics described in 9 CFR 113.53. 

Note to FDA Reviewers: For all animal derived products, enter the following 

information in the animal components database: source organism, 

supplier/vendor, country of origin, and stage of manufacture. Additionally, the 

information provided on bovine materials should be evaluated to determine 

whether, for informed consent that is adequate under 21 CFR Part 50, the subject 

should be informed of the potential risk that TSE agents have been introduced 

into the final product. 

• Reagent quality: We recommend that you use FDA-approved or cleared, 

or clinical grade reagents whenever they are available. 

Note to FDA Reviewers: If the reagent is regulated as a biological product, drug, 

or device, consider whether a consultative review should be obtained. See 

Section III.A.5.b below for further information about consultative review process. 

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• COA or cross-reference letters: If you are using a research grade (not 

FDA-approved or cleared) reagent as part of the manufacturing process, 

we recommend that you provide information verifying the source, safety, 

and performance of the reagent. If the vendor of the reagent has a 

regulatory file with the FDA, a cross-reference letter from the sponsor 

may be provided in the IND. If a COA from the reagent manufacturer is 

used, you may assess whether the testing performed is adequate (see 

“Qualification Program” below) and provide that information in the IND. 

Note to FDA Reviewers: For letters of cross-reference, include the regulatory file 

number and consider the need for a consultative review to determine whether 

there are any safety concerns or other outstanding issues. 

b. Qualification Program 

If the reagent is not FDA-approved or cleared, additional testing may be needed 

to ensure the safety and quality of the reagent. We recommend that you establish 

a qualification program that includes safety testing (sterility, endotoxin, 

mycoplasma, and adventitious agents), functional analysis, purity testing, and 

assays (e.g., residual solvent testing) to demonstrate absence of potentially 

harmful substances. The extent of testing will depend on how the specific reagent 

is used in the manufacturing process. 

c. Determination of Removal of Reagents from Final Product 

You should test the final product for residual manufacturing reagents with known 

or potential toxicities and describe the test procedures you use to detect residual 

levels of these reagents in the final product. We recommend that you determine 

whether a qualification study is sufficient to document their removal, or whether 

lot release testing is appropriate prior to initiation of clinical trials. 

d. Other Concerns 

Because some patients may be sensitive to penicillin, we recommend that you do 

not use beta-lactam antibiotics during the manufacturing of a therapeutic product 

for humans. If beta-lactam antibiotics are used, we recommend that you provide a 

rationale for their use and describe precautions to prevent hypersensitivity 

reactions. 

Note to FDA Reviewers: If beta-lactam antibiotics are used during 

manufacturing, consult with the clinical reviewer concerning appropriate 

exclusion criteria for the study and proper informed consent to address potential 

patient sensitivity. Discuss with the sponsor the rationale for using beta-lactam 

antibiotics and whether an alternative to beta-lactam antibiotics can be used as an 

appropriate substitute. 

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4. Excipients 

For the purpose of this guidance, an excipient is any component that is intended to be 

part of the final product, such as human serum albumin or Dimethyl Sulfoxide 

(DMSO). You must list in your IND all excipients used during manufacture of the 

product that are intended to be present in the final product (21 CFR 

312.23(a)(7)(iv)(b)). You should include the concentration and source of the 

excipients. Also, you must provide information regarding the qualification of all 

excipients that are present in the final product (21 CFR 211.84(a)). For further 

information, please see Section III.A.2 above on reagents. 

5. Additional Considerations 

a. Combination Products 

This guidance also applies to combination products containing a human gene 

therapy biological product in combination with a drug or device as part of the 

final product.3 When intended for a different use, the drug or device component 

may already have FDA marketing approval or clearance (e.g., new drug 

application (NDA), a premarket approval application (PMA), or a premarket 

notification (510(k)), or it may be previously unstudied. 

If information describing the drug or device component has already been 

submitted to FDA (e.g., in another IND, IDE, or Master File), you may submit a 

letter of authorization authorizing FDA to reference the previous submission for 

CMC or other information related to the drug or device component of your 

product. 

Note to FDA Reviewers: Determine the regulatory status of the drug or device 

either by contacting the RPM from the center with jurisdiction for the drug or 

device or the sponsor directly, if necessary. If the drug or device has been 

approved for any use, confirm and document this in your review. In most cases, 

request a consultative or collaborative review from the Center for Drug 

Evaluation and Research (CDER) or the Center for Devices and Radiological 

Health (CDRH) even if the drugs or device components were previously approved 

for another use. Confer with your supervisor if it is unclear whether a 

consultative or collaborative review is needed. Once an adequate letter of 

authorization is submitted, verify 

 

3

 Regulations on combination products are found in 21 CFR Part 3, which describes how we will determine which 

component of FDA has primary jurisdiction for the premarket review and regulation of a combination product. 

Note to FDA Reviewers: If you have any concerns regarding the appropriateness of the jurisdictional assignment or 

regulatory mechanism, you should contact the Office of Cellular, Tissues, and Gene Therapies’ jurisdictional 

officer. 

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that the referenced file contains the needed information. Inform the consultative 

or collaborative reviewer that the information referenced in the letter of 

authorization should be reviewed as part of the application. 

b. Consultative Reviews 

Note to FDA Reviewers: Follow the standard operating procedures and policies 

(SOPP) on the “Intercenter Consultative/Collaborative Review Process” (Ref. 9). 

Specify the questions the consultative reviewer should address, identify the 

specific sections of the IND applicable to those questions, and request a date for 

completion of the consultative review. The requested date should be determined 

by coordinating with the consulting review center and be based on timeframes 

mandated by statute, the priority of the consultative review request, and the 

workload of the designated reviewer. The RPM will request the consultative or 

collaborative review from the appropriate Center/Division using the form in 

Appendix 1 of the SOPP. Given the tight IND deadlines, work with the RPM to 

contact the appropriate Center/Division before sending the consult request to 

identify the appropriate reviewer and ensure that the review can be completed 

within the time requirements. Also, as described in the SOPP, the RPM should 

send to the Office of Combination Products a copy of the 

consultative/collaborative request for monitoring/tracking purposes. The RPM 

should follow up with the consultative reviewer to confirm that essential 

documents are received along with the consultative review request. If problems 

that affect the timeliness of the consultative review occur during the consult 

review period, discuss with your supervisor how to share these experiences with 

the Office of Combination Products, which is responsible for monitoring the 

efficiency and effectiveness of the intercenter consultative/collaborative review 

process. 

c. Review of Device Components 

Note to FDA Reviewers: In the device consultative/collaborative review request, 

describe the device component, and where to find relevant information in the 

submission. You should ask the consultative reviewer to identify concerns with 

how the device component will be used, to determine whether appropriate types 

of biocompatibility and other device testing were performed adequately, and to 

assess testing of any hardware and software controlling the hardware. In addition, 

if the sponsor asserts barrier or performance claims, identify information relative 

to these claims for the consultative reviewer to assess. You should document in 

the review basic information concerning the device, such as the device name, 

vendor or source, purpose, regulatory status, and a brief description of the device. 

When the consultative/collaborative review is completed, and signed off by the 

appropriate CDRH supervisory chain, attach it to your review and communicate 

any outstanding issues to the sponsor. 

