Guidance for Industry
 
Guidance for Human Somatic Cell 

Therapy and Gene Therapy
 
Comments and suggestions regarding this document may be submitted at anytime to Dano B. 
Murphy, HFM-17, Center for Biologics Evaluation and Research, Food and Drug Administration, 
1401 Rockville Pike, Rockville, MD 20852-1448.  For questions regarding this document, 
contact Suzanne L. Epstein, Ph.D., 301-827-0450, FAX 301-827-0449, e-mail 
Epsteins@CBER.FDA.GOV. 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Biologics Evaluation and Research 
March 1998  TABLE OF CONTENTS 

Note: page numbering may vary for documents distributed electronically. 
OVERVIEW (1998) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
 
I.	  INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
 
A.  Definitions of Somatic Cell Therapy and Gene Therapy  . . . . . . . . . . . . . . . . . . . . . . 3
 
B.  Types of Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
 
C.  Regulatory Considerations  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
 
D.  General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
 
II. 	 DEVELOPMENT AND CHARACTERIZATION OF CELL POPULATIONS FOR 

ADMINISTRATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
 
A.  Collection of Cells  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
 
B.  Cell Culture Procedures  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
 
C.  Cell Banking Systems Procedures: Generation and Characterization of Master Cell 

Banks (MCB), Working Cell Banks (WCB), and Producer Cells  . . . . . . . . . . . . . . . 9
 
