Source Animal, Product, Preclinical, and Clinical Issues Concerning the Use of Xenotransplantation Products in Humans: Guidance for Industry

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

Source Animal, Product,
Preclinical, and Clinical
Issues Concerning the Use of
Xenotransplantation Products
in Humans
______________________________________________________________________________
Guidance for Industry
Additional copies of this guidance are available from the Office of Communication, Outreach
and Development (OCOD), 10903 New Hampshire Ave., Bldg. 71, Rm. 3128, Silver Spring,
MD 20993-0002, or by calling 1-800-835-4709 or 240-402-8010, or email ocod@fda.hhs.gov, or
from the Internet at
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guida
nces/default.htm.
For questions on the content of this guidance, contact OCOD at the phone numbers or email
address listed above.
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Biologics Evaluation and Research
April 2003
Updated December 2016

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Table of Contents
I. 	INTRODUCTION............................................................................................................. 1
II. 	BACKGROUND ............................................................................................................... 2
III. 	DEFINITIONS AND ABBREVIATIONS ...................................................................... 3
IV. 	REGULATORY RESPONSIBILITY ............................................................................. 7
V. 	SOURCE ANIMAL CHARACTERIZATION .............................................................. 7
A. 	General Considerations ........................................................................................ 7
B. 	Animal Welfare Concerns .................................................................................... 8
C. 	Animal Origin........................................................................................................ 9
D. 	Animal Health and Husbandry.......................................................................... 11
E. 	Harvest of Nonhuman Live Cells, Tissues, or Organs for Use in Producing
Xenotransplantation Products ........................................................................... 17
F. 	Source Animal History for Xenogeneic Cell Lines .......................................... 20
G. 	Disposal of Animals and Use of Byproducts ..................................................... 21
VI. 	CHARACTERIZATION OF XENOTRANSPLANTATION PRODUCTS ............. 21
A. 	General Considerations ...................................................................................... 21
B. 	Considerations for Classes of Xenotransplantation Products ........................ 22
VII. 	MICROBIOLOGICAL TESTING OF XENOTRANSPLANTATION PRODUCTS .
........................................................................................................................................... 24
A. 	General Considerations ...................................................................................... 24
B. 	Considerations for Classes of Xenotransplantation Products ........................ 25
C. 	Assay Design for the Detection of Infectious Agents ....................................... 27
VIII. 	MANUFACTURING AND PROCESS-RELATED GMP CONSIDERATIONS FOR
HARVEST AND PROCESSING OF XENOTRANSPLANTATION PRODUCTS 31
A. 	General Considerations ...................................................................................... 31
B. 	Contamination/Cross-Contamination Precautions.......................................... 31
C. 	Validation and Qualification.............................................................................. 33
IX. 	PRECLINICAL CONSIDERATIONS FOR XENOTRANSPLANTATION ........... 35
A. 	General Considerations ...................................................................................... 35
B. 	Issues Related to Infectious Agents ................................................................... 36
C. 	Xenotransplantation Product-Host Interactions ............................................. 37
D. 	Considerations for the Use of Heterogeneous Xenotransplantation Products
............................................................................................................................... 39
E. 	In Vitro and In Vivo Tumorigenicity Models for Xenotransplantation
Products Intended for Transplantation ............................................................ 40
F. 	Combinations of Xenotransplantation Products with Devices ....................... 41

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X. 	CLINICAL ISSUES IN XENOTRANSPLANTATION ............................................. 43
A. 	General Considerations ...................................................................................... 43
B. 	Clinical Protocol Review .................................................................................... 43
C. 	Xenotransplantation Site .................................................................................... 43
D. 	Criteria for Patient Selection ............................................................................. 43
E. 	Risk/Benefit Analysis .......................................................................................... 44
F. 	Screening for Infectious Agents ......................................................................... 44
G. 	Patient Follow-up ................................................................................................ 50
H. 	Archiving of Patient Plasma and Tissue Specimens ........................................ 50
I. 	Health Records and Data Management ............................................................ 52
J. 	Informed Consent ............................................................................................... 54
K. 	Responsibility of the Sponsor in Informing the Patient of New Scientific
Information .......................................................................................................... 58
XI. 	REFERENCES ................................................................................................................ 59

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Source Animal, Product, Preclinical, and Clinical Issues Concerning
the Use of Xenotransplantation Products in Humans
______________________________________________________________________________
Guidance for Industry
This guidance represents the current thinking of the Food and Drug Administration (FDA or
Agency) on this topic. It does not establish any rights for any person and is not binding on FDA
or the public. You can use an alternative approach if it satisfies the requirements of the
applicable statutes and regulations. To discuss an alternative approach, contact the FDA staff
responsible for this guidance as listed on the title page.
I. 	INTRODUCTION
The Food and Drug Administration (FDA) is issuing this guidance to provide sponsors and
applicants of xenotransplantation products with updates concerning the production, testing, and
evaluation of products, during protocol development and during the preparation of submissions
to FDA, e.g., Investigational New Drug Applications (INDs) and Biologics License Applications
(BLAs). This guidance also includes updated references and Agency practices intended to
prevent the introduction and spread of infectious agents of animal origin into the human
population. This guidance amends the guidance of the same title dated April 2003 (April 2003
guidance).
For the purpose of this document, xenotransplantation refers to any procedure that involves the
transplantation, implantation, or infusion into a human recipient of either (a) live cells, tissues, or
organs from a nonhuman animal source; or (b) human body fluids, cells, tissues, or organs that
have had ex vivo contact with live nonhuman animal cells, tissues, or organs. Also, for the
purpose of this document, xenotransplantation products include live cells, tissues, or organs used
in xenotransplantation. However, for the purpose of this guidance, xenotransplantation does not
include transplantation, implantation, or other use of acellular animal tissues. These acellular
products derived from animal tissue, often referred to as xenografts, are beyond the scope of this
guidance (See Definitions in section III of this document).
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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II. 	BACKGROUND
In the mid 1990’s, xenotransplantation was suggested as a mechanism for circumventing the
shortage of human allographs for transplantation. Proposed xenotransplantation protocols
included implantation into humans of live organs, tissues, and cells from nonhuman animal
sources and procedures in which human cells have had ex vivo contact with nonhuman cells,
tissues, or organs. Examples of procedures involving xenotransplantation products include
transplantation of xenogeneic hearts, kidneys, or pancreatic tissue to treat organ failure,
implantation of neural cells to ameliorate neurological degenerative diseases, administration of
human cells previously cultured ex vivo with live nonhuman animal antigen-presenting or feeder
cells, and extracorporeal perfusion of a patient’s blood or blood component perfused through an
intact animal organ or isolated cells contained in a device to treat liver failure.
The use of xenotransplantation products presents potential public health risks, such as:
1. 	Transmission of infectious agents that are pathogenic for humans but may not be
pathogenic or even detectable in the source animal host;
2. 	Transmission of organisms that may not normally be pathogenic in humans but
can become so in the immunosuppressed or immunocompromised individual; and,
3. 	Recombination or reassortment of infectious agents, particularly viruses, with
nonpathogenic or endogenous human infectious agents, to form new pathogenic
entities.
It is difficult to predict the infectious agents that may cause disease in a recipient of a
xenotransplantation product solely on the basis of analysis of naturally occurring zoonoses
because there are major differences between normal contact of humans with animals and contact
of a recipient with a xenotransplantation product. For example, the physical barrier or distance is
eliminated in the recipient, due to transplantation and vascularization of xenotransplantation
products, or even due to implantation of nonvascularized cells or tissues, or ex vivo
manipulations that cause intimate proximity or contact of xenotransplantation product materials
with recipient cells, tissues, or fluids. The potential for viral adaptation in immunocompromised
or iatrogenically immunosuppressed hosts and the potential for undetected spread of previously
latent viral infections are of particular concern. For these reasons, during product development,
it is important to consider the safety not only of recipients and their contacts but also of the
public. Public discussion of these issues previously took place in the FDA Biological Response
Modifiers Advisory Committee (BRMAC) - Subcommittee on Xenotransplantation; the
Department of Health and Human Services (DHHS) Secretary’s Advisory Committee on
Xenotransplantation (SACX); and other public fora.i
i The BRMAC Subcommittee on Xenotransplantation (SACX) was initiated in 2001 and was discontinued in 2005.
Further discussions on xenotransplantation have taken place in the Cellular, Tissue, and Gene Therapy Advisory
Committee (CTGAC). In addition, a discussion took place in 2009 on the topic of “Animal Models for Porcine
Xenotransplantation Products Intended to Treat Type 1 Diabetes or Acute Liver Failure.”