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d. Review of Drug Components 

Note to FDA Reviewers: In the drug consultative/collaborative review request, 

describe the drug component of the combination product and state where to find 

relevant information on the component in the submission. Ask the 

consultative/collaborative reviewer to identify any concerns with how the drug 

will be used and also to evaluate the methods of manufacturing and the adequacy 

of results from testing of the drug substance and/or drug product. You should 

document in your review basic information concerning the drug component, such 

as the drug name, vendor or source, purpose, regulatory status, and a brief 

description of the use of the drug. When the consultative/collaborative review is 

completed and signed off by appropriate CDER supervisory chain, attach it to 

your review and communicate any outstanding issues to the sponsor. 

e. Summarize Any Areas of Concern to be Addressed 

Note to FDA Reviewers: Summarize any areas of concern identified during the 

review of the product components. Discuss these concerns with the sponsor 

and/or communicate them in a letter to the sponsor, as described in Section XI, 

“Comments to Sponsor Generated by FDA Reviewers”. 

B. Product Manufacturing – Procedures 

You should include a detailed description (list or summary) of all procedures used during 

the collection, production, and purification of the gene therapy product. We believe that 

a schematic of the production and purification process, and in-process and final product 

testing, helps to provide more clearly this information. 

Note to FDA Reviewers: If provided by the sponsor, append a copy of the process 

schematic to the IND review. If a schematic is not included, briefly summarize the 

processing steps used during product manufacturing. In addition, summarize any areas of 

concern identified during the review of any submitted product manufacturing procedures. 

Discuss these concerns with the sponsor and/or communicate to the sponsor in a letter, as 

described in Section XI, “Comments to Sponsor Generated by FDA Reviewers”. 

1. Vector Production/Purification 

You should include the following in your procedures: 

• all purification steps in order of processing, for example: centrifugation, 

column purification, and density gradients; 

• reagents/components used during vector manufacture; 

• describe cell lines used during production of vector product including; cell 

passage number and cell plating density; and 

• culturing procedures, and media components used, including serum, 

growth factors, and antibiotics used during cell propagation. 

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2. Preparation of ex Vivo Gene-Modified Autologous or Allogeneic Cells 

Autologous or allogeneic cells can be modified by using viral or plasmid vectors. 

We recommend that you describe the following procedures: 

a. Method of Cell Collection/Processing/Culture Conditions 

You should describe the cells used for ex vivo modification (i.e., cell lines, 

tumor cells, donor derived cells). If non-banked autologous or allogeneic cells 

are used, describe the volume and number of cells collected. You should 

include any mechanical or enzymatic digestion steps used. You should describe 

the use of any cell selection device or separation device, including density 

gradients, magnetic beads, or fluorescence activated cell sorting (FACS). You 

should include a description of culture systems (flasks, bags, etc.), and state 

whether the system is closed or open. Also, you should describe any in-process 

testing that will be performed during these procedures. 

b. Ex Vivo gene modification 

You should describe in detail the modification procedure, such as, transfection, 

or infection. You should describe in detail the selection of cells (methods, 

devices, reagents) as well as any other cell modification steps such as 

irradiation. If the cells are cultured after the genetic modification occurs, you 

should include the culture conditions used and time in culture. 

c. Irradiation of ex vivo gene modified cells 

If the autologous or genetically modified allogeneic cell product is irradiated 

before injection, you should provide data to demonstrate that the genetically 

modified cells are rendered replication-incompetent, but still maintain their 

desired characteristics after irradiation. You should describe the documentation 

of calibration of the cell irradiator source. 

d. Final Harvest 

You should provide a detailed description of the final harvest. You should 

inform FDA whether the final cell harvest is centrifuged prior to final 

formulation, and if so, describe the wash conditions and media used. You 

should also inform FDA in your IND submission whether the cells are 

cryopreserved after formulation or formulated immediately and given to the 

patient. If the final harvest is stored, you should describe the storage 

conditions and the length of storage, and provide appropriate supporting data 

(see Section VI below). 

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3. Process Timing and Intermediate Storage 

Report the approximate time elapsed for each step in vector production including 

vector purification, intermediate holding steps (i.e., storage of bulk harvest) and 

cryopreservation of final vector product. In addition, if the final product is ex vivo 

genetically modified cells, you should report the approximate time elapsed for each 

step from cell collection, to transduction, to final harvest. It is important to know the 

time limit of each step in production to determine what, if any, in-process testing to 

perform. If the final product is cryopreserved before injection into patients, you 

should include this information along with any stability studies initiated (see Section 

VI.A.1 below). Also, you should describe the time and conditions of storage prior to 

patient administration. 

Note to FDA Reviewers: Describe and assess the procedures in place to ensure the 

stability of the bulk harvest while in storage. 

4. Final Formulation 

You must describe the formulation of the final product, including excipients such as 

growth factors or human serum albumin (21 CFR 312.23(a)(7)(iv)(a)). You should 

state the source of these components (see Section II.A.3 above). You should identify 

the vendor and final concentration of these excipients used in the final product. If the 

final product is delivered to the clinical site frozen, you should include a description 

of how the product will be shipped and data to show that the product can be thawed 

with consistent results. In addition, if the product is shipped, you should provide data 

on product stability during shipping (see Section VI.A.2 below). 

IV. PRODUCT TESTING 

We recommend that product testing for gene therapies include, but not be limited to, 

microbiological testing (including sterility, mycoplasma, and adventitious viral agent testing) to 

ensure safety and assessments of other product characteristics such as identity, purity (including 

endotoxin), viability, and potency. We recommend that you perform this testing throughout the 

manufacturing process, including on the manufacture of cell banks, to evaluate the 

manufacturing process itself and to ensure the quality and consistency of the product. You 

should describe the specifications used for intermediate acceptance criteria and final product 

release criteria. Specifications are the quality standards (i.e., tests, analytical procedures, and 

acceptance criteria) that confirm the quality of products and other materials used in the 

production of a product. Acceptance criteria mean numerical limits, ranges, or other attributes or 

variables for the tests described. Specifications should be appropriate to the stage of product 

development, because release criteria should be refined and tightened as product development 

progresses toward licensure (see Appendix B). You should submit test results related to lot 

release, characterization testing, MCB/MVBs, and WCB/WVBs in tabular form including the lot 

number or identifier, date of manufacture, test, test method, the sensitivity and specificity of test 

methods and, when appropriate, release criteria. 

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Product testing is an integral part of ensuring control of the manufacturing process and lot-to-lot 

consistency. Therefore, it is important to identify critical parameters in the manufacturing 

process and critical product attributes to ensure the desired clinical effect of the final product. 

Refer to the “FDA Guidance Concerning Demonstration of Comparability of Human Biological 

Products, Including Therapeutic Biotechnology-Derived Products” (Ref. 10) for additional 

information. 

Note to FDA Reviewers: Document the testing performed. Assess the appropriateness of that 

testing and the acceptance criteria, based on any results previously obtained by the sponsor. 

A. Microbiological Testing 

We recommend that you perform microbiological testing on cell banks, in-process 

intermediates, and the final product, as appropriate. 