D.  Materials Used During Manufacturing  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
 
III. 	 CHARACTERIZATION AND RELEASE TESTING OF CELLULAR GENE 

THERAPY PRODUCTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
 
A.  Cell Identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
 
B.  Potency  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
 
C.  Viability  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
 
D.  Sterility  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
 
E.  Purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
 
F.  General Safety Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
 
G.  Frozen Cell Banks  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
 
IV. 	 ADDITIONAL APPLICATIONS: ADDITION OF RADIOISOTOPES OR TOXINS 

TO CELL PREPARATIONS  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
 
V.	  PRODUCTION, CHARACTERIZATION AND RELEASE TESTING OF 

VECTORS FOR GENE THERAPY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
 
A.  Vector Construction and Characterization  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
 
B.  Vector Production System  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
 
C.  Master Viral Banks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
 
D.  Lot-to-Lot Release Testing and Specifications for Vectors . . . . . . . . . . . . . . . . . . . 15
 
i  VI. 	 ISSUES RELATED TO PARTICULAR CLASSES OF VECTORS FOR GENE 

THERAPY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  18
 
A.  Additional Considerations for the Use of Plasmid Vector Products . . . . . . . . . . . . .  18
 
B.  Additional Considerations for the Use of Retroviral Vector Products  . . . . . . . . . . .  18
 
C.  Additional Considerations for the Use of Adenoviral Vectors. . . . . . . . . . . . . . . . . .  20
 
D.  Other Gene Delivery Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  22
 
VII. 	 MODIFICATIONS IN VECTOR PREPARATIONS  . . . . . . . . . . . . . . . . . . . . . . .  22
 
VIII. 	 PRECLINICAL EVALUATION OF CELLULAR AND GENE THERAPIES  . . .  23
 
A.  General Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  23
 
B.  Animal Species Selection and Use of Alternative Animal Models  . . . . . . . . . . . . . .  24
 
C.  Somatic Cell and Gene-Modified Cellular Therapies . . . . . . . . . . . . . . . . . . . . . . . .  24
 
D.  Direct Administration of Vectors In Vivo  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  25
 
IX. 	 CONCLUSION  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  27
 
ii  GUIDANCE FOR INDUSTRY:1
 
FDA GUIDANCE FOR HUMAN SOMATIC CELL THERAPY
 
AND GENE THERAPY
 
OVERVIEW (1998) 
Since the issuance of the "Points to Consider (PTC) in Human Somatic Cell Therapy and Gene 
Therapy"in 1991, the range of gene therapy proposals has expanded to include additional classes 
of vectors and use of vectors in vivo via direct vector administration to patients.  This guidance 
document updates and replaces the 1991 PTC with new information intended to provide 
manufacturers with current information regarding regulatory concerns for production, quality 
control testing, and administration of recombinant vectors for gene therapy; and of preclinical 
testing of both cellular therapies and vectors.  These guidances are not regulations, but rather 
represent issues that the Center for Biologics Evaluation and Research (CBER) staff believes 
should be considered at this time. 
Virus or DNA preparations used as preventive vaccines are not covered by this document, though 
there is some overlap in the issues.  Separate guidance on use of plasmid products to prevent 
infectious diseases is available from the Office of Vaccines Research and Review, CBER (301) 
594-2090.  One pertinent document is the "Points to Consider on Plasmid DNA Vaccines for 
Preventive Infectious Disease Indications (December, 1996)", (61 FR 68269).  Somatic cell 
therapies are affected by the evolving criteria for infectious disease testing.  For additional 
guidance refer to the following documents: 
1  This guidance document represents the Agencys current thinking on the development and regulation of 
somatic cell therapy and gene therapy products. It does not create or confer any rights for or on any person and does not 
operate to bind FDA or the public. An alternative approach may be used if such approach satisfies the requirements of 
applicable statutes, regulations, or both. For additional copies of this guidance, contact the Office of Communication, 
Training and Manufacturers Assistance, HFM-40, Center for Biologics Evaluation and Research, Food and Drug 
Administration, 1401 Rockville Pike, Rockville, MD 20852-1448. Send one self-addressed adhesive label to assist that 
office in processing your requests. The document may also be obtained by mail by calling the CBER Voice Information 
System at 1-800-835-4709 or 301-827-1800, or by calling the FAX Information System at 1-888-CBER-FAX or 301
827-3844. Persons with access to the Internet may obtain the document at http//www.fda.gov/cber/guidelines.htm. 
1
  Points to Consider in the Manufacture and Testing of Therapeutic Products for Human Use 
Derived from Transgenic Animals, (8/22/95), (60 FR 44036); 
PHS Guidelines on Infectious Disease Issues in Xenotransplantation, August, 1996, (61 FR 
49920) and January, 1997, (62 FR 3563); 
Guidance on Applications for Products Comprised of Living Autologous Cells Manipulated ex 
vivo and Intended for Structural Repair or Reconstruction (May, 1996), May 28, 1996, (61 FR 
26523). 
A Proposed Approach to the Regulation of Cellular and Tissue-based Products, February 28, 
1997, (62 FR 9721). 
2
  I.   INTRODUCTION 
A.  Definitions of Somatic Cell Therapy and Gene Therapy 
Recently, various innovative therapies involving the ex vivo manipulation and subsequent 
reintroduction of somatic cells into humans have been used or proposed.  Somatic cell 
therapy is the administration to humans of autologous, allogeneic, or xenogeneic living 
cells which have been manipulated or processed ex vivo.  Manufacture of products for 
somatic cell therapy involves the ex vivo propagation, expansion, selection (see: A 
Proposed Approach to the Regulation of Cellular and Tissue-based Products, Feb. 28, 
1997, (62 FR 9721)), or pharmacologic treatment of cells, or other alteration of their 
biological characteristics.  Such cellular products might also be used for diagnostic or 
preventive purposes.  Manufacturers should review policy and regulations to determine 
how a particular somatic cell therapy or gene therapy product is regulated. 
Recently, various innovative therapies involving the introduction of somatic cells into 
humans have been used or proposed.  For the purpose of this Guidance, the term somatic 
cell therapy refers to the administration to humans of autologous, allogeneic, or 
xenogeneic living non-germline cells, other than transfusable blood products, for 
therapeutic, diagnostic, or preventive purposes. 
Gene therapy is a medical intervention based on modification of the genetic material of 
living cells.  Cells may be modified ex vivo for subsequent administration to humans, or 
may be altered in vivo by gene therapy given directly to the subject.  When the genetic 
manipulation is performed ex vivo on cells which are then administered to the patient, this 
is also a form of somatic cell therapy.  The genetic manipulation may be intended to have a 
therapeutic or prophylactic effect, or may provide a way of marking cells for later 
identification.  Recombinant DNA materials used to transfer genetic material for such 
therapy are considered components of gene therapy and as such are subject to regulatory 
oversight. 
This document does not discuss genetic manipulation aimed at the modification of germ 
cells. 
B.  Types of Therapies 
Examples of somatic cell therapies include implantation of cells as an in vivo source of a 
molecular species such as an enzyme, cytokine or coagulation factor; infusion of activated 
lymphoid cells such as lymphokine activated killer cells and tumor-infiltrating lymphocytes 
(addressed in a separate Points to Consider document: see below); and implantation of 
manipulated cell populations, such as hepatocytes, myoblasts, or pancreatic islet cells, 
intended to perform a complex biological function. 
3
  Initial approaches to gene therapy have involved the alteration and administration of 
somatic cells.  However, additional approaches such as the direct administration to 
patients of retroviral vectors or other forms of genetic material have been used.  The 
concerns described below apply regardless of the method used, though the applicable tests 
may be different. 
Cells for therapeutic purposes may be delivered in various ways. For example, they may be 
infused, injected at various sites or surgically implanted in aggregated form or along with 
solid supports or encapsulating materials.  Any matrices, fibers, beads, or other materials 
which are used in addition to the cells may be categorized as excipients, additional active 
components, or medical devices. 
Because of the complexities of potential interactions with the cells and other constituents, 
additional components should be considered as part of the final biological product for 
purposes of preclinical evaluation. 
C.  Regulatory Considerations 
All gene therapy products and most somatic cell therapy products are regulated by the 
FDA.  See A Proposed Approach to the Regulation of Cellular and Tissue-Based 
Products, February 28, 1997, (62 FR 9721) as well as subsequent regulations and policy 
issued in this area. 
IND applications for somatic cell and gene therapies should follow the same format and 