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In 1996, the Public Health Service (PHS) published a “Draft Public Health Service (PHS)
Guideline on Infectious Disease Issues in Xenotransplantation” (61 FR 49920, September 23,
1996) that was revised and issued on January 29, 2001 (66 FR 8120), based on public comments
received and advances in fields relating to xenotransplantation [1] (hereafter referred to as “PHS
Guideline”). This FDA guidance document reiterates many of the concepts in the PHS
Guideline but, in addition, includes specific advice, regarding all aspects of xenotransplantation
product development and production and xenotransplantation clinical trials.
Since the publication of the PHS Guideline, the World Health Organization (WHO) published
the Second Global Consultation on Regulatory Requirements for Xenotransplantation Clinical
Trials.[2]
FDA anticipated that its approach to regulation of xenotransplantation products would evolve as
the scientific knowledge in the area of xenotransplantation continued to accumulate. Since the
issuance of the final guidance in 2003, FDA has issued additional guidance that contains
recommendation relevant to certain xenotransplantation products; see references. [3-8] FDA
realizes that it may not be appropriate to every aspect of the guidance to every
xenotransplantation product. For example, some of the recommendations for animal husbandry
may not be needed for xenotransplantation products consisting of well-characterized, long-
established animal cell culture lines or human cells co-cultured with such lines.
III. 	DEFINITIONS AND ABBREVIATIONS
Act: The Federal Food, Drug, and Cosmetic Act (21 U.S.C. 321 et seq.).
AAALAC: Association for Assessment and Accreditation of Laboratory Animal Care,
International. This organization inspects and accredits biomedical animal facilities.
Agents of concern: For the purpose of this document, agents of concern are infectious agents
that may pose a risk to the recipient and/or public, i.e., agents that can infect, potentially could
infect, or have an inadequately defined ability to infect humans.
ATCC: American Type Culture Collection.
BLA: Biologics license application. Approval of a biologics license application or issuance of a
biologics license constitutes a determination that the establishment(s) and the product meet
applicable requirements to ensure the continued safety, purity, and potency of such products.
BSL: BioSafety Level.
CDC: Centers for Disease Control and Prevention.
CDRH: Center for Devices and Radiological Health.

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cGMP: Current good manufacturing practice. For drugs, including biological drugs, cGMP
regulations can be found at 21 CFR Parts 210 and 211. For biological products, see
21 CFR Part 600 Subpart B and Part 610. For blood and blood components, additional
regulations can be found at 21 CFR Part 606. For devices, quality system regulations can be
found at 21 CFR Part 820.
Closed herd or colony: Herd or colony governed by standard operating procedures (SOPs) that
specify criteria restricting admission of new animals to assure that all introduced animals are at
the same or higher health standard, compared to the residents of the herd or colony.
CPE: Cytopathic effects. An effect on nucleated cells in vitro caused by some viruses that are
observable microscopically.
CVM: Center for Veterinary Medicine.
DPF: Designated pathogen free. This term is used to describe animals, animal herds, or animal
facilities that have been rigorously documented to be free of specified infectious agents and that
are maintained using well-defined routines of testing for designated pathogens and utilizing
rigorous SOPs and practices of herd husbandry and veterinary care to assure the absence of the
designated pathogens.
EM: Electron microscopy. A method used to visualize very small objects, such as subcellular
particles, or infectious agents, such as viruses.
FDA: Food and Drug Administration.
GE: Genetically engineered.
Gnotobiotic: The science of rearing laboratory animals, the microflora and microfauna of which
are specifically known in their entirety.
GVHD: Graft versus host disease.
HEPA: High-efficiency particulate air.
IACUC: Institutional Animal Care and Use Committee. A local institutional committee
established to oversee the institution’s animal program, facilities, and procedures. An IACUC
carries out semiannual program reviews and facility inspections and reviews all animal use
protocols and any animal welfare concerns. (See Public Health Service Policy on Humane Care
and Use of Laboratory Animals, revised 2015.)
IBC: Institutional Biosafety Committee. A local institutional committee, established to review
and oversee basic and clinical research conducted at that institution. The IBC assesses the safety
of the research and identifies any potential risk to public health or the environment. (See section
IV-B-2 of the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid
Molecules.)

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ICH: International Council for Harmonisation of Technical Requirements for Pharmaceuticals
For Human Use.
IDE: Investigational device exemption application. These are applications containing requests
to use an unapproved device in clinical tests using human subjects. The statutory requirement is
at section 520(g) of the Federal Food, Drug, and Cosmetic Act (the Act) (21 U.S.C. 360(g)), and
the implementing regulations can be found at 21 CFR Part 812.
IND: Investigational new drug application. These applications are required for persons who
intend to conduct clinical investigations involving unapproved drug products, including those
subject to section 505(i) of the Act (21 U.S.C. 355(i)) or to the licensure provisions of section
351 of the PHS Act (42 U.S.C. 262). The statutory requirement is at section 505(i) of the Act
(21 U.S.C. 355(i)), and the IND regulations are found at 21 CFR Part 312.
INAD: Investigational new animal drug.
IRB: Institutional Review Board. A board, committee, or other group, designated by an
institution established to review and approve biomedical and behavioral research involving
human subjects in order to protect the rights and welfare of human subjects (See 21 CFR Part 56,
Institutional Review Boards.)
Lot: Defined in 21 CFR 210.3(b)(10) as a batch or a specific, identified portion of a batch,
having uniform character and quality within specified limits, and in 21 CFR 600.3(x) as that
quantity of uniform material identified by the manufacturer as having been thoroughly mixed in
a single vessel. Each lot of final product is subjected to appropriate tests to ascertain adherence
to specifications prior to release of the product for clinical use. Licensed biological products
may be subject to lot release as described in 21 CFR 610.2(a). Often, in the case of
xenotransplantation products, an entire lot is used for treating a single recipient.
Master File: Master Files are submitted to FDA and contain information regarding a product,
such as product manufacture or general procedures. Procedures and information contained in the
Master File can be cross-referenced in INDs and IDEs on written permission from the Master
File sponsor, but confidentiality of the information within the Master File is maintained.
(See 21 CFR 314.420.)
NADA: New animal drug application.
PBMC: Peripheral blood mononuclear cells.
PCR: Polymerase chain reaction. An enzymatic technique, using a thermophyllic enzyme to
catalyze synthesis of short DNA sequences, that allows detection of nucleic acids by
amplification of specific DNA sequences.
PERV: Porcine endogenous retrovirus(es).

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PHS Act: The Public Health Service Act (42 U.S.C. 201 et seq.).
PMA: Premarket approval application. This is a marketing application for certain devices under
section 515 of the Act. The regulations for PMAs can be found at 21 CFR Part 814.
Recipient: An individual who receives or who undergoes ex vivo exposure to a
xenotransplantation product (as defined in xenotransplantation).
RT: Reverse transcriptase. An enzyme found particularly in retroviruses that catalyzes the
synthesis of DNA from RNA.
SAF: Source animal facility.
Sentinel animal: Those animals, usually of the same species as the resident herd or colony,
housed in direct contact with the animals being monitored and tested for the purpose of detecting
adventitious agents in the herd or colony, including viral, fungal, and bacterial diseases as well as
endo- and ectoparasites.
SOP: Standard operating procedure.
Source animal: An animal from which cells, tissues, and/or organs for use in xenotransplantation
are obtained.
TSE: Transmissible spongiform encephalopathy. TSEs are fatal, subacute degenerative diseases
of humans and animals with characteristic neuropathology (spongiform change and deposition of
an abnormal form of a prion protein present in all mammalian brains). TSEs are experimentally
transmissible by inoculation or ingestion of diseased tissue. The abnormal prion protein is
hypothesized to be the agent of transmission. Alternatively, other unidentified co-factors or an
as-yet-unidentified viral agent may be necessary for transmission.
USDA: United States Department of Agriculture.
WFI: Water for Injection.
Xenograft product(s): Xenograft products are acellular products derived from animal tissue.
These xenografts, are beyond the scope of this guidance. Please consult CDRH for guidance on
xenograft products.[9]
Xenotransplantation: For the purpose of this document, any procedure that involves the
transplantation, implantation, or infusion into a human recipient of either (a) live cells, tissues, or
organs from a nonhuman animal source; or (b) human body fluids, cells, tissues, or organs that
have had ex vivo contact with live nonhuman animal cells, tissues, or organs.
Xenotransplantation product(s): For the purpose of this document, xenotransplantation products
include live cells, tissues, or organs used in xenotransplantation (defined above).