1. Sterility Testing (Bacterial and Fungal Testing) 

Current practices for sterility testing. 

a. Test Method 

Suitable sterility tests include the test described in 21 CFR 610.12 and the test 

described in United States Pharmacopoeia (USP) <71> Sterility Tests (23rd

edition, 1995), (Ref. 11). Later versions of the USP<71> are also suitable. If you 

are using another test method, you should describe its suitability. Note that under 

21 CFR 610.9, prior to product licensing, the alternative method must be shown 

to provide assurances of the safety, purity, potency and effectiveness of the 

biological product equal to or greater that the assurances provided by the method 

or process specified in the 21 CFR 610.12 method. 

If you use antibiotics in manufacturing, you should provide documentation that 

the antibiotics were removed prior to sterility testing. If the antibiotics cannot be 

removed from the final product, we recommend that you assess the validity of the 

sterility assay using the bacteriostasis and fungistasis testing as described in USP 

<71> Sterility Tests. Use of this assay is designed to ensure that any residual 

antibiotic present in the product does not interfere with the results of sterility 

testing. 

Note to FDA Reviewers: If the Sponsor proposes to use an alternative method in 

place of the traditional sterility methods described in 21 CFR 610.12, then you 

will need to assess the adequacy of this alternative test method and the data 

provided to provide assurances of the safety, purity, potency and effectiveness of 

the biological product equal to or greater than the assurances provided by the 

method or process specified in the general standard (21 CFR 610.9). The 

completeness of the validation study for the alternative method used during the 

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product development should reflect an assessment of risk associated with the 

application of the method (e.g., stage of the manufacturing process). 

b. Test Timing 

Sponsors frequently perform in-process sterility testing at critical points during 

manufacturing, such as during purification, or after ex vivo gene modification or 

extended culture periods. You should identify when in-process sterility testing is 

performed during manufacturing and the test method used. The test method that 

you choose for in-process sterility testing should be adequate to provide assurance 

of product sterility. 

If you freeze the final product before its use, we recommend that you perform 

testing on 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 (e.g., washing, culturing) after thawing, particularly if 

procedures are performed in an open system, you may need to repeat sterility 

testing. You should incorporate the results of in-process sterility testing into your 

acceptance criteria for final product specifications. 

If your product has a short dating period and must be administered to patients 

before sterility test results of the final product are available, then you will need to 

develop an alternate approach to provide sterility assurance. As an alternative 

approach, we recommend that you perform all of the following tests: 

• in-process sterility testing on a sample taken 48 to 72 hours prior to final 

harvest or after the last re-feeding of the cultures 

• a rapid microbial detection test such as a Gram stain or other procedure on 

the final formulated product 

• sterility testing compliant with 21 CFR 610.12 on the final formulated 

product 

Under this alternative 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 in this situation 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 no-growth 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 culture on the final 

formulated product and when possible the in-process culture should be continued 

to obtain the full 14 day sterility test result even after the product has been given 

to the patient. 

In all cases where product release is prior to obtaining results from a full 14 day 

sterility test, the investigational plan should address the actions to be taken in the 

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event that the 14 day sterility test is determined to be positive after the product is 

administered to a subject. You should report the sterility failure, results of 

investigation of cause and any corrective actions, in an information amendment 

submitted to your IND in a timely manner, within 30 calendar days after initial 

receipt of the positive culture test result (21 CFR 312.31). 

The 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 experience that could be from administration of the 

sterility failure of the 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). If you determine that an 

investigational drug presents an unreasonable and significant risk to subjects, you 

must discontinue those investigations that present the risk, notify FDA, all 

Institutional Review Boards, and all investigators (21 CFR 312.56(d)). 

2. Mycoplasma 

There are several potential sources of mycoplasma contamination. Two major 

sources include animal serum products used in culture and the culture facility 

environment, particularly with open culture systems. We recommend that you 

perform mycoplasma testing on the product at the manufacturing stage when the test 

is most likely to detect contamination, such as after pooling of cultures for harvest but 

prior to cell washing. Testing should be conducted on both cells and supernatant. 

We recommend that you inform FDA whether there is in-process testing for 

mycoplasma during extended culture procedures. Due to the limited dating period of 

many ex vivo genetically modified cellular products, it is frequently not feasible for a 

sponsor to perform the recommended culture-based assay (see Ref. 4) for release 

testing. In those cases, we recommend the use of polymerase chain reaction (PCR)-

based mycoplasma assays or another rapid detection assay during product 

development. As part of your BLA, you should submit appropriate data to 

demonstrate that the PCR or alternative test has adequate sensitivity and specificity. 

3. Adventitious Agent Testing 

As appropriate, you should perform and describe in your IND adventitious agent 

testing as set out below. For more information on adventitious agent testing, refer to 

“Points to Consider in the Characterization of Cell Lines Used to Produce 

Biologicals” and ICH guidance Q5A: “Guidance on Viral Safety Evaluation of 

Biotechnology Products Derived From Cell Lines of Human or Animal Origin” (Refs. 

4 and 12). 

a. In Vitro Viral Testing 

When cell lines are used, you should describe the cell lines and perform in vitro 

viral testing. In vitro viral testing should be performed on the MCB, WCB, MVB, 

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WVB, and final vector product, and as a one time test on the EOP. Testing should 

be conducted by inoculating the test sample (MCB, WCB, MVB, WVB, final vector 

product) onto various susceptible indicator cell lines such as the human cell line 

MRC-5 or Vero cells which are primate in origin. The choice of cells used would 

depend on the species of origin of the product to be tested. An appropriate test 

should include monolayer cultures of the same species and tissue as that used for 

production of the product, as well as a human and a non-human primate cell line 

susceptible to human viruses. In addition, the test would include a measure of both 

cytopathic and hemadsorbing viruses. 

In the event that the product is a cytolytic virus, it will need to be neutralized using 

specific antibodies prior to in vitro adventitious testing of the MVB, WVB and final 

product. If neutralizing antibodies are not available, then only in vivo adventitious 

agent testing needs to be performed on the MVB, WVB and final product until 

appropriate neutralizing antibodies are developed. 

b. In Vivo Viral Testing 

When cell lines are used, we recommend that you perform and submit data on in 

vivo viral assays carried out by inoculating the test sample (MCB, MVB) into 

animals such as adult and suckling mice and embryonated hen eggs. You should 

consider whether to include additional testing of guinea pigs, rabbits, or monkeys. 

Such studies would assess the test animals for any indication of illness. If such 

additional testing is appropriate, you should describe and explain the suitability of 

the animals used. 

c. Selected Testing for Adventitious Viruses 

We recommend that you test your MCB and MVB for appropriate, species-specific 

viruses. You should describe the testing that is performed, the different stages of 

manufacturing where those tests are performed (e.g., cell banks, viral banks, final 

product), and the test methods used. 

1) Species-Specific Viruses 

You should describe all species-specific virus testing performed on the MCB 

and MVB. We believe that all rodent cell lines used during product 

manufacturing should be tested for rodent specific viruses. These viruses are 

usually detected by antibody production tests, murine antibody production 

(MAP), rat antibody production (RAP), or hamster antibody production (HAP). 

If human cell lines are used in the therapeutic product, we recommend that you 

perform testing for human pathogens (CMV, HIV-1 & 2, HTLV-1 & 2, EBV, 

HBV, HCV, and B19) and other human viral agents, as appropriate. We 

recommend such testing because these cells are manipulated (cultured for 

extended time periods) and human pathogens can be introduced or propagated 

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during the extended culture periods. Human viral agents may be tested using a 

PCR-based test system.