contain the same sections as IND's for any investigational biological product, as described 
in 21 CFR 312.23.  Forms and guidance documents are available from CBER by phone, 
FAX or E-mail as listed at the end of this document.  The particular information required 
will depend upon the experimental system and the phase of study.  For those therapies for 
which patient entry criteria include results of a genetic test, information should be 
submitted to the IND documenting and validating the test method. 
Biological products are often complex mixtures that cannot be completely defined. 
Quality control of the manufacturing process as well as the final product is necessary. 
Poor control of production processes can lead to the introduction of adventitious agents 
or other contaminants, or to inadvertent changes in the properties or stability of the 
biological product that may not be detectable in final product testing.  For these reasons, 
the methods and reagents involved in the production process should be defined.  Also, cell 
banks and key intermediates in the production process should be subject to quality control. 
Lot-to-lot reproducibility of both the final product and of critical materials such as vector-
containing supernatants should be examined.  Existing general regulations (21 CFR 210, 
211, 312, and 600) may be relevant and should be consulted for guidance. 
4
  Exploratory phase I trials for somatic cell and gene therapy products should be based on 
data that assure reasonable safety and rationale.  Less data may be submitted to support 
beginning exploratory trials than may be submitted at later stages of product development, 
especially in the case of severe or life-threatening diseases.  The review of data to support 
initiation of phase I trials focuses on safety, although some demonstration of rationale 
should also be provided. 
Data from further product testing should be available at later stages of product 
development.  A quantitative potency assay reflective of bioactivity in vivo should be 
developed and product stability should be studied to assure product integrity.  In addition 
to safety, evidence of clinical efficacy is required for licensure. 
If product formulation is changed during product development, a comparison of the 
different formulations should be made by quantitative assays of biological potency and, 
when appropriate, preclinical safety evaluation.  If the product used in later phase trials 
differs in major ways from that used during earlier trials and if the results of the earlier 
trials are essential to the final product evaluation, product comparability should be 
demonstrated or the sponsor should assess whether earlier trials may need to be repeated 
(see: "FDA Guidance Concerning Demonstration of Comparability of Human Biological 
Products, Including Therapeutic Biotechnology-derived Products", 4/26/96, (61 FR 
10426)). 
For vectors intended to be used in a number of different IND's, manufacturing information 
can be submitted in a master file to simplify the filing process.  Neither a master file nor 
the product it describes is "approved" or "disapproved".  Rather the master file contains 
information and data that supplement the IND.  Use of the same product for different 
patient populations may raise different issues or may indicate different levels of acceptable 
risk, but use of the master file can help identify common issues and facilitate their efficient 
resolution.  Multiple IND sponsors can be authorized to cross-reference a master file, thus 
reducing redundant submissions as well as retaining desired confidentiality. 
D.  General Considerations 
Some of the issues regarding cellular and gene therapy products overlap with those 
discussed in other Points to Consider documents. It is suggested that the most recent 
versions of Points to Consider or other Guidance documents be reviewed.  Other 
Guidance documents should be consulted as is appropriate, for example: 
Application of Current Statutory Authorities to Human Somatic Cell Therapy Products 
and Gene Therapy Products, October 14, 1993, (58 FR 53248). 
5
  Points to Consider in the Production and Testing of New Drugs and Biologicals Produced 
by Recombinant DNA Technology (1985), and Supplement: Nucleic Acid 
Characterization and Genetic Stability (1992), July 27, 1992, (57 FR 33201). 
Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals 
(1993),  August 12, 1993, (58 FR 42974). 
Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for 
Human Use (1997), February 28, 1997, (62 FR 9196). 
Points to Consider in the Collection, Processing, and Testing of Ex-Vivo-Activated 
Mononuclear Leukocytes for Administration to Humans (1989), November 2, 1989, (54 
FR 46303). 
The following sections indicate areas of concern and questions to be addressed by 
manufacturers of such products when filing applications.  Initial clinical trials should be 
preceded by submission of data adequate to assure a reasonable degree of safety. A 
description of the methods used, actual data from appropriate tests, and evidence of assay 
validation should be included. 
It may not be practical or possible to address all of the issues discussed below for a given 
system.  In some instances, tests mentioned will be inapplicable or inappropriate, or 
alternative procedures may be more appropriate.  The methods and procedures mentioned 
are suggestions:  sponsors may propose alternative techniques which will be acceptable if 
these issues are adequately addressed and supported by data and rationale.  In addition, all 
of the information discussed below may not be necessary before clinical trials are initiated. 
Sponsors are encouraged to consult with CBER staff for discussion. 
The guidance suggested in this document is based in part on an assessment of the 
available experience with cell and gene therapy products and methods of production. 
Modifications of procedures will occur with time, and alternate control procedures will be 
needed.  The principles included here can serve as guidance for developing these 
procedures. 
6
  II. 	 DEVELOPMENT AND CHARACTERIZATION OF CELL POPULATIONS FOR 
ADMINISTRATION 
A.  Collection of Cells 
The following information should be provided: 
1.  Cell types:  The type(s) of cell to be used should be classified as autologous, 
allogeneic, or xenogeneic in origin.  The tissue source and other relevant 
identifying information should be provided. 
2.  Donor selection criteria:  Any relevant characteristics of the donor(s) should be 
specified, including age and sex.  As stated in the "Points to Consider in the 
Collection, Processing, and Testing of Ex-Vivo-Activated Mononuclear 
Leukocytes for Administration to Humans," as a minimum, allogeneic donors 
should meet the standards for blood donors (21 CFR 640.3), the testing and 
acceptance procedures should be described, and any deviations should be justified. 
Where applicable, additional Public Health Service recommendations regarding 
organ and tissue donors should be incorporated.  Exclusion criteria should focus 
on the presence or likelihood of infection by HIV-1 and HIV-2, hepatitis B and C 
viruses, HTLV-1, and other infectious agents.  Serological, diagnostic, and clinical 
history data to be obtained from donors should be specified.  Provision for follow-
up of donors will be appropriate in some cases and methods of obtaining donor 
data and record keeping should be thoroughly described. 
If autologous cells are used, please refer to A Proposed Approach to the 
Regulation of Cellular and Tissue-based Products, February 28, 1997, (62 
FR 9721) for additional guidance on adventitious agent testing and 
labeling.  If animal species other than humans are used, a description 
should be provided of the origin, relevant genetic traits, husbandry, and 
health status of the herd or colony (see also "PHS Guidelines on Infectious 
Disease Issues in Xenotransplantation", August, 1996, 61 FR 49920 and 
January, 1997, (62 FR 3563). 
3.  Tissue typing:  If allogeneic donors are to be used, typing for polymorphisms 
such as blood type should be included when appropriate.  The importance of 
matching for histocompatibility antigens (HLA class I and/or II, and perhaps minor 
antigens in some cases) between donor and recipient should be addressed, and 
typing procedures and acceptance criteria provided. 
Should it be indicated or necessary to use mixtures of cells from multiple donors, 
special attention should be paid to possible cell interactions that could result in 
7
  immune responses or other changes that might alter the performance of the cells. 
Characterization of multiple-donor cell mixtures may be problematic.  Multiple-donor cell mixture 
products would not meet the criteria set forth in the Proposed Approach to the Regulation of 
Cellular and Tissue-based Products, February 28, 1997, (62 FR 9721) for regulation as human 
cellular or tissue-based products under section 361 of the Public Health Service Act (the PHS 
Act).  Such products  would be subject to regulation under the Federal Food, Drug, and Cosmetic 
Act and section 351 of the PHS Act. 
4.  Procedures:  The procedures for the collection of cells, including the location of 
the facility, and any devices or materials used, should be submitted. 
B.  Cell Culture Procedures 
1.  Quality control procedures:  In general, cell culture operations should be 
carefully managed in terms of quality of materials, manufacturing controls, and 
equipment validation and monitoring.  See I, C, General Considerations. 
2.  Culture media:  Acceptance criteria should be established for all media and 