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Zoonosis: A disease of animals (e.g., brucellosis, rabies) that may be transmitted to humans
under natural conditions.
Zoonotic: Relating to diseases that arise from the transfer of infectious agents by normal
contacts between animals and humans.
IV. 	REGULATORY RESPONSIBILITY
FDA has regulatory oversight of xenotransplantation products, including live organs, tissues, or
cells from a nonhuman animal source or xenotransplantation product materials used in
encapsulated form or in which nonhuman live organs, tissues, or cells have ex vivo contact with
human body fluids, cells, tissues, or organs that are subsequently given to a human recipient. If
xenotransplantation products are to be used in clinical investigation, they require an appropriate
investigational application be in place (e.g., 21 CFR Part 312). FDA will regulate most
xenotransplantation products as biological products. CBER regulates biological products,
including cellular therapies, under authority of section 351 of the PHS Act (42 U.S.C. 262) and
the Act (21 U.S.C. 321 et seq.). You will find regulations for drugs, biological products, and
devices in Title 21 of the Code of Federal Regulations (e.g., 21 CFR Part 312 for regulations
governing INDs, 21 CFR Part 812 for regulations governing IDEs, and 21 CFR Part 601 for
regulations governing licensing of biological products). To assist developers of cell therapy
products in preparing IND submissions to the Agency, FDA has published guidance on
regulatory considerations in the development of somatic cell therapy and gene therapy and the
content and review of chemistry, manufacturing, and control information for cell therapy product
INDs. [3, 10] In addition, for products derived from GE animals, please refer to 21 CFR Part 511
and Guidance for Industry: Regulation of Genetically Engineered Animals Containing Heritable
Recombinant DNA Constructs [4] for recommendations regarding regulations governing
investigational new animal drug that are regulated by FDA’s Center for Veterinary Medicine
(CVM). The primary responsibility for designing and monitoring the conduct of clinical trials
rests with the sponsor (e.g., 21 CFR 312.23(a)(6)(iii)(d) and 312.50). In this document, “you”
refers to the sponsor, the clinical investigator, or to any party designated by the sponsor to fulfill
the recommended function (e.g., SAF staff, laboratory personnel, etc.).
Some products may be combination products consisting of a biologic and a device, such as
xenogeneic cells contained in a device used for extracorporeal hemoperfusion. Refer to
21 CFR Part 3 for issues regarding the regulation and assignment for premarket review of
combination products.
V. 	SOURCE ANIMAL CHARACTERIZATION
A. 	General Considerations
The cross-species infectious potential of specific animal pathogens should be a major
consideration in the selection of source animal species. Anatomic and physiologic
considerations are also important. For example, whether an organ is of appropriate size

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and will function adequately across species barriers are considerations, as are certain
immunologic concerns, including the suitability of current regimens in prevention of
rejection of the nonhuman animal live cells, tissues, or organs. Please note that the
species you use may be subject to other federal laws and regulations (see, e.g., 16 U.S.C.
1538), such as those covering endangered or protected species. You should consult all
relevant PHS and FDA guidance documents on this subject prior to submitting an
application and, specifically, should consult the document, “Guidance for Industry:
Public Health Issues Posed by the Use of Nonhuman Primate Xenografts in Humans”[11]
before submitting an application to FDA that involves the use of nonhuman primates as
sources of a xenotransplantation product. (The term “xenograft” as used in the above
referenced document is synonymous with the term “xenotransplantation product”
however, these terms are no longer synonymous in current use (see Definitions)).
Due to potential infectious disease risks associated with the use of xenotransplantation
products, you should develop appropriate source animal qualifications. These
qualifications should include herd management and programs for prevention and
screening for infectious agents. Although testing of the final xenotransplantation product
for infectious agents is crucial, appropriate control of animal sources and husbandry
provides important additional assurance for the safety of such products by controlling
infections by both known and potentially even unknown agents. Therefore, the specific
information supplied by the sponsor, regarding animal husbandry including housing,
feeding, veterinary care, and drug and biologic treatment of source animal herds and
individual source animals, will be crucial for FDA evaluation of the potential for safe use
of cells, tissues, or organs from such source animals.
The SAF, production process, and records are subject to FDA inspection under section
704 of the Act (21 U.S.C. 374) and section 351(c) of the PHS Act (42 U.S.C. 262(c)).
B. 	Animal Welfare Concerns
Another area of consideration for SAFs and manufacturers of xenotransplantation
products is the welfare of the source animals. Procedures for animal husbandry, tissue
harvesting, and termination of animals should be approved by an appropriate Institutional
Animal Care and Use Committee, in accordance with the Animal Welfare Act (7 U.S.C.
2131, et seq.). In cases where funds are received from the PHS, procedures must also
comply with the PHS Policy on Humane Care and Use of Laboratory Animals, according
to section 495 of the PHS Act (42 U.S.C. 289(d)). We recommend that the SAF be
accredited by the AAALAC. Standards for accredited facilities for when funds are
received from the National Institutes of Health are provided in the National Research
Council’s Institute for Laboratory Animal Research, Guide for the Care and Use of
Laboratory Animals.

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C. 	Animal Origin
1. 	Animal and Herd Qualification
You should derive source animals only from closed herds with documented health
screening programs. Individuals with expertise in infectious diseases of the
species involved should develop a list of viruses, bacteria (including the
rickettsiae), mycoplasma, fungi, transmissible spongiform encephalopathies
(TSEs), and parasites for which the herd is screened and supply this information
to FDA as part of the application to FDA (e.g., IND). You should consider all
infectious agents known to infect the source species. You should justify the
rationale for omitting agents that are found in the source animal species from the
herd screening program in the application to FDA requesting investigational use
(e.g., IND). For example, the geographic location of the herd may allow
exclusion of certain infectious agents. You should obtain source animals from
TSE-susceptible species only from closed herds that are documented to be free
from TSE diseases or TSE-associated agents [12] (see also section V.C.3.c.). You
should not use source animals obtained from geographic areas in which TSEs are
known to exist in the source species. In the application to FDA requesting
investigational use of the xenotransplantation product (e.g., IND), you should
describe and justify the frequency of the screening, the method of assay, and the
method of identifying which and what proportion of animals are sampled. As
data are accumulated that demonstrate product safety, you may modify the
screening program in consultation with the FDA.
2. 	Animal History
The sponsor should document the geographic origin, species, strain, and
genealogy of the source animal(s) and herd(s). The documentation of source
animal history should describe factors that may pose risks to recipients, such as
possible exposure of the predecessor animals to sources of TSEs or other
adventitious or infectious agents of concern (see Definitions, section III.). Source
animals should be bred and raised in captivity and be derived from closed herds.
You may use artificial insemination, embryo transfer, cloning, or hysterotomy
plus foster feeding to establish animal herds with fewer endemic pathogens. In
particular, the PHS Guideline suggests that breeding programs use caesarean-
derived animals, whenever possible.[1] You should document the use of these
procedures in the animal history.
3. 	Source Animals from Outside the U.S.
a. 	You should not use animals from outside the U.S. or their first
generation offspring as sources for the production of
xenotransplantation products unless they are of a species or strain
not available in the United States or have specific qualities that

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provide a unique and scientifically justified clinical advantage,
such as transgenic animals.
b. 	If the use of source animals from outside the U.S. is necessary and
justifiable, you should apply the same considerations for these
animals as for source animals bred in the U.S. (e.g., see section
V.D. for Animal Health and Husbandry). You should use a
quarantine period of sufficient length to demonstrate the absence
of infectious agents of concern, and you should perform extensive
screening of the animals. In the application to FDA requesting
investigational use of the xenotransplantation product (e.g., IND),
you should submit thorough documentation to demonstrate that
such source animals have been derived from closed herds, have
been housed under appropriate conditions and subjected to
recommended health maintenance procedures and screenings, and
have not been fed rendered or recycled mammalian materials for at
least two generations. You should include in the screening agents
that are endemic in the country of origin. In the application to
FDA requesting investigational use of the xenotransplantation
product (e.g., IND), you should describe methods and conditions
of transport of imported animals. Descriptions should include
means of transport and husbandry during transport, including
isolation, caging, handling, animal treatment, and presence of other
animals of the same or different species. If animals from countries
outside the U.S. are needed, you should use them as founders for a
domestic herd that will be well-characterized for an extended
period of time before use, using procedures sufficient to validate
the herd’s acceptability as source animals.
c. 	You should not import source animals from any country or
geographic region where TSEs are known to be present in the
source species. For these purposes, only countries that are listed by
the World Organisation for Animal Health (OIE) as having
“negligible” risk of BSE in cattle should be considered
acceptable.[13] Use of the OIE BSE risk status for source animal
importation is consistent with current USDA regulations
(9 CFR Parts 92-96 and 98) with respect to importation of bovines
and bovine products. [14]
d. 	You should consult the USDA and, when appropriate, CVM and
CDC for their requirements regarding importation of animals or
animal tissues.