When the gene therapy product is produced in a human cell line, e.g., an 

adenoviral vector produced in human 293 cells, you should test for the presence 

of additional human viruses such as adenovirus and adeno-associated virus 

(AAV), and describe those tests in your IND. 

2) Testing for Retroviruses 

When the MCB and MVB are used for production of vectors other than 

retroviral vectors, you should test the MCB and MVB for retroviral 

contamination using Reverse Transcriptase (RT) assays and electron 

microscopic analysis, and include a description of those tests in your IND. 

We recommend that you perform testing for replication competent retrovirus 

(RCR) in the production of retroviral vectors at multiple points in production, 

including MVB, WVB, vector supernatants, end of production cells, and ex vivo 

modified cells. For further information on retroviral testing refer to the 

“Guidance for Industry: Supplemental Guidance on Testing for Replication 

Competent Retrovirus in Retroviral Vector Based Gene Therapy Products and 

During Follow-up of Patients in Clinical Trials Using Retroviral Vectors” (Ref. 

13). 

For cells that produce vectors containing amphotropic murine leukemia virus 

envelope, we recommend that you test for RCR using a permissive cell line such 

as Mus dunni and describe that testing in the IND. If an ecotropic packaging 

cell line is utilized during retroviral vector production, you should conduct and 

describe an ecotropic retroviral assay for the detection of low-level viral 

contamination in the MCB. Murine ecotropic viral contamination can be 

detected using either XC or D56 plaque assay methods. 

You should describe how vector supernatant is tested. An appropriate test of

vector supernatant lots would be by amplification on a permissive cell line such 

as Mus dunni, followed by detection in an appropriate indicator cell assay such 

as PG-4 S+L-. An appropriate test of the pooled End of Production (EOP) cells 

would be by co-culture with a permissive cell line such as Mus dunni for 

amphotropic virus, and the amplified material assayed in an appropriate 

indicator cell assay. We have determined that detection of RCR in ex vivo 

modified cells requires cells to be cultured for at least 4 days post transduction. 

Therefore, ex vivo gene modified cells that are cultured for ≥ 4 days should use 

RCR testing as a release assay. If ex vivo gene modified cells are cultured for < 

4 days, archiving cells would be appropriate in place of active RCR testing. If it 

is not possible to have results from the RCR assay prior to treatment, we 

recommend that you initiate the culture assay and perform an alternative method 

(such as PCR) for product release. 

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3) Adenoviruses 

For studies using adenoviral vectors, we recommend that you conduct tests for 

replication competent adenovirus (RCA) on MVB as well as the final vector. 

We believe that an appropriate maximum level of RCA contamination would be 

< 1 in 3 x 1010 viral particles, and that the adenoviral particle to infectious unit 

(iu) ratio would be ≤ 30 to 1. 

4) Adeno-Associated Virus (AAV) 

For studies using AAV, we recommend that you conduct tests to determine 

the amount of replication competent AAV present in the final vector product 

and report the results in your IND. 

B. Identity 

We recommend that you verify the identity of the MCB/MVB, WCB/WVB, and the final 

product by assays that will identify the product and distinguish it from other products 

being processed in the same facility. For the final product, identity testing is important to 

ensure that the contents of the vial are labeled appropriately. For additional information 

on labeling, refer to Section VII.B below. 

If the final product is an ex vivo genetically modified cell product, we recommend that you 

test the final cell product for identity. Testing should include an assay to measure the 

presence of vector (i.e., expression assay, restriction digest) and an assay specific for the 

cellular component of the final product (i.e., cell surface markers). 

C. Purity 

Product purity is defined as relative freedom from extraneous material in the finished 

product, whether or not harmful to the recipient or deleterious to the product (21 CFR 

610.3(r)). Purity testing includes assays for pyrogenicity/endotoxin (see below), residual 

proteins or reagents/components used during vector manufacture, such as cytokines, growth 

factors, antibodies, and serum, and in the case of ex vivo modified cells any unintended 

cellular populations or cell debris. 

1. Residual Contaminants 

The appropriate purity testing should include assays for residual proteins, DNA, 

RNA, and solvents used during production and purification, and reagents used 

during manufacture such as cytokines, growth factors, antibodies, and serum. If the 

product is a genetically modified ex vivo cell therapy product, appropriate purity 

testing should include a measurement of contaminating cell types or cell debris. For 

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further information, refer to ICH Q3 on “Impurities” (Ref. 14). You should describe 

in your IND the purity testing you conduct and your specifications for release. 

2. Pyrogenicity/Endotoxin 

The rabbit pyrogen test method is the currently required method for testing 

biological products for pyrogenic substances (21 CFR 610.13). Although the 

pyrogenicity test is required, there may be specific cases where this test method can 

not be performed for release due to properties of the final product (i.e., short product 

shelf life, toxicity of product in rabbits). Under these circumstances, a test method 

such as the Limulus Amebocyte Lysate test method (LAL) may be used as an 

alternative method, but prior to licensure must be shown to provide equal or greater 

assurances of safety, purity, and potency (see 21 CFR 610.9). The 1987 FDA 

Guideline on Validation of the Limulus Amebocyte Lysate (LAL) Test as EndProduct Endotoxin Test for Human and Animal Parenteral Drugs, Biological 

Products, and Medical Devices (Ref. 15) sets forth acceptable conditions for use of 

LAL. 

For any parenteral drug, except those administered intrathecally, we recommend that 

the upper limit of acceptance criterion for endotoxin be 5 EU/kg body weight/hour. 

For intrathecally-administered drugs, we recommend an upper limit of acceptance 

criterion of 0.2 EU/kg body weight/hour. You should describe in your IND the 

pyrogenicity/endotoxin testing you conduct, and your acceptance criterion for 

release. 

Note to FDA Reviewers: Document in your review the specification for 

pyrogenicity/endotoxin testing and verify that testing is on the final product and 

that results are available prior to lot release. 

D. Potency 

You should describe and justify all assays you will use to measure potency. We 

recommend that these assays be quantitative, but in addition, they may include a 

qualitative biological assay. For a Phase 1/Phase 2 study, we recommend that the assay 

quantify the expression of a gene therapy vector product. For a Phase 3 study, we 

recommend that the potency assay consist of in vivo or in vitro tests that measure an 

appropriate biological activity. If development of a quantitative biological assay is not 

possible, then a quantitative physical assay which correlates with and is used in 

conjunction with a qualitative biological assay can be used. Note that potency assays are 

part of lot release and should be validated prior to licensure. 

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E. Other 

1. General Safety Testing 

Testing for general safety is required for licensure of all gene therapy vector products, 

unless the product is exempt under 21 CFR 610.11(g). General safety testing is 

performed on biological products intended for administration to humans and specific 

tests are described in 21 CFR 610.11. You should inform FDA whether general safety 

testing is being performed during product development. 

Note to FDA Reviewers: Advise the sponsor regarding the applicability of the general 

safety test for the product under review. (Revisions to the General Safety Requirements 

for Biological Products (68 FR 10157; March 4, 2003) (Ref. 16)). 

2. Viability 

You should establish minimum release criteria for viability. For genetically 

modified cellular therapies, the minimum acceptable viability specification is 

generally set at 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. 