components, including validation of serum additives and growth factors, as well as 
verification of freedom from adventitious agents.  Records should be kept detailing 
the components used in the culture media, including their sources and lot numbers. 
Medium components which have the potential to cause sensitization, for example 
certain animal sera, selected proteins, and blood group substances, should be 
avoided.  For growth factors, measures of identity, purity, and potency should be 
established to assure the reproducibility of cell culture characteristics.  More 
detailed discussions of specifications for medium components and biologicals 
added to cultures are presented in the "Points to Consider in the Characterization 
of Cell Lines Used to Produce Biologicals (1993)" and the "Points to Consider in 
the Collection, Processing, and Testing of Ex-Vivo-activated Mononuclear 
Leukocytes for Administration to Humans (1989)." 
As stated in the "Points to Consider in the Characterization of Cell Lines Used to 
Produce Biologicals," it is recommended that penicillin and other beta-lactam 
antibiotics be avoided during production, due to the risk of serious hypersensitivity 
reactions in patients. 
3.  Adventitious agents in cell cultures:  Documentation should be provided that 
cells are handled, propagated, and subjected to laboratory procedures under 
conditions designed to minimize contamination with adventitious agents.  During 
long term culturing, cells should be tested periodically for contamination.  Testing 
should ensure that cells are free of bacteria, yeast, mold, mycoplasma, and 
adventitious viruses.  For a discussion of adventitious agent testing and details 
8
  regarding virus testing and mycoplasma testing, the "Points to Consider in the 
Characterization of Cell Lines Used to Produce Biologicals" (1993), should be 
consulted. 
4.  Monitoring of cell identity and heterogeneity:  Both manufacturing and testing 
procedures should be implemented which ensure the control of cell  cultures with 
regard to identity and heterogeneity. 
Cell culturing practices and facilities should be designed to avoid contamination of 
one cell culture with another. 
During cell culturing, extensive drift in the properties of a cell population, or 
overgrowth by a different cell type originally present in low numbers, may occur. 
To detect such changes, cell identity should be assessed quantitatively, for 
example, by monitoring cell surface antigens or biochemical  markers.  The method 
of identification chosen should also be able to detect contamination or replacement 
by other cells in use in the facility.  Acceptable limits for culture composition 
should be defined.  Quantitative assays of functional potency may sometimes 
provide a method for population phenotyping.  The desired function should be 
monitored when the cells are subjected to manipulation, and the tests carried out 
periodically to assure that the desired trait is retained.  Identity testing should in 
some cases include verification of donor-recipient matching and immunological 
phenotyping. 
5.  Characterization of therapeutic entity:  If the intended therapeutic effect is 
based  on a particular molecular species synthesized by the cells, enough structural 
and biological information should be provided to show that an appropriate and 
biologically active form is present. 
6.  Culture longevity:  The essential characteristics of the cultured cell population 
(phenotypic markers such as cell surface antigens, functional properties, activity in 
bioassays, as appropriate) should be defined, and the stability of these 
characteristics established with respect to time in culture.  This profile should be 
used to define the limits of the culture period. 
C.  Cell Banking System Procedures: 	 Generation and Characterization of Master Cell 
Banks (MCB), Working Cell Banks (WCB), and Producer Cells 
Cell banking systems are appropriate for use with some somatic cell therapy products that 
are made repeatedly from the same cell source, and with packaging or producer cells used 
to make gene therapy vectors, for example, bacterial cells producing a plasmid or 
mammalian cells producing a recombinant viral vector.  These cell stocks should be 
handled by a formal cell banking system (often a two-tiered system).  Specific guidance 
9
  for the establishment of Master Cell Banks and Working Cell Banks is provided in the 
"Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals 
(1993)", 58 FR 42974.  In addition, 21 CFR 610.18 may be applicable.  The cell bank 
system used should be described as follows: 
1.  Origin and history of cells:  A description should be provided. 
2.  Procedures:  The procedure for freezing and for recovering the cells should be 
described.  Components used (such as DMSO or glycerol) should be specified. 
The number of vials preserved in a single lot and the storage conditions should be 
specified. 
3.  Characterization:  The identity of the cells should be confirmed by appropriate 
genotypic and/or phenotypic markers, and the fraction of the cell population 
having such identity markers measured as an indication of purity.  In the case of 
transduced or vector-producing cells, vector retention and identity should be 
confirmed by restriction mapping or assay of the bioactivity of protein expressed 
by an inserted gene. 
4.  Testing for contaminating organisms:  MCB's should be shown to be free of 
contaminating biological agents, including fungi, viruses other than a vector, 
mycoplasma, bacteria other than an intended bacterial host strain, and replication-
competent viruses related to vector in certain cases (see section VI, B and C). 
In the case of MCB's consisting of bacteria carrying plasmids of interest, testing 
for bacteriophage is not required but the possible presence of bacteriophage should 
be considered, since it could adversely affect stability and yield. 
5.  Expiration dating:  Product development plans should include accumulation of 
data demonstrating how long and under what conditions the cells can remain 
frozen and still be acceptably active when thawed. 
6.  Tests on thawed cells:  Tests of viability, cell identity, and function should be 
repeated after thawing and/or expansion.  The yield of viable cells and of 
quantitative functional equivalents should be compared to those values before 
freezing.  Sterility should be confirmed using aliquots of the frozen cells. 
Working Cell Banks, if used, should undergo limited testing for identity by phenotypic or 
genotypic markers.  Vector retention and identity should be confirmed as in MCB's by 
restriction mapping or assay of secreted protein activity.  They should also be shown to be 
free of microbial and viral contamination. 
10
  For producer cells, extended culture of end-of-production cells should be performed on a 
one-time-basis to evaluate whether new contaminants are induced by growth conditions or 
if vector integrity is compromised (See "Points to Consider in the Manufacture and 
Testing of Monoclonal Antibody Products for Human Use," 1997 revision, 62 FR 9196). 
Sponsors should propose a schedule of testing at steps which will be most informative and 
sensitive. 
It may not be feasible to use cell banking practices with cell therapies made differently for 
each patient, for example autologous cells for treatment of individual patients.  However, 
consideration should be given to testing of the final cellular product for crucial 
characteristics. 
D.  Materials Used During Manufacturing 
Materials used during in vitro manipulation procedures, for example antibodies, cytokines, 
serum, protein A, toxins, antibiotics, other chemicals, or solid supports such as beads can 
effect the safety, purity, and potency of the final therapeutic product. These components 
should be clearly identified and a qualification program with set specifications should be 
established for each component to determine its acceptability for use during the 
manufacturing process.  When using reagent grade material, the qualification program 
should include testing for safety, purity, and potency of the component where appropriate. 
Abbreviated testing may be appropriate for use of clinical grade components.  Materials of 
animal origin will in some cases need to be tested for adventitious agents.  The country of 
origin should be certified when there is risk of transmissible agents causing spongiform 
encephalopathy 
Limits should be established for the concentrations of all production components that may 
persist in the final product.  The methods used to remove them and the results of 
quantitative testing (including a description of methods and sensitivity) to show the 
effectiveness of their removal should be provided.  Some added components, by virtue of 
binding or uptake, may be present in measurable amounts when the cells are administered. 
In such cases, consideration should be given to assessing toxicity of these components in 
animals or other appropriate systems. 
11
  III. 	 CHARACTERIZATION AND RELEASE TESTING OF CELLULAR GENE 
THERAPY PRODUCTS 
Applies to cellular products including ex vivo transduced cells for gene therapy. 
The final biological product to be administered, as well as the production process and materials 
used, should be subjected to quality control testing.  The specifications to be applied to the final 
product and to other elements of the production process, along with the range of acceptable 
values for each, should be specified. 
One lot of a biological product is considered to be a quantity of material that has been thoroughly 