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4. 	Range and Wild Animals
You should not use source animals that are raised under free-ranging conditions.
Such animals have a higher likelihood of harboring infectious agents, due to
potential exposure of the source animal to other animals, birds, insects, or other
uncontrolled environmental factors. You should not use wild-caught animals as
source animals.
5. 	Animal Sources Obtained from Slaughterhouses or Abattoirs
Animals from slaughterhouses or abattoirs are unsafe for use as source animals.
Appropriate documentation and histories of animals from slaughterhouses may
not be available because the animals are often obtained from geographically
divergent farms or markets, and exposure to other animals or potential sources of
infectious agents during transit or after arrival at the slaughterhouse are unknown.
Therefore, you should not use such animals as source animals.
6. 	Semen Donors
You should apply the same source animal considerations to semen donors,
whether or not they are members of the herd, including, for example, screening
for infectious agents that may be transmitted by semen.
D. 	Animal Health and Husbandry
Production of animals as sources of live cells, tissues, or organs for use in
xenotransplantation products involves an adequately designed facility and a program for
the operation of the facility to minimize the animals’ exposure to infectious agents.
You should obtain source animals exclusively from SAFs. Your application or
submission to FDA should include detailed plans for maintaining source animals. These
plans should include SOPs, detailing the containment and housing of animals, feeding
and obtaining feed, water and bedding, performance and monitoring of the health
screenings, removal from production and disposal of the animals and their byproducts,
and identifying individual animals and recording their movements to, through, and out of
the facility. These procedures should take into consideration the source animal species
and xenotransplantation product(s) as appropriate.
1. 	Facilities
You should house the animals in facilities built and operated in accordance with
recommendations described in the National Research Council’s Institute for
Laboratory Animal Research, Guide for the Care and Use of Laboratory Animals
and accredited by the AAALAC. You should not locate SAFs in geographic
proximity to manufacturing or agricultural activities that could compromise the
facility’s biosecurity by providing or enabling a source of infections.

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SAFs are subject to the regulations in 21 CFR Part 600, Subpart B, on
establishment standards, including the requirements regarding animals and
personnel in 21 CFR 600.10 and 600.11. SAFs also are subject to the regulations
in 21 CFR Part 600, Subpart C, regarding inspections. These facilities are subject
to inspection by designated representatives of the clinical protocol sponsor and
public health agencies.
You should include a detailed description of the facilities and procedures for
housing source animals with the FDA submission (e.g., IND or Master File). The
information provided should include plans for the shelters, the feeding areas, the
washing areas, the fencing, air handling systems (particularly in quarantine areas),
lighting, temperature, and other physical attributes of the animal environment.
Facility descriptions should also include information on physical barriers and
operational measures intended to eliminate or minimize exposure to insects, birds,
or other animals that may transmit disease to the source animals. You should
keep records of any biological or physical compromise of the animal environment
as well as measures taken in response to this problem.
These descriptions should also cover the procedures and schedules followed for
cleaning and other routine maintenance of the animal enclosure. You should
include procedures for elimination of animal wastes. Include in the description
how qualified source animals will be housed (for example, as a batch or as
individuals) and the methods used to decontaminate the housing after the source
animals are used. The SAF staff should include veterinarians with expertise in the
infectious diseases and agents prevalent in the particular animal species being
raised in the facility. If a veterinarian with expertise in infectious diseases is not
on staff, you should provide documentation in the investigational application
(e.g., IND) that an individual with the appropriate expertise is available for
consultation. Staff should also include adequate numbers of caretaker personnel
with appropriate training in the care and health of the species being housed (e.g.,
21 CFR 600.10 and 600.11). Developers of xenotransplantation products should
consider becoming ISO 9001:2015[15] certified.
2. 	Maintenance of Source Animals
a. 	General
You should maintain source animals in accordance with standard
operating procedures appropriate to the species, xenotransplantation
product, and the intended clinical application. SOPs should provide for
admission of new animals to the SAF and source animal pool, for
quarantine, and for removal, isolation, or elimination of diseased animals.
You should provide this information to FDA in the application for
investigational use (e.g., IND). You should not reintroduce animals that
have been removed from the source animal pool due to illness or infection.

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You should develop procedures to identify incidents that negatively affect
the health of the herd or colony. This information is relevant to the safety
review of every xenotransplantation product application. You should
report such information to FDA as well as procedures to collect the
information as part of the application for investigational use (e.g., IND).
b. 	Health Screening
i. 	You should maintain source animals in barrier facilities that
are considered free of designated pathogens. For the
purpose of this document, such facilities are termed
“Designated Pathogen Free” (DPF), and animals derived
from them are termed “DPF animals.” Initial screening and
routine monitoring are important to validate that such
facilities maintain DPF status. Protocols for monitoring the
herd for disease and infectious agents should exist, and you
should include a copy or a summary of the SOPs in the
FDA submission requesting investigational use (e.g., IND).
You may modify the frequency of testing as the reliability
of the production system is established using data from
earlier screens. You should consult appropriate experts,
such as infectious disease consultants, virologists,
microbiologists, accredited microbiological laboratories,
and veterinarians, to generate a list of agents for which all
source animals should be screened and a list of appropriate
diagnostic tests. In addition to screening for specific
infectious agents, you should use more general assays for
detection of classes of agents. For example, you should
examine feces from source animal herds on a regular basis
for evidence of parasitic infections. If infectious agents,
including normal flora that could potentially be infectious
in an immunosuppressed recipient, have been identified in
source animals, the use of such animals should be avoided.
You should consult with CBER if the use of such animals
is contemplated (see, for example, section VII.C.4.d.).
Techniques for introducing new animals, such as artificial
insemination, caesarean section, cloning, or novel
gnotobiotic techniques, should be fully described.
ii. 	Subclinical infections of source animals may not be
apparent at the time of harvest of the nonhuman live cells,
tissues, or organs and may be identified only
retrospectively. Sampling of individual animals from the
herd of origin for screening and the use of sentinel animals
should help minimize this problem and may help identify

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infections in recipients, post transplantation. You should
consider establishing a sentinel animal program that
includes periodic necropsy and extensive histology and
pathology evaluations. The screening procedures should be
appropriate for the animal species, the xenotransplantation
product, and the clinical application. Specific screening
procedures should include appropriate physical
examination and laboratory tests, and should be able to
detect zoonoses known to exist in the species or
geographical regions in which the source animals originate
and are maintained. For example, a sentinel animal
program for ruminants might specify that the program
maintain some animals into old age and test all diseased
animals eliminated from the herd as well as all sentinels at
necropsy for evidence of BSE using USDA-approved
postmortem diagnostic tests for BSE with brainstem tissue
collected as specified by USDA. [16]
iii. 	You should quarantine and screen individual source
animals before harvest of cells, tissues, or organs, as
discussed in section V.D.4.b., below, and in the PHS
Guideline[1] .
c. 	Health Care
The herd health surveillance system should include comprehensive
documentation of all veterinary care received by source animals. This
includes documentation of all illnesses, medical care, procedures, drugs
administered, vaccinations, routine physical exams, and any treatments
received by each animal. You should carefully document use of
antimicrobial agents due to potential risk to allergic recipients receiving
unprocessed nonhuman animal live cells, tissues or organs. You should
validate residual drug levels to be insignificant in cells, tissues, or organs
taken from source animals that previously have received medications.
Exclusive use of killed vaccines generally is warranted both in the source
animal and in the herd with which it is associated. You should use live
vaccinations only when alternative immunogens for vaccinations are not
available and only if scientific evidence exists to support that the live
cells, tissues, or organs from the vaccine-treated animal will not pose a
risk of infection for the human recipient. You should describe the
procedures to deal with illnesses or other incidents that affect the health of
the herd in your application to FDA for investigational use. You should
not use as source animals those animals requiring treatment with blood,
blood products, or tissues obtained from animals outside the closed herd,
and you should remove these animals from the herd. You should use
aseptic techniques and sterile equipment for all parenteral interventions,