3. Cell Number/Dose 

If your final product is a genetically modified cell therapy, you should develop 

specifications for the minimum number of viable and functional cells as part of 

product testing and release. We recommend that you inform FDA whether a 

maximum number/dose of cells to be administered has been established, and the 

basis for that level. For administration of a gene vector, you should describe your 

dose as the concentration of plasmid DNA, viral particle number, or titer. 

V. FINAL PRODUCT RELEASE CRITERIA TESTING 

The final product is the final formulated product used for administration to human subjects. 

Final product release criteria testing should be performed on each lot of product manufactured. 

In some situations, each dose could be considered a single lot, depending on the manufacturing 

process. The results from final product release criteria testing should be available prior to 

administration to a human subject. If results from final product testing will not be available prior 

to release, we recommend that you clearly indicate this in the IND, together with your 

specifications, and include a description of the reporting notification process if the acceptance 

criteria are not met. We recommend that you provide, in table format, all of your proposed 

specifications (tests for safety, purity, potency, and identity as described in Section IV, test 

methods, and acceptance criteria), including test sensitivity and specificity, where appropriate, 

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for the final product. (Note that, before the product may be licensed, these parameters must be 

validated (21 CFR 211.165(e)). 

VI. PRODUCT STABILITY 

Stability testing must be performed during early phases of the clinical trial to establish that the 

product is sufficiently stable for the time period required by the study (21 CFR 312.23(a)(7)(ii)). 

Data supporting a final formulation and dating period will be necessary for licensure. You 

should describe the stability measures you will use to support your studies. For further 

information, refer to ICH Q5C: “Quality of Biotechnological Products: Stability Testing of 

Biotechnological/Biological Products,” (Ref. 17), ICH Guideline Q1A(R): “Stability Testing of 

New Drugs and Products” (revised guideline) (Ref. 18), ICH Guideline Q1E: “Evaluation of 

Stability Data” (Ref. 19). 

Note to FDA Reviewers: Assess the product development plan in the IND review to determine 

how much stability data are needed for the current phase of investigation and whether sufficient 

data are included in the submission. If submitted, include preliminary data in your review. 

Assess whether proposed expiration dating is appropriate. 

Stability Testing 

Under 21 CFR 312.23(a)(7)(ii), you must conduct stability testing in all phases of the 

IND, to demonstrate that the product is within acceptable chemical and physical limits 

for the planned duration of the proposed clinical investigation. If a very short term 

clinical investigation is proposed, the stability data submitted may be correspondingly 

limited. You should submit a stability protocol and data for both in-process material and 

the final gene product. A proposed stability protocol should include a measure of 

product sterility, identity, purity, quality, and potency. For each test conducted, you 

should describe the test method, sampling time points (there should be a zero-time point), 

testing temperature, and other appropriate information, including your justification of the 

assays used to indicate product stability, measuring those parameters for the duration of 

storage required by the clinical protocol. We recommend sterility testing be performed at 

zero-time, end of stability study, and at an intermediate point during the study.

Note to FDA Reviewers: If the sponsor plans to use the product past the duration of the 

clinical trial (e.g., for a separate trial being conducted after the initial trial), verify that 

testing establishes stability throughout the relevant time period. 

1) In-Process Stability Testing 

You should describe the stability protocol that will be used to ensure that the final 

product is stable during the period of cryopreservation, measuring the parameters 

described in Section VI.A, as appropriate. A comparison is often made of 

analyses carried out pre-freeze and post-thaw. You should describe any stability 

25 
Contains Nonbinding Recommendations 

testing performed on the product during the holding steps, such as 

cryopreservation of final product and storage of bulk product. 

2) Final Product Stability Testing 

You should include any data that demonstrate that the product is stable between 

the time of product formulation and infusion to subjects to aid in establishing an 

expiration-dating period. We recommend that you conduct the testing at the 

appropriate temperatures and at time points consistent with predicted storage 

times. If the product is shipped from the manufacturing site to the clinical site, 

describe the time and shipping conditions (e.g., packaging, temperature). Your 

stability protocol should be adequate to demonstrate that product integrity, 

sterility, and potency are maintained under the proposed shipping conditions. We 

further recommend that validation studies using conditions that stress the system 

be initiated by Phase 3 and completed prior to submission of a BLA. 

VII. OTHER ISSUES 

A. Product Tracking 

For all products, you should establish a product tracking and segregation system. An 

adequate system should allow identification of the therapeutic product from collection to 

administration of the product and should include procedures to ensure that the product is 

segregated from other products in incubators, hoods, and cryopreservation units. You 

must establish and maintain a system of tracking that enables the tracking of all products 

from the donor to the consignee or final disposition, and from consignee or final 

disposition to donor (21 CFR 1271.290(b)). 

B. Labeling 

You should describe the product labeling used throughout the manufacturing process and 

on the final product container. As described in 21 CFR 312.6(a), the label for an 

investigational product must contain the following statement: “Caution: New Drug – 

Limited by Federal law to Investigational Use.” To minimize the potential for mix-ups 

for products manufactured in multi-use facilities and for patient-specific products, we 

recommend that the product label contain the date of product manufacture, storage 

conditions, expiration date and time (if appropriate), product name, and two non-personal 

patient identifiers. As HCT/Ps, labeling for ex vivo genetically modified cell therapy 

products must meet the requirements in 21 CFR 1271.250. For 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 applicable required labeling in 21 

CFR 1271.90(b). For example, for autologous cells intended for autologous use you 

must label the product “FOR AUTOLOGOUS USE ONLY” (21 CFR 1271.90(b)(1)), 

and “NOT EVALUATED FOR INFECTIOUS SUBSTANCES” if donor testing and 

26 
Contains Nonbinding Recommendations 

screening is not performed (21 CFR 1271.90(b)(2)). For more information, refer to Ref. 

3. 

C. Container/Closure 

You should describe the types of container and closure used, and that you have 

determined that the containers and closures are compatible with the product. For more 

information, refer to “Guidance for Industry: Container Closure Systems for Packaging 

Human Drugs and Biologics; Chemistry, Manufacturing, and Controls Documentation” 

(Ref. 20) and “Guidance for Industry: Container Closure Systems for Packaging Human 

Drugs and Biologics, Questions and Answers” (Ref. 21).

D. Environmental Impact 

Under 21 CFR 312.23(a)(7)(iv)(e), you must submit either a claim for categorical 

exclusion under 21 CFR 25.30 or 25.31, or an environmental assessment under 21 CFR 

25.40. Such categorical exclusion is ordinarily granted, absent extraordinary 

circumstances indicating that the specific proposed action might significantly affect the 

quality of the human environment. Extraordinary circumstances are described in 40 CFR 

1508.27 and may include actions that create a potential for serious harm to the 

environment and actions that adversely affect a species or the critical habitat of a species 

determined to be endangered, threatened, or entitled to special protection (21 CFR 

25.21). See the “Guidance for Industry: Environmental Assessment of Human Drug and 

Biologics Applications” (Ref. 22) for additional information. 

Note to FDA Reviewers: Document in your review whether the sponsor has claimed a 

categorical exclusion or evaluate the sponsor’s assessment of any extraordinary 

circumstances using this product. 