mixed in a single vessel.  This concept can be applied to somatic cell and gene therapy for 
purposes of planning lot testing procedures.  This means that each cell population, vector 
preparation, or other product for such therapies prepared as a unique final mixture should be 
subjected to appropriate lot release testing.  Preparations intended solely for individual recipients 
differ from products prepared as large batches, and appropriate lot release criteria should be 
chosen to fit the practical constraints of each protocol.  Lot-to-lot variation provides a measure of 
the reproducibility of the procedures. 
A.  Cell Identity 
Quantitative testing by phenotypic and/or biochemical assays should be used to confirm 
cell identity and assess heterogeneity (21 CFR 610.14). 
B.  Potency 
The relevant function of the cells, if known, and/or relevant products biosynthesized by 
the cells should be defined and quantitated as a measure of potency (21.CFR 610.10). 
C.  Viability 
The viability of the cells should be quantitated and a lower limit for acceptability 
established. 
D.  Adventitious Agent Testing 
Tests should demonstrate that the cells are not contaminated with adventitious agents such 
as bacteria, fungi, (21 CFR 610.12), and mycoplasma, (Points to Consider in the 
Characterization of Cell Lines Used to Produce Biologicals, (1993), Attachment #2, (58 
FR 42974) and viruses.  The agency is considering proposed rulemaking to allow for 
validation of a mycoplasma free manufacturing process in cases where the final cell 
therapy product is too short lived to complete adequately sensitive testing prior to 
administration to patients. 
12
  E.  Purity 
Purity (21 CFR 610.13) or validation of endotoxin testing by LAL or other acceptable 
assays should be established.  The suitability and appropriateness of methods of endotoxin 
testing should be considered on a case-by-case basis.  The test used should be validated to 
show that the cell preparation does not interfere with endotoxin detection.   See 
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, 
December, 1987. 
F.  General Safety Test 
The general safety test (21 CFR 610.11) must be performed on the final product. When 
appropriate, modified procedures may be developed according to 21 CFR 610.9.  Please 
note that the agency is considering proposed rulemaking to amend the GST rules and 
scope of applicability especially for cell therapy products. 
G.  Frozen Cell Banks 
When cell populations frozen for subsequent administration are thawed, expanded, and 
then administered to patients, lot release testing on the thawed cells is needed, and can be 
adapted from Part II, C on cell banking practices. 
IV. 	 ADDITIONAL APPLICATIONS:  ADDITION OF RADIOISOTOPES OR 
TOXINS TO CELL PREPARATIONS 
Therapeutic or diagnostic applications may be proposed involving cells which are modified by 
radiolabeling or pre-loading with bioactive materials such as toxins.  Thus, the cell implant may be 
used as a delivery system not only for its own products and functions but also for other products. 
Novel safety concerns may arise related to the site of cell implantation and localization of the 
radionuclide or toxin, or due to metabolic properties of the cells.  These should be anticipated and 
addressed where possible. 
Similar special issues have been raised in the past by use of radiolabeled or toxin-conjugated 
antibodies, and are addressed in the "Points to Consider in the Manufacture and Testing of 
Monoclonal Antibody Products for Human Use" (1997).  Although the application to somatic cell 
therapies may differ, that document should be consulted. 
13
  V.	  PRODUCTION, CHARACTERIZATION AND RELEASE TESTING OF 
VECTORS FOR GENE THERAPY 
The information requested below may be difficult to acquire in some systems.  Sponsors may 
present alternative methods and data to CBER staff for review.  The types of information which 
will assure adequate safety depend in part on the nature of the proposed clinical trial, such as: 
route and frequency of administration, and the intended patient population. 
A. Vector Construction and Characterization 
Vector source materials should be characterized and documented thoroughly.  Viral 
vectors or plasmids should be generated from cloned and characterized constructs, and 
subjected to confirmatory identity tests.  Information supplied should include vector 
derivation, including descriptions of any vectors, helper viruses, and producer cell lines 
used for preparation of the final construct.  Known regulatory elements such as promoters 
or enhancers contained within the construct should be identified. 
Early in product development, vector characterization consisting of sequence data of 
appropriate portions of vectors and/or restriction mapping supplemented by protein 
characterization is acceptable.  For later phases of product development and licensure, 
more extensive sequencing information should be provided.  When sequencing of the 
entire vector is not feasible due to the size of the construct, it may be sufficient to 
sequence the genetic insert plus flanking regions and any significant modifications to the 
vector backbone or sites known to be vulnerable to alteration during the molecular 
manipulations.  Vector sequences which modulate vector-host interactions should be 
described if known, and stability of the host cell/vector system considered. 
B.  Vector Production System 
The vector production system is composed of the host cell, final gene construct or when 
appropriate vector intermediate, used to produce the vector (for example, retroviral 
producer cell).  The procedure for selection of the final gene construct, method of transfer 
of the gene construct into the host cell and selection and characterization of the 
recombinant host cell clone including vector copy number, and the physical state of the 
final vector construct inside the host cell (i.e. integrated or extra chromosomal), should be 
described in detail.  In addition, a detailed description of procedures for the propagation 
and expansion of the recombinant host cell clone, establishment of the seed stock and 
qualification of the seed stock should be provided.  For information on cell banking 
procedures refer to Section II, C. 
14
  C.  Master Viral Banks 
When a virus, with or without a therapeutic gene, is used as a seed in the manufacture of a 
therapeutic vector, it is recommended that a Master Viral Bank be created and 
characterized.  This would include vectors derived from adenovirus, adeno-associated 
virus, herpes virus, poxviruses, and other lytic and non-lytic viruses.  The sponsor should 
describe the source materials, (i.e. plasmids, vectors, oligomers, etc.) and molecular 
methods used to produce the source or seed vector.  The genetic integrity and stability 
(i.e. identity) of the seed vector should be confirmed and bioactivity of the vector seed 
should be demonstrated.  In the absence of bioactivity data, expression of the gene should 
be assessed. 
Master seed stocks should also be demonstrated to be free of adventitious agents, 
including virus, bacteria, fungi, and mycoplasma.  In the case of replication-defective or 
replication-selective vectors, Master Viral Banks should be demonstrated to be free of 
replication-competent viruses, which may arise as a result of contamination or 
recombination during the generation of the MVB.  Testing for other inappropriate viruses 
will depend upon the vector and feasibility of assays in the presence of vector virus.  The 
"Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals 
(1993)" (58 FR 42974), should be considered as background information. 
D.  Lot-to-Lot Release Testing and Specifications for Vectors 
General testing recommendations are discussed in the sections that follow.  Not all tests 
listed will be applicable to every vector class.  Sponsors should choose appropriate testing 
protocols, and consult CBER if there are questions as to the applicability of a specific test. 
Note that if drug substance (defined as bulk product not necessarily in final formulation) 
and drug product (defined as product in its final formulation) are the same, then only a 
single set of tests is necessary. 
Any standard assays for the properties listed below can be used if they are quantitative, 
and are of adequate specificity and sensitivity.  Assay methods should be validated by 
testing of known amounts of reference lots, spiked samples, or other appropriate 
measures, and data documenting assay performance submitted to the IND. 
1.  Tests of drug substance (bulk product not necessarily in final formulation): 
a.  Purity (21 CFR 610.13) 
i.  Test for total DNA or RNA content if appropriate to vector 
composition, e.g. A  /A  .
260  280 
ii.  Test for homogeneity of size and structure, supercoiled vs. 
linear, e.g. agarose gel electrophoresis. 
15
  iii.  Test for contamination with RNA or with host DNA, e.g. gel 
electrophoresis, including test with bacterial host-specific probe. 
iv.  Test for proteins if present as a contaminant, e.g. silver stained 
gel. 
v. 	 Test for non-infectious virus in cases in which that would be a 
contaminant, such as empty capsids.  See: Section VI C. 
vi.  Tests for toxic materials involved in production. 
b. 	 Identity (21 CFR 610.14) 
Test for vector identity by methods such as restriction enzyme mapping 
with multiple enzymes or PCR should be performed on the drug substance 
(see 21 CFR 610.14).  In the case of a facility making multiple constructs, 
it should be verified that the identity testing is capable of distinguishing the 
constructs and detecting cross-contamination. 