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including vaccinations, treatment with drugs or biologics, phlebotomy,
and biopsies. If animals within the closed herd have been treated with a
biological product (e.g., vaccine, monoclonal antibody), you should
document the treatment in the application to FDA requesting
investigational use (e.g., IND), and you should provide copies of package
inserts or labeling. You should document and maintain records showing
the treatment of animals with drugs for any reason, including the
withdrawal period following drug treatment. You should develop
procedures for disposal of dead animals (see section V.G.).
d. 	Feed
You should describe the storage and delivery of feed, water, and other
consumables in the application to FDA for investigational use (e.g., IND).
Records should include manufacturer, batch numbers, and other pertinent
information, and you should describe recordkeeping procedures in an
SOP. You should record in the individual source animal’s records the
vendor and contents of feed given to a source animal for at least two
generations before use as a source for live cells, tissues, or organs used in
xenotransplantation. You should not use feeds containing animal proteins
or other cattle materials that are prohibited by the FDA feed ban as
expanded in 2008 as source animals (21 CFR 589.2000).[17] You should
not use feeds containing significant drug contamination or pesticide or
herbicide residues for source animals (21 CFR 589.2001). [9] You should
not use natural, non-sterile foods, such as hay, to minimize potential risks
of exposure to pests or infectious agents. Water should be of sufficient
quality to prevent unnecessary exposure of animals to infectious or
adventitious agents, drugs, pesticides, herbicides, and fertilizers. You may
include pasteurized milk products in feeds. You should feed newborn
animals colostrum or milk from dams only if the dams have been
specifically qualified by the same procedures used for herd qualification.
e. 	Caretakers
You should provide SOPs for animal caretakers in the FDA submission
requesting investigational use (e.g., IND), and you should include entry
and exit procedures, clothing requirements, and all interactions with the
animals, e.g., feeding, watering, exercising, delivery of immunizations and
medications, etc. (e.g., 21 CFR 600.11). There should be a documented
training program for personnel as described in the cGMP regulations
(21 CFR 211.25).
You should monitor the health of humans in contact with animals on a
routine basis.[18] You should predetermine and customize the program for
screening and monitoring of caretaker and other staff to maximize
screening information, and you should describe the program in an SOP.

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Health monitoring of humans who come into contact with the animals
should include physical exams with baseline and, if indicated, either
periodic sampling and storage of serum or plasma for individuals having
frequent and close contact with source animals or less rigorous monitoring
for those with occasional contact. You should obtain baseline samples
from all caretakers.
3. 	Animal and Personnel Traffic through the Source Animal Facility
You should develop SOPs for entry and exit of animals, and they should include
transportation of animals to and from the facility. You should subject all animals
entering the facility to a defined quarantine period, allowing for completion of
any screening procedures. The minimum quarantine period for animals used in
manufacture is seven days (21 CFR 600.11(f)(2)). However, you should use
longer quarantine periods that extend beyond the incubation period for infectious
agents in the source animal species for animals entering a SAF. You should
devise a tracking system that allows unique identification of each individual
animal in the facility. You should minimize entry and exit of animals and human
staff to avoid exposures to transmissible infectious agents. We encourage the use
of an ‘all in/all out’ or batch approach for moving qualified source animals as a
method of minimizing the potential for infectious agent transmission.
You should describe personnel traffic patterns in the FDA submission requesting
investigational use (e.g., IND) and should minimize transmission of infectious
agents. Caretakers should not work in more than one animal facility or with more
than one species of animal. Caretakers should not work with more than one
isolated group of animals or more than one herd within any given day unless
validated SOPs for caretaker decontamination and disinfection are used.
4. 	Individual Source Animal Qualification
a. 	Testing for infectious agents
You should screen all individual source animals for presence of the same
infectious agents used for herd qualification. In addition, you should
perform further laboratory tests for infectious agents as described in
section V. for testing of the xenotransplantation product (e.g., viral co-
cultivation assays) on appropriate samples of source animal blood or
tissue. When fetal or neonatal animals will be used as source animals, you
should conduct testing of the mothers, which may supplant testing of the
fetus or neonate if technical and temporal difficulties render such testing
unfeasible.
When feasible, you should examine a biopsy of the live animal cells,
tissue, or organ or other relevant tissue (e.g., contra-lateral kidney as a
surrogate for a kidney graft or thoracic tissue as a surrogate for a heart) by

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histopathology and tested for infectious agents by appropriate assays. You
should take care in performing biopsies to use fastidious sterile technique
so as not to introduce new microbial contaminants. You should archive
remaining biopsy tissue as described in section V.E.3.
You should perform all tests at a time as close as possible to the date of
harvest of the live cells, tissues, or organs but which allows the results to
be obtained before their use. If more than 3 months have elapsed since the
initial testing or biopsy of the source animal, you should repeat tests
before harvest.
You should consider the nature, timing, and results of surveillance of the
herd from which the individual animal is procured in designing
appropriate screening of individual animals.
b. 	Quarantine
You should generally quarantine individual source animals for a minimum
of three weeks before harvest of their live cells, tissues, or organs. It may
be appropriate to modify individual quarantine periods, depending on the
characterization and surveillance of the source animal herd, the design of
the facility, and the clinical indication. If the quarantine is shorter, you
should provide justification in the application to FDA for investigational
use (e.g., IND). 	During the quarantine period, in addition to tests for
infectious agents, source animals should undergo physical examination by
a veterinarian, including complete blood count, peripheral blood smear,
and fecal exam for parasites.
E. 	Harvest of Nonhuman Live Cells, Tissues, or Organs for Use in Producing
Xenotransplantation Products
1. 	Harvest and Documentation
You should describe the procedures and physical facilities used for harvesting of
live cells, tissues, or organs from source animals in detail in the application to
FDA requesting investigational use (e.g., IND). Qualified and controlled
procedures for avoiding the introduction of infectious agents during harvesting
should be in place. Qualification of the procurement and screening procedures
should include documented performance of the processes, with documented
results supporting successful harvest of live cells, tissues, or organs from source
animals that meet lot release criteria including identity, potency (or activity), and
safety (e.g., microbiological sterility). Source animal anesthetic agents should not
be harmful to the human recipient. A summary of the health records (e.g., health
status and microbiological screening reports, results of lot release tests, and

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anesthetic used, if relevant) regarding the source animal should accompany the
xenotransplantation product, and you should incorporate them into the recipient’s
records.
SOPs should permit rapid, accurate, and facile tracking of tissue from an
individual source animal to the recipient.
2. 	Transportation
Transportation of source animals may expose them to risks not encountered in
closed herds and should be avoided if possible. Therefore, we recommend that,
when feasible, and particularly in cases where source animal tissues or cells are
going to be processed further prior to use, live cells, tissues, or organs should be
procured at the animal facility prior to shipping. In some cases, particularly when
the xenotransplantation product is a whole organ intended for immediate
transplantation, it may be necessary to ship live animals. In those cases where
transportation is necessary, you should maintain barriers, equivalent to or better
than those in place at the SAF during transit, to ensure that source animal
contamination does not occur en route. Transportation should occur in dedicated
vehicles in which source animals are not exposed to any other animals, and you
should document the method in the submission to FDA (e.g., in the IND). If there
is any question regarding the effectiveness of the transportation and containment
procedures, you should quarantine and rescreen animals in a fashion comparable
to that used for entry of new animals into a closed herd.
You should develop and implement procedures for avoiding shipping errors,
avoiding contamination, and documenting transfer of animal materials to the
correct patient. You should describe in detail the method of transporting the live
animal cells, tissues, or organ from harvest site to the clinical xenotransplantation
site in the application to FDA requesting investigational use (e.g., IND).
3. 	Source Animal Sample Archive
a. 	Timing of Sample Acquisition
If the source animal is sacrificed at the time its live cells, tissues, or organs
are harvested, you should conduct a full necropsy, including gross,
histopathological, and microbiological evaluation, and you should obtain
archival samples, including portions of the product for storage as
described in section V.E.3.b.
If the source animal is not sacrificed at the time its cells, tissues, or organs
are harvested, you should archive portions of the harvested material and
plasma and leukocytes from the source animal, and you should monitor
the health of the source animal for life.