E. Qualification of the Manufacturing Process 

The manufacturing process for gene therapy products entails the use of reagents and 

source materials of differing complexity, variability and risk for introduction of 

adventitious agents. Qualification of reagents and source materials, as well as ensuring 

that appropriate controls are in place for monitoring manufacturing consistency and 

product quality, are key elements in ensuring that subjects receive a product that is 

consistently safe, pure, and potent. We recommend that, prior to production of clinical 

lots and initiation of clinical studies, you establish and implement written procedures to 

ensure proper manufacturing oversight. This includes the responsibilities and procedures 

applicable to the quality control unit. We recommend that you establish a quality control 

(QC) plan and document that plan in writing. For example, a sound QC plan must 

provide for the following functions: 

• Responsibility for examining the various components used in the production of a 

product (e.g., containers, closures, in-process materials) to ensure that they are 

appropriate and meet defined, relevant quality standards (21 CFR 211.84(a)); 

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Contains Nonbinding Recommendations 

• Responsibility for review and approval of production procedures, testing 

procedures and acceptance criteria (21 CFR 211.22(c)); 

• Responsibility for releasing or rejecting each clinical batch based on a cumulative 

review of completed production records and other relevant information (21 CFR 

211.22(a), 21 CFR 211.165, 21 CFR 211.192); and 

• Responsibility for investigating and initiating corrective actions if unexpected 

results or errors occur during production (21 CFR 211.22(a), 21 CFR 211.192). 

You should summarize the QC plan that is in place to prevent, detect, and correct 

deficiencies that may compromise product integrity or function, or that may lead to the 

possible transmission of adventitious infectious agents. We recommend that QC 

responsibilities be performed independently from production responsibilities by 

dedicated QC personnel who are familiar with QC principles. You should conduct 

internal audits at planned intervals to evaluate effective implementation of the quality 

plan and to determine if processes and products meet established parameters. You should 

develop and document audit procedures to ensure that the planned audit schedule takes 

into account the relative risk of the various QC activities, the results of previous audits 

and corrective actions, and the need to audit the entire operation at least annually. 

Note to FDA Reviewers: Document that you have reviewed the summary of the QC 

plan. Note each individual who has authority over the QC unit and the individual’s 

assigned duties. Document the date of the most recent internal audits of the 

manufacturing operations and those of contract manufacturers, vendors, or other parties. 

You should describe the changeover procedures that are followed to ensure that no crosscontamination occurs among ex vivo genetically modified cells intended for an individual 

subject and other products stored or produced in the same facility. You should describe 

the use of PCR assays for detecting area clearance, cleaning and decontamination 

reagents and segregation of activities, and the qualification of aseptic processing steps. 

Because most ex vivo genetically modified cell therapy products are not subject to final 

sterile filtration prior to infusion, you should manufacture these products under aseptic 

conditions. A media fill is an appropriate method to qualify that the process produces a 

sterile product. Refer to the “Guidance for Industry: Sterile Drug Products Produced by 

Aseptic Processing – Current Good Manufacturing Practice” (Ref. 23) for further 

information. 

Note to FDA Reviewers: Obtain consultative reviews from the Division of 

Manufacturing and Product Quality, CBER, to assess any data submitted by the sponsor 

on facilities and environmental issues such as decontamination and cleaning validation. 

F. Biostatistics 

Note to FDA Reviewers: Obtain consultative reviews for relevant portions of the CMC 

section from the Division of Biostatistics to ensure the adequacy of proposed 

experimental designs and analytic plans. There are many significant design and analysis 

issues in the areas of assay validation, establishing specifications, evaluation of product 

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Contains Nonbinding Recommendations 

potency, and evaluation of product stability. Proper statistical design and analysis of 

such studies are essential to ensure reliable documentation of the safety, purity, and 

potency of the product. If applicable, document in your review recommendations from 

the Biostatistics consult. 

VIII. PRECLINICAL STUDIES TO BE DOCUMENTED BY FDA REVIEWERS 

A. Summary of Concept Studies 

Note to FDA Reviewers: Document information provided by the sponsor to support the 

scientific rationale underlying the proposal. Include a brief summary of preclinical data 

that was generated using in vivo animal studies and/or in vitro studies to assess the 

product’s activity, efficacy, and any safety issues observed during the study. Some issues 

specific to gene therapy that should be documented include localization or trafficking of 

vectors, and level and persistence of gene expression. 

B. Gonadal Distribution 

Note to FDA Reviewers: For gene therapy vectors used for direct in vivo administration, 

work in consultation with the pharmacology/toxicology reviewer to document data that 

demonstrate the extent to which a vector is able to disseminate out of the administration 

site and distribute to the gonads. In most cases, this information is not available at the 

beginning of the Phase 1 study but would become available in the course of product 

development. Such data are usually obtained by using a PCR assay. In cases where a 

novel vector, route of administration, indication, or vector delivery system is proposed, 

preclinical studies to assess vector dissemination may be appropriate prior to initiation of 

the Phase 1 study. Document in your review the sensitivity of this assay (amount 

vector/μg cellular DNA), including assay controls (positive, negative and spiked 

controls). We believe that the PCR assay sensitivity should be less than 50 copies of 

vector genome per μg cellular DNA. 

IX. CLINICAL STUDIES TO BE DOCUMENTED BY FDA REVIEWERS 

Note to FDA Reviewers: Provide a brief description of the following in the CMC review: 

A. Protocol Title 

Name of the protocol. 

B. Subject Population 

Define the population; e.g., size, gender, race, age, etc. 

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Contains Nonbinding Recommendations 

C. Route of Administration 

Define; e.g., oral, intravenous, intramuscular, etc. 

D. Dose 

Include the dosing regimen and whether there is a dose escalation. Document the dosing 

range and number of subjects enrolled in each dose. Describe the plans for dose 

escalation and what time interval/data evaluations occur between dose increases. 

E. Frequency 

Include the frequency of dose injections per treatment cycle and the number of proposed 

cycles. 

F. Genetic, Biochemical, and Immunological Testing 

Assess, in conjunction with the clinical reviewer, whether all genetic and/or productspecific biochemical and immunological testing of subjects is appropriate for the stage of 

clinical investigation. Evaluate the sensitivity and specificity of the test methods used to 

demonstrate biological activity (e.g., immunological assay, PCR) and document this 

information in your review. In conjunction with the clinical reviewer, verify and 

document that serum from a patient on a retroviral gene therapy protocol is analyzed for 

the presence of RCR at 3, 6, and 12 months after treatment. If all post-treatment assays 

are negative during the first year, then yearly patient samples may be archived (Ref. 13). 

G. Informed Consent 

If the informed consent document is submitted for your review, verify that the product is 

described accurately and completely. 

H. Recombinant DNA Advisory Committee (RAC) Review 

If the sponsor or the sponsor’s institution receives NIH funding for DNA recombinant 

studies or if any clinical sites used in the study receive NIH funding for Recombinant DNA 

studies, you should remind the sponsor that under the NIH Recombinant DNA Guidelines, 

NIH will not allow the protocol to be initiated until the Recombinant DNA Advisory 

Committee (RAC) review has occurred. Confirm that the sponsor has submitted the 

protocol to RAC for review. For additional information refer to the “NIH Guidelines for 

Research Involving Recombinant DNA Molecules” (Ref. 24). 

X. RECOMMENDATION TO BE DETERMINDED BY FDA REVIEWERS 

Note to FDA Reviewers: Describe any information that is missing or incomplete, and issues that 

require additional clarification. Provide an overall assessment from the CMC perspective of 

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Contains Nonbinding Recommendations 

whether the trial may proceed or should be placed on clinical hold. Document all information 

obtained from the sponsor through telephone conversations or faxes. Note this documentation in 

the Recommendation Section of the Product Review Template, throughout the review document, 

or as an attachment to the review, as appropriate. Upon completion, sign and date the review 

and then obtain concurrence from your supervisor. 