c.  Adventitious agents 
As methods of testing for adventitious agents become increasingly sensitive 
and specific over time, sponsors are encouraged to accumulate data 
validating testing methods other than those indicated, to permit future 
updating of this policy.  In cases in which a vector product interferes with 
appropriate assays, for example, a lytic viral vector that kills indicator cells 
in an assay for adventitious virus, some information may be obtained by 
parallel mock cultures using the same media and other reagents to allow 
outgrowth of a contaminant, or by assays in the presence of neutralizing 
antibody.  The following tests should be performed: 
i. 	 Sterility test (21 CFR 610.12), for aerobic and anaerobic 
bacteria and fungi. 
ii. 	 Mycoplasma testing, as specified in the "Points to Consider in 
the Characterization of Cell Lines Used to Produce Biologicals 
(1993)", Attachment #2, (58 FR 42974), which specifies the 
procedures for detecting mycoplasma contamination. 
iii.  Testing for adventitious viruses, in some cases, source materials 
or cell lines used in vector production introduce the risk of 
contamination with adventitious viruses.  In other cases, 
adventitious virus can be introduced during product 
manufacture.  Testing for an appropriate range of possible 
contaminating viruses is recommended, as discussed extensively 
in the "Points to Consider in the Characterization of Cell Lines 
Used to Produce Biologicals (1993)" 58 FR 42974, 
16
  and the ICH draft guideline Q5A, "Viral Safety Evaluation of 
Biotechnology Products Derived from Cell Lines of Human or 
Animal Origin," Step 4, approved by ICH, 3/5/97.  Testing for 
replication-competent retrovirus and adenovirus is discussed 
below. 
d.  Potency (21 CFR 610.10) 
Potency assays should be validated during the product development 
process.  Expression of the inserted gene can be determined by transfection 
of appropriate cells and demonstration of active gene product by an 
appropriate assay, characterized as to its sensitivity and specificity. 
Whenever possible, a potency assay should measure the biological activity 
of the expressed gene product, not merely its presence.  For example, if 
enzymatic activity is the basis of the proposed therapy, an enzyme activity 
assay detecting conversion of substrate to product would be preferred over 
an immunological assay detecting epitopes on the enzyme.  If no 
quantitative potency assay is available, then a qualitative potency test 
should be performed. 
2.  Tests of drug product (product in its final formulation):  The vector product in 
final container form should be tested for the properties listed below by 
quantitative, validated assays.  Tests for endotoxin and general safety if performed 
on a drug product (final product) need not be performed on drug substance (bulk 
product). 
a.  Sterility (21 CFR 610.12), identity (21 CFR 610.14), and potency (21 
CFR 610.10). 
b.  Purity (21 CFR 610.13) or validation of endotoxin testing by LAL or 
other acceptable assay (see "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, 
December, 1987). 
c.  General safety, as per 21 CFR 610.11.  Note that this test is not needed 
for therapeutic DNA plasmid products because they are among the 
specified biotechnology products (Federal Register notice, Vol. 61, No. 94, 
May 14, 1996), even if liposomes are added. 
17
  VI. 	 ISSUES RELATED TO PARTICULAR CLASSES OF VECTORS FOR GENE 
THERAPY 
A.  Additional Considerations for the Use of Plasmid Vector Products 
Many product and quality control considerations covered in the general sections above are 
appropriate to plasmid DNA products. The "Points to Consider on Plasmid DNA 
Vaccines for Preventive Infectious Disease Indications (1996)", 61 FR 68269, may also be 
a useful reference.  In general, complete sequencing of the plasmid should be performed. 
Plasmids should be characterized and specifications set with regard to the presence of 
RNA, protein, and bacterial host DNA contaminants, quantities of linear and supercoiled 
DNA in the preparation, and presence of toxic chemicals.  Toxic chemicals such as 
ethidium bromide should be avoided during production. 
As stated in the "Points to Consider in the Characterization of Cell Lines Used to Produce 
Biologicals," it is recommended that penicillin and other beta-lactam antibiotics be avoided 
during production, due to the risk of serious hypersensitivity reactions in patients.  If 
antibiotic selection is used during production, it is preferable not to use selection markers 
which confer resistance to antibiotics in significant clinical use, in order to avoid 
unnecessary risk of spread of antibiotic resistance traits to environmental microbes.  Also, 
residual antibiotic in the final product should be quantitated when possible, and the 
potential for allergy considered.  See 21 CFR 610.61(m) concerning labeling requirements 
for approved biological products if antibiotics are used during manufacture.  Concerning 
environmental impact and the use of drug resistance traits, consult the NIH Guidelines for 
Research Involving Recombinant DNA Molecules, Section III-A-1-a (59 FR 34496, 
amended 61 FR 59732).  Non-antibiotic selection systems can also be used. 
Plasmid vectors may be administered in conjunction with lipid preparations, local 
anesthetics, or other chemicals intended to facilitate DNA uptake.  If such a facilitating 
agent is added during formulation, a specification for its amount and identity in the final 
product should be established.  If toxic organic solvents such as chloroform are used in 
producing a lipid component, then processing should remove them and lot release 
specifications should include testing for residual solvent. 
B.  Additional Considerations for the Use of Retroviral Vector Products 
1.  Testing for replication competent retrovirus:  The following testing scheme for 
detection of replication competent retroviruses (RCR) summarizes current 
recommendations based on information available at this time.  This scheme is 
currently being reevaluated, and modified guidance will be made public when 
available.  Testing at multiple stages in production is recommended due to the 
limited knowledge of the risks of retrovirus exposure and the possibility of 
18
  generation of recombinant RCR at any point in the production process. 
Alternative assays (e.g. marker rescue) are acceptable if sensitivity is comparable 
to the PG4 S + L assay.  Testing should be complete prior to patient administration, 
particularly if cells can be cryopreserved; otherwise testing should be performed 
concurrently.  In order to gain biological information about events during 
production, molecular characterization of any RCR detected in clinical lots is also 
recommended. 
a.  Master Cell Bank of vector-producing cells (one time testing): 
i.  Supernatant testing, 5% of the total supernatant from culture of 
cells for a master cell bank should be tested by amplification on 
a permissive cell line (e.g., Mus dunni) including several blind 
passages followed by the PG4 S + L- or alternative assay.
ii.  Producer cell testing, 1% of pooled producer cells or 108  cells,
whichever is fewer, should be cocultured with a permissive cell 
line (e.g., Mus dunni) including several blind passages. 
Supernatant from the coculture should be tested by PG4 S+  L- or
alternative assay. 
b.  Working cell bank (one time testing):  Either supernatant testing or 
cocultivation of cells is recommended, using conditions described for 
master cell bank testing. 
c. 	 Lot testing of vector products: 
i. 	 Clinical grade supernatant, 5% of the supernatant should be 
tested by amplification on a permissive cell line (e.g., Mus 
dunni) including several blind passages, followed by the PG4 
S+  L or alternative assay.
ii. 	 Testing of end of production cells, 1% of total pooled end of 
production cells or 108 cells, whichever is fewer, should be
cocultured with a permissive cell line (e.g., Mus dunni), and 
then amplified by several blind passages.  Supernatant from the 
coculture should be tested by S + L or alternative assay.
d. 	 Lot testing of ex vivo transduced cells: 
i. 	 1% of pooled transduced cells or 10 8cells, whichever is fewer,
should be cocultured with a permissive cell line (e.g., Mus 
dunni) including several blind passages.  Supernatant from the 
coculture should be tested by PG4 S+  L or alternative assay.
19
  ii. 	 5% of supernatant from the transduced cells should be tested by 
amplification on a permissive cell line (e.g., Mus dunni) 
including several blind passages.  Supernatant from the 
coculture should be tested by PG4 S+  L assay.
2.  Patient monitoring:  Patients given retrovirus-related products should be 
monitored for RCR exposure.  Please consult CBER for guidance. 
C. 	 Additional Considerations for the Use of Adenoviral Vectors. 
1.  Measurement of particles vs. infectious units:  Patient doses of adenovirus
based gene therapy vectors are presently based upon some method of enumerating 
viral particles, plaque forming units (PFU), or infectious units (IU) measured in 
cell lines complementing the replication defect (not to be confused with 
measurement of RCA; see Section 2 below).  Given the potential toxicity of the 
adenoviral particles themselves, CBER recommends that patient dosing be based 
on particle number.  This recommendation also reflects that particle number can be 
readily and reproducibly measured.  However, since it is quite possible that some 
outcomes are a function of the number of infectious units administered, it is 
important for investigators or sponsors to develop in vitro infectivity assays which 