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When source animals die or are euthanatized, you should perform a full
necropsy, and you should obtain archival samples for storage as described
in section V.E.3.b.
b. 	Samples to be Archived and Storage Conditions
You should store archived samples of source animal tissues and body
fluids at -70 degrees Celsius or lower temperatures, as appropriate for
preserving the sample, or you should maintain fixed samples at room
temperature. Section 3.7.1 of the PHS Guideline[1] recommends that you
cryopreserve at least ten 0.5 cc aliquots of citrated- or EDTA-
anticoagulated plasma and at least five aliquots of viable leukocytes
(1x107
/aliquot, for subsequent isolation of nucleic acids and proteins or for
use as a source of viable cells for co-culture or other tissue culture assays).
You should select the conditions of cryopreservation and storage for
viable samples to maintain cell viability for the period of storage (see
section V.E.3.c.). You should collect appropriate tissue samples for
formalin fixation and paraffin-embedding and for cryopreservation from
source animals at the time the live cells, tissues, or organs are procured.
You should collect and cryopreserve tissue samples representative of
major organ systems of source animals (e.g., spleen, liver, bone marrow,
central nervous system, lung,) at necropsy. As appropriate to the
xenotransplantation product, you should archive other body fluids, such as
cerebrospinal fluid, at the time of procurement of the product and/or
necropsy. If sentinel animals are used, you should also archive tissue
samples and body fluids obtained at necropsy.
c. 	Archive Rationale, Duration, and Responsibility
The PHS Guideline[1] recommends that a sufficient quantity of materials
be harvested and cryopreserved for three different uses:
i. 	dedicated sample(s) for use by the PHS,
ii. 	for use if needed for recipient diagnosis and care, and
iii. 	for use by the sponsor, as appropriate.
You should include detailed plans for obtaining and storing the archive
samples in the application to FDA requesting investigational use (e.g.,
IND). The PHS Guideline[1] recommends that samples should be stored
for 50 years from the time of sample acquisition. You should clearly
designate responsibility for the archives and access to the specimens.

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4. 	Herd Records
You should keep records pertaining to the source animals and facilities. These
records are subject to inspection, and you should maintain the records for 50 years
beyond the date of procurement of the nonhuman animal live cells, tissues, or
organs for use in xenotransplantation.
5. 	Disposition of Records on Closing of a Source Animal Facility
If a SAF ceases operation, all records and archived samples should be transferred
to the respective sponsors, or the sponsors should be notified of a new archive
site. You should make provisions for all records to be maintained for the
requested period in the event that the establishment ceases operation. If you are a
sponsor who plans to cease doing business, you should consult FDA regarding the
disposition of records and archive samples.
F. 	Source Animal History for Xenogeneic Cell Lines
Cell lines from animals may be established and used in the production of
xenotransplantation products. You should include the history of the cell line in the
application to FDA requesting investigational use. Especially for long-term cultures, it
need not always include all the detailed information about the source animal and source
animal facility mentioned above. However, it should include, at a minimum, the species
and tissue of derivation. You should include, whenever possible, information such as age
and sex of the source animal, laboratory of derivation, date of derivation, and the
immediate provider of the cell line. For short-term cultures, (e.g., derived less than one
year previously), you should also include a description of the husbandry and health status
of the particular source animal and herd or colony used to derive the cells. The history of
the cell line should also include the above information on feeder cells or animals used for
passage in vivo if such techniques were used to develop the cell line. Information
regarding the commercial source and country of origin of any animal products used in
deriving or maintaining the cell line should also be included in the history. You should
characterize and test the final product as described in section VII. You may also consult
the “Points to Consider in the Characterization of Cell Lines Used to Produce Biological
Products”[19] for pertinent recommendations regarding the production, identification, and
characterization of cell lines used in manufacture. Additional information may be found
in relevant ICH Guideline.[20]
FDA has considered how this guidance relates to human embryonic stem (hES) cell lines
that were in existence prior to August 9, 2001. These hES cell lines had used murine
feeder layer cells and, thus, fit the definition of xenotransplantation used in this guidance
and the PHS Guideline. FDA does not intend that the Agency’s regulation of
xenotransplantation will preclude the use of these hES cell lines. It may be necessary for
a sponsor who wishes to investigate a stem cell product derived from existing hES cell
lines in a clinical trial to demonstrate to FDA that the hES cell line is free from infectious
agents, including murine infectious agents. Given current technology, a sponsor should

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be able to do this without undue burden. For example, it will not be necessary for a
sponsor to provide FDA with complete animal husbandry information for mice from
which the mouse feeder layers were derived. If continued expansion on murine feeder
cell layers is planned, FDA may request that a sponsor demonstrate that the mouse
colonies, from which such murine feeder layers are to be obtained, are maintained
appropriately to ensure safety. (See section V. of this guidance and section 3 of the PHS
Guideline[1] ). The same recommendations apply to other xenotransplantation products
that contain human cells with a history of co-culture with nonhuman animal cells.
G. 	Disposal of Animals and Use of Byproducts
There is a need for advance planning for the ultimate disposition of source animals,
including those animals in which the insertion of genetic information failed (“no-takes”)
and sentinel animals bred for use in producing xenotransplantation products, especially
animals of species ordinarily used to produce food. Food or feed derived from such
animals may be adulterated under the Act. Generally, you should not use such animals as
sources of human food via milk or meat or as ingredients of feed for other animals. You
should not use such animals as pets or breeding animals because of the potential for them
to enter the food/feed chain, either directly or through the rendering process. You should
dispose of source animals in a manner consistent with the disposal of medical waste in
compliance with federal, state, and local requirements.
There may be infrequent situations where animals from xenotransplantation facilities can
be considered safe for human food use or as feed ingredients when disposed of through
rendering. Persons wishing to offer animals into the human food or animal feed supply
or who have food safety questions should first consult with FDA’s Center for Veterinary
Medicine. CBER will refer food safety issues from sponsors to CVM, or you may
contact CVM directly through the Division of Compliance, HFV-235, FDA, Center for
Veterinary Medicine, 7500 Standish Place, Rockville, MD 20855, 240-276-9227.
VI. 	CHARACTERIZATION OF XENOTRANSPLANTATION PRODUCTS
A. 	General Considerations
In general, you should test the xenotransplantation product for safety, identity, purity, and
potency. Part 610 (21 CFR Part 610) describes types of assays that are required for
licensed biologics. You should use similar tests during investigational stages of product
development. We discuss in more detail assays for safety testing, including infectious
agent tests and tests for endotoxin, in section VII. of this document. Assays for testing
identity and potency will depend on the product, itself. Assays for purity should include
tests for endotoxin or pyrogen and, for certain xenotransplantation products, should
include measurements of cell populations in the xenotransplantation product. For further
guidance in this section, see references.[20-22]

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Additional recommendations and comments regarding microbiologic tests are found in
section VII. of this document.
B. 	Considerations for Classes of Xenotransplantation Products
1. 	Xenotransplantation Products Used Immediately after Procurement from
the Source Animal
When xenotransplantation products are transplanted directly after removal from a
source animal, it may not be possible to perform all tests on the final product and
have the results available before use. However, you should use a biopsy of the
organ or a relevant surrogate sample (e.g., adjacent tissues or contra-lateral
organs) for assay of the xenotransplantation product. Safety analyses should
include fungal and bacterial sterility, mycoplasma, and virus testing. You should
also perform tests for endotoxin or pyrogen. Although we realize that results of
these tests will not be available before transplantation, you should still complete
assay or culture periods and record the results. As appropriate, you may use
histology, performed on a retention sample or biopsy of the xenotransplantation
product, to document identity of the product.
2. 	Stored or Processed Xenotransplantation Products
For live xenogeneic cells or tissues that are stored, processed, or expanded ex
vivo, in addition to safety testing, you should perform additional product
characterization to measure identity, purity, and potency. As much as possible,
results of these assays should be available before xenotransplantation and used for
lot release. You should also apply these same product characterization steps to
xenotransplantation products comprised of human cells that have had ex vivo
contact, for example, by co-culture, with cells or tissues of nonhuman origin.
a. 	Safety
We discuss tests for bacterial and fungal sterility, mycoplasma, and viral
agents, generally considered safety tests, in detail in section VII. of this
document.
b. 	Identity
You should develop a means to assess identity of the active component of
the xenotransplantation product. This may include identification of
relevant cell or tissue types, using immunological, immunohistological, or
biochemical cell markers. In some cases, it may be possible to use
histological evaluation. Depending on the manufacturing process,
verification of the species or strain identity of the final product may be
necessary, such as when the SAF handles more than one strain or species
of animal.