XI. COMMENTS TO SPONSOR GENERATED BY FDA REVIEWERS 

Note to FDA Reviewers: Draft letter comments on unresolved issues as discussed below. Refer 

to SOPP 8201, “Issuance of and Response to Clinical Hold Letters for Investigational New Drug 

Applications” (Ref. 25), for additional information. After you have obtained supervisory 

concurrence on your review, forward your comments to the RPM for inclusion in a letter to the 

sponsor. 

A. Clinical Hold 

Clinical hold comments are comments that the sponsor must satisfactorily address prior 

to allowing clinical studies to proceed after FDA has imposed a clinical hold. These 

comments must meet the criteria listed in 21 CFR 312.42(b). 

B. Non-Clinical Hold 

Non-clinical hold comments are comments that the sponsor should address as product 

development progresses. In some cases, a sponsor may need to address specific 

manufacturing issues by a certain point in clinical development, such as prior to initiation 

of Phase 3 studies. 

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Contains Nonbinding Recommendations

XII. REFERENCES 

1. Guidance for Industry: Content and Format of Investigational New Drug Applications 

(INDs) for Phase 1 Studies of Drugs, Including Well-Characterized, Therapeutic, 

Biotechnology-Derived Products, November 1995, 

http://www.fda.gov/cder/guidance/phase1.pdf. 

2. Final Rule: Eligibility Determination for Donors of Human Cells, Tissues, and Cellular 

and Tissue-Based Products, (69 FR 29786, May 25, 2004), 

http://www.fda.gov/cber/rules/suitdonor.pdf. 

3. Guidance for Industry: Eligibility Determination for Donors of Human Cells, Tissues, and 

Cellular and Tissue-Based Products (HCT/Ps), August 2007, 

http://www.fda.gov/cber/gdlns/tissdonor.pdf. 

4. Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals, July 

12, 1993, http://www.fda.gov/cber/gdlns/ptccell.pdf. 

5. ICH Guidance on Quality of Biotechnological/Biological Products: Derivation and 

Characterization of Cell Substrates Used for Production of Biotechnological/Biological 

Products, (63 FR 50244, September 21, 1998), http://www.fda.gov/cber/gdlns/qualbiot.pdf.

6. Guidance for Industry: Source Animal, Product, Preclinical and Clinical Issues 

Concerning the Use of Xenotransplantation Products in Humans, April 2003, 

http://www.fda.gov/cber/gdlns/clinxeno.htm. 

7. PHS Guideline on Infectious Disease Issues in Xenotransplantation, January 19, 2001, 

http://www.fda.gov/cber/gdlns/xenophs0101.htm. 

8. Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for 

Human Use, February 28, 1997, http://www.fda.gov/cber/gdlns/ptc_mab.pdf. 

9. Manual of Standard Operating Procedures and Policies: Intercenter 

Consultative/Collaborative Review Process, Version 4, dated June 18, 2004, 

http://www.fda.gov/oc/ombudsman/intercentersop.pdf. 

10. FDA Guidance Concerning Demonstration of Comparability of Human Biological Product, 

Including Therapeutic Biotechnology-derived Products, April 1996, 

http://www.fda.gov/cber/gdlns/comptest.pdf. 

11. United States Pharmacopoeia (USP), Chapter <71> Sterility Tests, 23rd edition, 1995, 

http://www.usp.org. 

12. ICH Guidance on Viral Safety Evaluation of Biotechnology Products Derived From Cell 

Lines of Human or Animal Origin, (63 FR 51074, September 24, 1998), 

http://www.fda.gov/cber/gdlns/virsafe.pdf. 

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Contains Nonbinding Recommendations 

13. Guidance for Industry: Supplemental Guidance on Testing for Replication Competent 

Retrovirus in Retroviral Vector Based Gene Therapy Products and During Follow-up of 

Patients in Clinical Trials Using Retroviral Vectors, dated October 2006,

http://www.fda.gov/cber/gdlns/retrogt1000.htm. 

14. ICH Topic Q3. Impurities. 

 Guidance for Industry: Q3A Impurities in New Drug Substances, February 2003, 

http://www.fda.gov/cber/gdlns/ichq3a.pdf. 

 Guidance for Industry: Q3B(R2) Impurities in New Drug Products, July 2006, 

http://www.fda.gov/cber/gdlns/ichq3br.pdf. 

 Guidance for Industry: Q3C Impurities: Residual Solvents, December 1997, 

http://www.fda.gov/cber/gdlns/q3cresolvent.pdf. 

15. Guideline on Validation of the Limulus Amebocyte Lysate test as an End-Product 

Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products and Medical 

Devices, 1987, http://www.fda.gov/cber/gdlns/lal.pdf. 

16. Revisions to the General Safety Requirements for Biological Products, 68 FR 10157, 

March 4, 2003. 

17. Guideline for Industry: Quality of Biotechnological Products: Stability Testing of 

Biotechnological/Biological Products, July 1996 (ICH Q5C), 

http://www.fda.gov/cder/guidance/ichq5c.pdf. 

18. Guidance for Industry: Q1A(R2): Stability Testing of New Drug Substances and Products 

(Revision 2), November 2003, http://www.fda.gov/cber/gdlns/ichstab.pdf. 

19. Guidance for Industry: Q1E: Evaluation of Stability Data, June, 2004, 

http://www.fda.gov/cber/gdlns/ichstabdta.pdf. 

20. Guidance for Industry: Container Closure Systems for Packaging Human Drugs and 

Biologics; Chemistry, Manufacturing, and Controls Documentation, May 1999,

http://www.fda.gov/cber/gdlns/cntanr.pdf. 

21. Guidance for Industry: Container Closure Systems for Packaging Human Drugs and 

Biologics, Questions and Answers, May 2002, 

http://www.fda.gov/cber/gdlns/cntanrq&a.pdf. 

22. Guidance for Industry: Environmental assessment of Human Drug and Biologics 

Applications, Revision 1, July 1998, http://www.fda.gov/cber/gdlns/environ.pdf.

23. Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current 

Good Manufacturing Practice, September 2004, 

http://www.fda.gov/cber/gdlns/steraseptic.htm. 

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Contains Nonbinding Recommendations 

24. NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines), 

April 2002, http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html. 

25. Manual of Standard Operating Procedures and Policies Investigational New Drug; Issuance 

of and Response to Clinical Hold Letters for Investigational New Drug Applications, SOPP 

8201, Version #3, April 27, 1999, http://www.fda.gov/cber/regsopp/8201.htm. 