are reproducible and informative, and to set appropriately tight specifications on 
the ratio of infectious particles to total viral particles. 
Adenovirus particle measurement is commonly based on genomic DNA 
quantitation.  Using the absorbance at 260 nm in the presence of sodium dodecyl 
sulfate or other virus lysing agents, the maximum number of adenoviral particles 
can be calculated from OD .  The presence of non-adenovirus nucleic acid may 
260
yield inaccurate particle numbers and should be minimized during the 
manufacturing and viral purification process.  Electron microscope particle count 
has also been used for viral particle enumeration. 
Presently the titer of an adenovirus vector preparation usually refers to the 
infectious titer.  The cell used for determining the titer is often the producer cell 
line.  Differences in viral vectors may lead to changes in growth properties and 
kinetics.  The plaque method is efficient for adenoviral vectors with an easily 
complemented replication defect.  Vectors with multiple replication defects may be 
more readily titered by alternative methods such as use of fluorescent antibodies. 
Both assays require optimization for time of adsorption, need for deaggregation of 
virus, stability, etc.  Inclusion of a standard wild type virus control may facilitate 
the comparison of titers between different vectors and laboratories. 
20
  It is currently recommended that a ratio in the product of viral particles to 
biologically active virus of less than 100:1 be employed in phase I studies.  The 
purpose of this specification is to ensure the consistent manufacture of 
recombinant viruses and the highest bioactivity/particle/ patient dose and to limit 
possible toxicity due to viral structural proteins.  As new assays are developed and 
validated, their comparison to old ones and their use in product characterization is 
encouraged. 
2.  Detection of replication-competent adenovirus:  The presence of RCA in 
clinical lots of adenovirus vector raises a variety of safety concerns, including the 
possibility of adenovirus infection, unintended vector replication due to the 
presence of wild-type helper function, and exacerbation of host inflammatory 
responses.  The safety risks entailed by these events and other potential adverse 
events will differ depending on the indication and the patient population. 
Preclinical safety studies are inherently limited in assessment of RCA-related risks 
since there are no animal models that support extensive replication of human wild-
type or replication competent recombinant adenovirus. 
Therefore, adenovirus vectors intended to be replication-defective should be 
examined for the presence of replication-competent adenovirus (RCA).  RCA may 
arise at multiple steps during the manufacturing process, through recombination 
with host sequences or by contamination.  The amount of RCA generated during 
manufacture will be influenced by the overall design of the vector.  Use of 
replication-selective adenovirus vectors raises additional considerations and may 
call for additional or different testing strategies.  Such cases should be discussed 
with CBER. 
Detection of RCA in final vector product by a cell culture/cytopathic effect method 
is preferred at this time.  Validation of assay sensitivity by spiking decreasing 
numbers of wild type adenovirus particles into the test inoculum is recommended. 
Input multiplicity of infection (MOI) should be carefully chosen because of toxicity 
of higher doses of virus inoculum unrelated to the presence of RCA.  It should also 
be noted that too high an input MOI may lead to suppression of RCA outgrowth 
by the vector.  One or two blind passages on cells permissive for growth of the 
RCA, for example A549 cells, may be performed to amplify RCA before reading 
out on an indicator cell line.  In addition, it is recommended that the assay be 
quantitative, that is, able to determine the number of RCA present in any patient 
dose. 
Previous recommendations from the FDA have been that patient doses should 
contain no more than 1 pfu of RCA or equivalent in patients in whom adenovirus 
infection would be considered a potential risk.  However, the agency recognizes 
that current production techniques in combination with proposed dosing schemes 
21
  may make this recommendation prohibitively burdensome.  Therefore, if sponsors 
wish to propose a different specification, data should be provided demonstrating 
that the level of RCA present represents an acceptable risk for the intended patient 
population, route of administration, and dose.  In order to gain biological 
information about events during production, molecular characterization of any 
RCA present in clinical lots is also recommended at this time, and should be as 
thorough as is practical until more is known about the types of recombination that 
are occurring. 
3.  Adeno-associated virus:  Because of the association of AAV with adenovirus, 
testing for AAV is currently recommended in the Master Cell Bank, the Master 
Virus Seed Stock, and the final product. 
D.  Other Gene Delivery Systems 
Other gene delivery systems including additional types of viral or nucleic acid vectors are 
currently under development.  Sponsors using new systems are encouraged to contact 
CBER early in product development, to facilitate a safe and efficient development process. 
VII.  MODIFICATIONS IN VECTOR PREPARATIONS 
In the past, CBER has considered any change in a vector to result in a new product, and has 
requested submission of a new IND.  There is now an accumulating body of scientific evidence 
that will permit flexibility in this policy.  CBER's goal is to facilitate progress towards effective 
therapies by abbreviating testing and reducing documentation, whenever this can be done while 
preserving patient safety. 
Two aspects of IND submission are affected by consideration of a product as a modified vector: 
the decision as to whether a new IND should be submitted, and the decision as to what data 
should be submitted for review.  Certain changes, for example minor modifications in the genetic 
insert or changes in the antibiotic resistance gene, do not necessarily call for a new IND or for full 
product retesting.  In all cases, derivation of a new vector should be described and the vector 
should meet the specifications for release testing.  The other data which should be collected will 
vary with the degree and nature of the modifications to the vector.  The need for additional 
preclinical testing is determined by the likelihood of altered vector biology, not just the number of 
nucleotide changes.  In some cases, tissue localization, germ line alteration, and animal 
pharmacology/ toxicology studies may be optional.  Instead, the relevant safety studies could 
focus on specific safety concerns related to changes in the vector. 
When a number of related vectors involving minor modifications are studied, they may be 
considered members of a panel, analogous to panels of monoclonal antibodies described in the 
22
  "Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human 
Use," 1997 revision, 62 FR 9196.  As stated, such panels could be studied under a single IND and 
submitted for approval in a single license application.  Phase 3 clinical trials should include some 
experience with all panel members, and efficacy established for the overall panel. 
Vector modifications should be discussed with CBER case by case.  If a sponsor wishes to 
abbreviate testing and IND submission for a product or product series, the sponsor should verify 
with CBER the adequacy of the proposed abbreviated testing scheme prior to initiating clinical 
trials.  Data forthcoming from sponsors can help establish whether particular changes in vectors 
alter their behavior when they are compared in vivo, and therefore whether complete testing 
should continue to be performed. 
VIII.  PRECLINICAL EVALUATION OF CELLULAR AND GENE THERAPIES 
A.  General Principles 
Preclinical studies are intended to define the pharmacologic and toxicologic effects 
predictive of the human response, not only prior to initiation of clinical trials, but also 
throughout drug development.  The goals of these studies include the following: to define 
safe starting doses and escalation schemes for clinical trials, to identify target organs for 
toxicity and parameters to monitor in patients receiving these therapies, and to determine 
populations which may be at greater risk for toxicities of a given cellular or gene 
therapeutic. 
Design of preclinical studies should take into consideration:  1)  the population of cells to 
be administered or the class of vector used, 2)  the animal species and physiologic state 
most relevant for the clinical indication and product class, and 3) the intended doses, route 
of administration, and treatment regimens.  Parameters which should be studied will be 
discussed below. 
Due to the unique and diverse nature of the products employed in cellular and gene 
therapies, conventional pharmacology and toxicity testing may not always be appropriate 
to determine the safety and biologic activity of these agents.  Issues such as species 
specificity of the transduced gene, permissiveness for infection by viral vectors, and 
comparative physiology should be considered in the design of these studies.  Available 
animal models mimicking the disease indication may be useful in obtaining both sufficient 
safety and efficacy data prior to entry of these agents into clinical trials. 
The ICH Draft Guideline S6, "Preclinical Safety Evaluation of Biotechnology-Derived 