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c. 	Purity
If the final product is a heterogeneous xenotransplantation product, i.e., a
tissue, possessing several types of cells, or a cellular implant, containing
extraneous tissues or cells which may be incompletely removed during
tissue dissection, cell processing, or ex vivo culture, the determination of
the purity of the cell population is especially important. You should
develop a quantitative method to assess the presence of the putatively
active cell type as well as contaminating cell types in the final product.
You may achieve this, for example, using morphologic, histologic,
molecular genetics, biochemical and/or immunocytochemical techniques
to identify contaminating cells and/or their products. For
xenotransplantation products comprised of human cells that have had ex
vivo contact with cells or tissues of nonhuman origin, you should perform
quantitative assays to assess the presence of nonhuman cells in the final
product. You should validate purity assays. Purity assays are important
for production of a consistent product. You should use results of such
tests as a lot release specification if possible. In those cases where the
final product is a purified population of cells of a single or few types, such
as an established cell line, you should still test the product for purity, and
you should develop tests for identity of the cells.
You should measure endotoxin levels on the final product, and results
should be available for use as a lot release specification. We also discuss
tests for endotoxin in the context of tests for infectious agents (section
VII.C.3.).
d. 	Potency
You should perform potency assays that measure and reflect the intended
biological activity of the final product. For example, potency assays may
measure biologically active molecules secreted/produced by the
xenotransplantation product, such as cytokines, hormones, or
neurotransmitters. If necessary, development of appropriate potency
assays may proceed along with product development. In addition, you
should assay cell viability and use the results for lot release. [5]

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VII. 	MICROBIOLOGICAL TESTING OF XENOTRANSPLANTATION PRODUCTS
A. 	General Considerations
1. 	Framework
This section of the guidance document provides a general framework for the
microbiological testing of xenotransplantation products. We suggest some
specific examples of tests and infectious agents. However, we encourage
sponsors to consider all available up-to-date information, regarding potential
pathogens and testing strategies, to evaluate their own systems, to perform
experiments to identify potentially infectious agents, and to propose and validate
appropriate tests in consultation with CBER. During the initial stages of
investigations, complete validation for all assays may not be necessary, with the
exception of standard sterility tests. However, you should establish the
specificity, sensitivity, and reproducibility for all procedures used to detect
infectious agents to the extent possible.
2. 	General Biological Products Standards
For general standards on testing of biologics for infectious agents, refer to
21 CFR Part 610 (see references).[3, 10, 20-22]
You can also find additional guidance on these issues as they relate to
xenotransplantation in section 3.3 of the PHS Guideline.[1]
3. 	Inactivation or Removal of Infectious Agents
Whenever possible, without compromising the integrity and effectiveness of the
xenotransplantation product, we encourage you to develop and incorporate
validated procedures for inactivation or removal of adventitious agents, infectious
agents, or other microbiological contaminants into the manufacture of the
xenotransplantation product.
4. 	Archiving
You should cryopreserve and archive for further testing, as needed, samples of all
final xenotransplantation products (i.e., cells or tissues or biopsies of organs),
whether fresh or from culture ex vivo. In some cases, for example if the
xenotransplantation product is a whole intact organ, it may be acceptable to
archive a relevant surrogate sample (e.g., adjacent tissues or contra-lateral organ).
If the final product consists of human cells, you should archive tissues or organs
that have been in contact ex vivo with live nonhuman animal cells, tissues, or
organs and samples of the final product and the nonhuman animal cells, tissues, or

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organs. As in the case of the animal source samples (see section V.E.3.c.), you
should harvest and cryopreserve sufficient quantities and numbers of replicates of
the xenotransplantation product for three different uses:
a. 	dedicated sample(s) for use only by PHS [1] ;
b. 	for use if needed for recipient diagnosis and care; and
c. 	for use by the sponsor, as appropriate.
You should include detailed plans for obtaining and storing archive samples in the
application to FDA requesting investigational use (e.g., IND). You should store
samples for 50 years from the time of manufacture of the xenotransplantation
product. You should clearly describe responsibility for the archives and access to
the specimens.
If you are the sponsor, you should provide for all samples and attendant records to
be maintained for the requested period of time in the event that an establishment
ceases operation.
B. 	Considerations for Classes of Xenotransplantation Products
1. 	Xenotransplantation Products Used Immediately after Procurement from
the Source Animal
In procedures in which the xenotransplantation product is transplanted
immediately after removal from the source animal, such as xenotransplantation of
whole organs, results of testing of the xenotransplantation product may not be
available before its clinical use. In such cases, testing of the source animal, itself,
may be all the testing that is possible before the procedure. Testing of samples
taken from such xenotransplantation products or appropriate relevant biological
surrogates, e.g., adjacent tissues or contra-lateral organs, is also warranted, even
though the results will not be available before use of the xenotransplantation
product because results may contribute to patient management. (See also section
VI.B.1.)
2. 	Stored or Processed Xenotransplantation Products
For xenogeneic cells or tissues that are stored, processed, or expanded ex vivo,
you should accomplish or, at a minimum, initiate testing for infectious agents
before xenotransplantation. If cells or tissues are maintained in culture, you
should validate cell culture procedures and reagents for maintenance of microbial
sterility, including both xenogeneic infectious agents and other cell culture
adventitious agents. You should perform testing periodically during the culture
period. Performance of all tests at every time point may not be necessary, but you

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should provide FDA, as part of the application for investigational use (e.g., IND),
with a scientific rationale to support the selection of tests performed at each given
time. As an example, you may test samples:
a. 	at the initiation of culture ex vivo;
b. 	before cryopreservation if performed as a step in manufacture;
c. 	as late as possible during culture, such that final results (or useful
preliminary results) will be available before the release and use of
the product;
d. 	two to three days before clinical use (e.g., for microbiological
cultures used in lot release); and
e. 	at the time of final product harvest, though results may not be
available before clinical use.
3. 	Xenotransplantation Product/Device Combination Products
In certain biologic/device combination products, physical barriers may separate
the xenogeneic component from human fluids or tissues and might prevent or
reduce transmission of certain classes of infectious agents. If such claims are to
be made or are implicit, or if the existence of the physical barrier is to be used in
lieu of certain other precautions to lower the risk of transmission, you should
provide to FDA, as part of the application for investigational use (e.g., IND), the
results of validation studies that demonstrate the inhibition of transmission of
specific infectious agents and the maintenance of device/barrier integrity. For
specific guidance on the design of these types of studies, see reference. [23] For
example, if such claims are to be made in the patient informed consent document,
you should provide the results of these studies in the application to FDA
requesting investigational use; if such claims are to be made during marketing,
you should provide the data in the marketing application. The design of these
studies should take into consideration the following parameters:
a. 	Conditions of normal physiologic use of the xenotransplantation
product/device combination product and conditions under which
the combination is subjected to physical and biological stress;
b. 	Use of infectious agents that are representative of those potentially
present in the xenotransplantation product. Note that the size and
plasticity of selected agents should be considered; and
c. 	Use of agents that would demonstrate the physical properties of the
barrier (i.e., permeability to viruses or other particles with differing
properties, such as size, charge, hydrophobicity, shape, etc.).

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Without supporting data obtained from such studies, you should not assume that
xenotransplantation products contained within a barrier present lesser risk of
infection to humans than xenotransplantation products not contained within a
barrier.
C. 	Assay Design for the Detection of Infectious Agents
1. 	General
The choice of tests will vary, depending on the animal source, including the
species, strain, and geographic origin, the type of tissue, the processing of tissue
before use, and the proposed use or clinical indication. You should give special
consideration to infectious agents known to infect the source animal and those
known to cause zoonoses. You should base the list of infectious agents for which
you will test on that used for individual source animal qualification. We
encourage discussions with CBER. You should include data in the FDA
application for investigational use to document the specificity, sensitivity, and
reproducibility of novel assays used to detect infectious agent(s).
2. 	Tests for Bacteria, Fungi, and Mycoplasma
You can find standards concerning the types of methods used for detection of
bacteria, fungi, and mycoplasma in licensed biologics in 21 CFR Part 610. You
may use alternative methods during product development, but you should support
use of such methods by data on the sensitivity, specificity, and reproducibility of
the method. For xenotransplantation products, you should obtain such data using
infectious agents appropriate to the source animal species, geographic origin of
the source animal, and the cells, tissues, or organ(s) to be used. You should
submit these data to FDA in the application for investigational use.
In addition to testing the final product for viable organisms, you should perform
Gram stains on appropriate samples of all final xenotransplantation products. The
results of these stains should be available before use of the product in humans,
and you should set a negative Gram stain as a lot release criterion.
3. 	Endotoxin Test
During the product development phase, you may perform a bacterial endotoxin
test in lieu of the rabbit pyrogen test as described for licensed products
(21 CFR 610.13(b)). You should describe the type of endotoxin assay and its
specificity and sensitivity in the application for investigational use submitted to
FDA. If you intend to use an endotoxin assay in lieu of the rabbit pyrogen test
after licensure, you should demonstrate equivalency with the pyrogen test for the
specific xenotransplantation product at or before the time of license application
(21 CFR 610.9).