34 
Contains Nonbinding Recommendations 

APPENDIX A – PRODUCT REVIEW TEMPLATE (GENE THERAPY) 

PRODUCT REVIEW (GENE CELL) 

 Supervisor Concurrence/Date 

 IND: XXXX Sponsor’s Submission Date: Month DD, YYYY 

30 Day Review Due Date: Month DD, YYYY 

STATUS: Pending 

 DATE: Month DD, YYYY 

 REVIEWER: Your Name 

 Your Title, OCTGT/DCGT/Your Branch 

 THROUGH: Branch Chief Name 

 Branch Chief, OCTGT/DCGT/Branch 

 SPONSOR: Name: 

 Address: 

 Title: 

 Phone: 

 Fax: 

 SPONSOR POINT OF CONTACT: 

Name: 

 Address: 

 Title: 

 Phone: 

 Fax: 

 TITLE OF IND: 

 PROPOSED USE: 

 REVIEW TEAM: Clinical: 

 Pharm-Tox: 

 RPM: 

 Consults: 

 PRODUCT DESCRIPTION: 

 PHASE OF STUDY: 

 CROSS-REFERENCED INDS, IDES, MFS: 

 KEY WORDS: 

INTRODUCTION / RATIONALE: 

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Contains Nonbinding Recommendations 

STUDY OBJECTIVES: 

PRODUCT MANUFACTURING AND CHARACTERIZATION: 

Product Manufacturing - Components 

Cells 

Allogeneic or Autologous Cell Components 

Cell Source: 

Method of Collection: 

 Donor Screening: 

 Description 

 Tabulation of Testing 

Cell Bank System - If Applicable 

 Master Cell Bank (MCB) 

 Description 

 Tabulation of Testing 

 Working Cell Bank (WCB) 

 Description 

 Tabulation of Testing 

Reagents 

Tabulation of Reagents Used in Manufacture 

Reagent/Excipient Concentration at use Source Grade Vendor COA 

 Qualification Program 

Determination of Removal of Reagents from Final Product 

Combination Products - If Applicable 

 Drug or Device Components - If Applicable 

36 
Contains Nonbinding Recommendations 

Consult Review Issues: 

Areas of Concern for Components: 

Product Manufacturing - Procedures 

Preparation of Autologous or Allogeneic Cells 

Method of Cell Collection/Processing/Culture Conditions 

Irradiation - If Applicable 

Process Timing & Intermediate Storage 

Final Harvest 

 Timing/Methods/Wash Procedure 

Final Formulation 

 Formulation/Infusion Buffer 

 Excipients 

 Cell Density/Concentration in the Final Product 

 Storage Method Prior to Use 

Areas of Concern for Manufacturing 

PRODUCT TESTING 

In-Process Testing and Criteria 

 

Tabulation of Tests, Manufacturing Step, Test Methods, Criteria, and Test Sensitivity & 

Specificity 

37 
Contains Nonbinding Recommendations 

Test Manufacturing 

Step Where 

Performed 

Method Criteria Sensitivity Specificity 

Sterility 

Mycoplasma 

Purity 

(endotoxin) 

Purity (other 

contaminants) 

Identity 

Potency 

Others (cell 

dose) 

Others (cell 

viability) 

Description of Test Methods 

FINAL PRODUCT RELEASE CRITERIA/SPECIFICATIONS 

Tabulation of Final Product Release Criteria Tests, Test Methods, Criteria, Test Sensitivity & 

Specificity 

 

Test Method Criteria Sensitivity Specificity Results Available 

Prior to Release 

Description of Test Methods 

PRODUCT STABILITY 

 In-Process Stability Testing 

 Cryopreserved Cells 

 Other Intermediate Holding Steps 

 Final Product Stability Testing 

Product Formulation to Patient Infusion 

Shipping Conditions 

OTHER ISSUES 

Product Tracking 

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Contains Nonbinding Recommendations 

Labeling and Containers 

In-Process Labeling 

Final Product Labeling 

 

Container Closure & Integrity 

Environmental Impact 

Validation and Qualification of the Manufacturing Process 

 QA/QC Program 

 

 Manufacturing Process Validation 

Biostatistics 

 

PRECLINICAL STUDIES 

CLINICAL STUDIES 

Protocol Title 

Subject Population 

Route of Administration 

Dose 

Frequency 

Genetic, Biochemical, and Immunological Testing 

Informed Consent 

RECOMMENDATION 

COMMENTS TO SPONSOR 

Clinical Hold 

Non-Clinical Hold 

 Signature Date:____________________ 

Reviewer Name 

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Contains Nonbinding Recommendations 

APPENDIX B – CONSIDERATIONS FOR DEVELOPMENT OF FINAL PRODUCT 

RELEASE CRITERIA SPECIFICATIONS AND STABILITY PROTOCOLS 

Specifications are the quality standards (i.e., tests, analytical procedures, and acceptance criteria) 

that confirm the quality of products and other materials used in the production of a product. 

Acceptance criteria are the numerical limits, ranges, or other criteria for the tests described. For 

additional information, see ICH Guideline Q6B: “Test Procedures and Acceptance Criteria for 

Biotechnological/Biological Products”.4 We believe that certain release specifications, such as 

those related to product safety, should be in place prior to initiating Phase 1 clinical studies. As 

product development proceeds, additional specifications for product quality and manufacturing 

consistency are developed and implemented. For additional discussion of manufacturing quality 

control, see “Guidance for Industry: Guideline on the Preparation of Investigational New Drug 

Products”5 and “Guidance for Industry: IND Meetings for Human Drugs and Biologics; 

Chemistry, Manufacturing and Controls Information”.6 

A. Development of Release Acceptance Criteria 

We recommend that proposed release acceptance criteria for the final product be based 

on scientific data and manufacturing experience obtained during development of the 

product as described below: 

• Phase 1 – Based on data from lots used in preclinical studies. 

• Phase 2 – Refine and tighten based on data generated during Phase 1. 

• Phase 3 – Based on information collected during product development. 

• Licensure – Based on information collected during product development 

using validated assays.

B. Development of Acceptance Criteria Analytical Procedures 

We recommend that proposed analytical procedures be based on scientific data and 

manufacturing experience as described below: 

• Phase 1-3 – Usually based on Code of Federal Regulation (CFR) methods 

or alternative methods, if appropriate. 

• Phase 2 – If an alternative to the CFR method is used, we recommend that 

the sponsor initiate validation of the alternative by Phase 3. 

• Licensure – The product specification should be in place and established 

under a validated assay. 

 

4

 Guidance for Industry: Q6B Specifications: Test Procedures and Acceptance Criteria for 

Biotechnological/Biological Products, August 1999, http://www.fda.gov/cder/guidance/Q6Bfnl.pdf. 5

 Guidance for Industry: Guideline on the Preparation of Investigational New Drug Products (Human and Animal), 

dated March 1991, reprinted November 1992, http://www.fda.gov/cder/guidance/old042fn.pdf. 6

 Guidance for Industry: IND Meetings for Human Drugs and Biologics; Chemistry, Manufacturing and Controls 

Information, May 2001, http://www.fda.gov/cber/gdlns/ind052501.htm 

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Contains Nonbinding Recommendations 

C. Development of Stability Protocols 

In order to develop adequate stability data for timely submission in a license application, 

we recommend that a sponsor implement and expand the stability program as described 

below: 

• Phase 1 - 3 – Preliminary data on product stability must indicate whether 

the product or components are likely to remain stable for the duration of 

the clinical trial. Note: the regulations require that the IND contain these 

data at each stage of the clinical trial (21 CFR 312.23(a)(7)(ii)). 

• Phase 2 – We recommend that the sponsor initiate a stability protocol to 

accumulate additional data. 

• Phase 3 – We recommend that the sponsor begin to establish the dating 

period, storage conditions, and shipping conditions based on data derived 

from the stability protocol.

41