Pharmaceuticals," (Step 4, approved by ICH, 7/16/97) discusses in Section 3.1 the flexible 
application of Good Laboratory Practices in testing of biotechnology products. Although 
pivotal safety studies in support of marketing (e.g. carcinogenicity, reproductive 
23
  toxicology) are expected to be conducted in compliance with the regulations as outlined in 
21 CFR part 58, it is recognized that studies in support of entry into clinical trials may not 
always strictly adhere to GLP.  In these cases, the principles of the regulation should be 
followed as closely as possible, and where deviations occur, they should be evaluated for 
impact on the expected clinical application, and discussed in the report submitted to the 
agency. 
If a product is comparable to agents for which there is wide previous clinical experience, 
or for which the insertion of a different expression cassette is not expected to influence the 
toxicity or the dissemination of the vector, less extensive preclinical testing may suffice 
(see Section VII, above). 
It is recommended that plans for preclinical studies be discussed with representatives from 
CBER prior to their initiation.  Clinical plans requiring rapid enrollment of patients should 
be anticipated and preceded by adequate preclinical testing. 
B.  Animal Species Selection and Use of Alternative Animal Models 
It is recognized that animal models of disease may not be available for every cellular or 
gene therapy system.  Preclinical pharmacologic and safety testing of these agents should 
employ the most appropriate, pharmacologically relevant animal model available.  A 
relevant animal species would be one in which the biological response to the therapy 
would be expected to mimic the human response.  For example, a vector expressing a 
human cytokine would best be tested in an animal species in which that cytokine binds to 
the corresponding cytokine receptor with affinity comparable to that seen with human 
receptors, and initiates a pharmacologic response comparable to that expected in humans. 
C.  Somatic Cell and Gene-Modified Cellular Therapies 
1.  In vivo biological/pharmacological activity:  The transduction procedure, dose 
of expanded or genetically modified cells, and route of administration planned for 
the clinical trial should be evaluated preclinically.  Pharmacologic studies in 
animals may provide useful information regarding the in vivo function, survival 
time and appropriate trafficking of the modified cells. 
2.  Toxicologic testing:  Safety testing of expanded, activated, or genetically 
modified somatic cells should be conducted in an appropriate animal model.  Data 
on distribution, trafficking, and persistence of these cells in vivo mentioned above 
should be evaluated for safety implications as well.  At a minimum, treated animals 
should be monitored for general health status, serum biochemistry, and 
hematologic profiles.  Target tissues should be examined microscopically for 
histopathological changes. 
24
  D.  Direct Administration of Vectors In Vivo 
A number of different vectors are currently in development for direct administration to 
human subjects.  Direct administration of any of these vectors presents a number of safety 
concerns that will be addressed here.  All toxicity and localization studies, including 
studies of gonadal tissue described below, should use the final formulated product, since 
added materials such as liposomes, or changes in pH or salt content, may alter the toxicity 
or distribution pattern. 
Specific concerns for each vector subclass will generally be handled on a case-by-case 
basis, and discussion with CBER is encouraged. 
1.  Route of administration:  The route of administration of vectors can influence 
toxicity in vivo.  Safety evaluation in the preclinical studies should be conducted by 
the identical route and method of administration as in the clinical trial whenever 
possible.  When this is difficult to achieve in a small animal species, a method of 
administration similar to that planned for use in the clinic is advised.  For example, 
intrapulmonary instillation of adenoviral vectors by intranasal administration in 
cotton rats or mice is an acceptable alternative to direct intrapulmonary 
administration through a bronchoscope. 
2.  Selection of animal species:  The species of animal chosen for preclinical 
toxicity evaluations should be selected for its sensitivity to infection and pathologic 
sequelae induced by the wild-type virus related to a vector, as well as its utility as a 
model of biologic activity of the vector construct.  Rodent models, rather than 
non-human primates may be useful if they are susceptible to pathology induced by 
the virus class.   When evaluating the activity of a vector in an animal model of the 
clinical indication, safety data can be gathered from the same model to assess the 
contribution of disease-related changes in physiology or underlying pathology to 
the response to the vector. 
3.  Selection of dose to be employed:  The doses of vectors studied preclinically 
should be selected based on preliminary activity data from studies both in vitro and 
in vivo.  A no-effect level dose, an overtly toxic dose, and several intermediate 
doses should be determined, and appropriate controls, such as naive or vehicle-
treated animals, should be included.  For products in limited supply or with 
inherently low toxicity, a maximum feasible dose may be administered as the 
highest level tested in the preclinical studies.  Preclinical safety evaluations should 
include at least one dose equivalent to and at least one dose escalation level 
exceeding those proposed for the clinical trial; the multiples of the human dose 
required to determine adequate safety margins may vary with each class of vector 
employed and the relevance of the animal model to humans.  Scaling of doses 
based on either body weight or total body surface area as appropriate facilitates 
25
  comparisons across species.  Information generated can be used to determine the 
margin of safety of the vector for use in the clinical trial, as well as to gauge an 
acceptable dose-escalation scheme. 
4.  Toxicologic testing:  Treated animals should be monitored for general health 
status, serum biochemistry, and hematology, and tissues should be examined for 
pathological changes in histology. 
5.  Distribution of vector out of the site of administration:  Localization studies, 
designed to determine the distribution of the vector after administration to its 
proposed site, are also recommended.  Whenever possible, the intended route of 
administration should be employed.  Additional groups of animals may be treated 
intravenously, as a "worst-case" scenario representing the effects of widespread 
vector dissemination.  Transfer of the gene to normal, surrounding, and distal 
tissues as well as the target site should be evaluated using the most sensitive 
detection methods possible, and should include evaluation of gene persistence. 
Dose levels selected should follow those used in toxicity testing.  When aberrant or 
unexpected localization is observed, studies should be conducted to determine 
whether the gene is expressed and whether its presence is associated with 
pathologic effects. 
a.  Expression of gene product and induction of immune responses 
Expression of the therapeutic gene product in intended or unintended 
tissues may result in unexpected toxicities, which should therefore be 
addressed in preclinical studies.  Inflammatory, immune, or autoimmune 
responses induced by the gene product may be of concern.  Animal studies 
should be conducted over a sufficient duration of time to allow 
development of such responses.  Host immune responses against viral or 
transgene proteins may limit their usefulness for repeated administration in 
the clinic. 
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  b.  Vector localization to reproductive organs 
With vectors for direct administration, the risk of vector transfer to germ 
cells should be considered.  Animal testicular or ovarian samples should be 
analyzed for vector sequences by the most sensitive method possible.  If a 
signal is detected in the gonads, further studies should be conducted to 
determine if the sequences are present in germ cells as opposed to stromal 
tissues, using techniques that may include but are not limited to cell 
separations or in situ PCR, or other techniques.  Semen samples for 
analysis can be collected from mature animals including mice (G.D. Snell, 
et. al., Anat. Rec., 90:243-253, 1944; J.C. Kile, Jr., Anat. Rec., 
109:109-117, 1951), for determination of vector incorporation into germ 
cells. 
6.  Host immune status and effects on gene therapy vectors:  Immune status of the 
intended recipients of a gene therapy should be considered in the risk-benefit 
analysis of a product, particularly for viral vectors.  If exclusion of 
immunocompromised patients would unduly restrict a clinical protocol, immune 
suppressed, genetically immunodeficient, or newborn animals may be used in 
preclinical studies, to evaluate any potential safety risks. 
IX.  CONCLUSION 
As new classes of gene therapies and somatic cell therapies are developed, concerns and methods 
of testing will most likely change.  The above guidance outlines the types of issues that should be 
examined and provides a framework for analysis of new technologies as they emerge.  CBER 
encourages comments and suggestions from the academic and commercial communities and other 
interested parties during the evolution of policy in this area. 
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