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Since it is possible to perform an endotoxin assay within a few hours, you should
select and perform an appropriate assay on the final product. Results should be
available before use for any xenotransplantation product that has been cultured,
stored, or processed for more than the few hours required to perform the assay.
You should use these results as a lot release specification. Consult the USP <85>
and the FDA “Guidance for Industry: Pyrogen and Endotoxins Testing:
Questions and Answers”[6] for information on bacterial endotoxins testing.
4. 	Viruses
a. 	Culture Assays
You should test xenogeneic cells used for xenotransplantation (fresh or
cultured) by co-culture with a panel of appropriate indicator cells to
amplify potential viral contaminants. The panel of cells used in this
analysis should include a cell line representative of the source animal
species, a cell line representative of the animal tissue(s) type used in the
manufacture of the xenotransplantation product, and a human cell line.
For additional guidance, see the “Points to Consider in the
Characterization of Cell Lines Used to Produce Biological Products.”[19]
This reference provides especially useful information for testing rodent
xenotransplantation products for viral adventitious agents. You may find
additional information in “Viral Safety Evaluation of Biotechnology
Products Derived From Cell Lines of Human or Animal Origin
Q5A(R1).”[23] When possible, you should also co-cultivate manipulated
and/or unmanipulated source animal cells with recipient cells, such as
peripheral blood cells. You should routinely observe co-cultivation
cultures for CPE, focus formation, RT activity, and changes in cell growth
or other unexpected changes. We recommend visualization of co-cultures
by EM to identify morphologic changes or to recognize certain viruses.
You should try to identify any viruses detected using immunoassay, PCR,
or other assays using virus-specific probes. At the end of the culture
period, you should test cultures for hemagglutination and hemadsorption
with erythrocytes of three different species [19] . Additional efforts may be
necessary to characterize viruses that are detected that may be novel or for
which specific probes may not yet be available.
You should base lot release specifications on available data. You should
use these specifications for release of xenotransplantation products for
which results can be available before administration of the product to
humans, such as for products that can be cryopreserved. For cells that are
manipulated ex vivo, if time allows, you should perform viral tests during
the period of culture or manipulation so that the results are available
before delivery of the product to the recipient. If it is not possible to
obtain the results prior to use, you should still test samples of each product

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lot. In these instances, you should qualify assay procedures and obtain
data on a number of representative final product lots before beginning
human trials.
b. 	Activation of Latent Viruses
You should give special consideration to testing for viruses known to
occur in the latent state. Transmission of viruses with long clinical
latencies is of concern, due to the possibility of transmission of these
viruses from the recipient to the recipient’s contacts in the absence of
symptoms or signs of disease. Immunosuppression and transplantation,
either alone or in combination, may activate latent viruses. [24]
Manipulation or culture of cells ex vivo may also activate latent viruses
(see references).[25, 26] Which experiments might be appropriate to detect
latent viruses in animal cells, tissues, or organs would depend upon the
tissue type and the virus in question. Examples of experiments that have
been used to detect viral activation and may be useful in the
xenotransplantation product setting include the following:
i. 	the expression of endogenous retroviruses is induced by
culturing in vitro; or
ii. 	by treatment with iododeoxyuridine or demethylating
agents, such as 5-aza-cytidine[27] ; and
iii. 	cultivation in vitro of ganglia latently infected with Herpes
simplex virus results in the production of infectious
virus [28] .
In certain cases, positive results may not necessarily preclude use of such
tissue (see section VII.C.4.d. for information regarding
xenotransplantation products containing porcine endogenous retrovirus(es)
(PERV)), but the identification and characterization of the resulting virus
may provide useful information and materials for monitoring the recipient
of the xenotransplantation product for the presence of the activated virus
(see section X.F.3.).
You should attempt to evaluate the potential of the processing or clinical
use of the xenotransplantation product to activate latent viruses (e.g.,
PERV) before use.
c. 	In Vivo Assays for the Detection of Viruses
You should test xenotransplantation products by assay in vivo for
detection of certain viruses that may not be found by culture methods in
vitro. For example, many serotypes of Coxsackie A virus are only

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detected upon inoculation of newborn mice. [28] Therefore, we recommend
that if there are no reliable in vitro assays, appropriate in vivo assays
should be applied (see “Points to Consider in the Characterization of Cell
Lines Used to Produce Biological Products”[19] and “Viral Safety
Evaluation of Biotechnology Products Derived From Cell Lines of Human
or Animal Origin Q5A(R1).”[23] Certain in vivo assays, such as antibody
production assays, may be particularly useful for testing for rodent
viruses.[19, 23]
d. 	Assays Suitable for the Detection of Porcine Endogenous
Retrovirus(es) (PERV)
All live cells, tissues, or organs derived from pigs contain sequences for
PERV in their genome[28]
. It has been demonstrated in some primary
porcine cells these sequences are expressed, resulting in production of
infectious retroviruses. In light of data demonstrating that PERV can
infect human cell lines in vitro[19, 23] we recommend that all porcine-
derived xenotransplantation products be evaluated using appropriate
assays for the production of infectious retrovirus. You should assess
several lots of each xenotransplantation product. Each time major
manufacturing changes occur, such as use of a new source or herd or
method of product procurement, you should repeat testing for infectious
PERV.[29]
You should test xenotransplantation products (e.g., a fresh sample of the
xenotransplantation product or relevant surrogate tissue, e.g., adjacent
tissues or contra-lateral organs or the cultured xenogeneic cells) by co-
culture with appropriate indicator cells to amplify any infectious
retrovirus(es). Indicator cells that have been demonstrated to be
permissive for PERV replication include the human embryonic kidney cell
line 293 (American Type Culture Collection (ATCC CRL-1573)), mink
lung fibroblasts (ATCC CCL-64), certain feline cell lines (such as PG-4,
ATCC CRL-2032), and a swine testis (ST) cell line (ATCC CRL-1746).
You should choose one or more of these cell lines for initial analysis of the
porcine xenotransplantation product or appropriate relevant surrogate
tissue. After co-culture for a period of at least 30 days or 10 cell passages,
you should analyze the cells for the transfer of PERV from the porcine
cells to the indicator cell by either an optimized RT assay or use of PERV-
specific primers to amplify, by PCR, reverse-transcribed viral RNA or
cellular RNA. Evidence for virus production will not necessarily result in
the xenotransplantation product’s being considered unsuitable for clinical
use. Rather, you should pursue additional characterization of the virus in
consultation with Center for Biologics Evaluation and Research (CBER)
in order to ensure appropriate reagents are available for recipient follow-
up (section X.F.). Additional characterization may include analysis for the
cell substrate most sensitive to infection by the particular strain of PERV

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present in the xenotransplantation product and sequence analysis of the
infectious virus produced by the xenotransplantation product. These steps
will provide important information and development of diagnostic tools to
optimize the protocols for follow-up of recipients for evidence of infection
(section X.F.).
VIII. 	MANUFACTURING AND PROCESS-RELATED GMP CONSIDERATIONS FOR
HARVEST AND PROCESSING OF XENOTRANSPLANTATION PRODUCTS
A. 	General Considerations
You should use facilities for the harvest and/or processing of xenotransplantation
products that have been designed to minimize the potential for contamination of the
harvested and/or processed xenogeneic cells, tissue, or organs and cross-contamination
between lots of these cells, tissues, or organs.
For sponsors of investigational trials, you should phase in the validation activities
described in this section, during the investigational phase, as the clinical studies progress
toward submission of an application for approval to market (e.g., BLA). The exception
to this is sterility assurance validation, which you should complete before initiating
clinical trials. Manufacturing process controls should be in adherence to cGMP
regulations (21 CFR Parts 210 and 211). The IND regulations (21 CFR 312.23(a)(7))
allow that some controls may be introduced as appropriate for the phase of development.
B. 	Contamination/Cross-Contamination Precautions
You should take precautions to prevent contamination/cross-contamination during
harvest and manipulation of xenogeneic cells or tissues. You should give consideration
to:
• 	personnel, animal, material and waste flow into and out of the facility;
• 	proposed air cleanliness classifications;
• 	cleaning/sanitizing agents used and demonstration of their efficacy in relation to
facility isolates, viruses, and other potential adventitious agents; and
• 	environmental monitoring and gowning procedures.
1. 	Flows
Your facilities should be designed such that personnel, animal, material, product,
and waste flows into and out of the facility exclude mixing of “clean” and “dirty”
activities. Ideally, flows should be one way so that personnel, animals, materials,
and product enter and exit separately. Using this design, waste would only exit
through designated airlocks, pass-throughs, and/or autoclaves. Alternatively, you
may accomplish segregation of activities procedurally and/or temporally. In this
case, you should take special care to avoid contamination or cross-contamination.

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For example, more stringent cleaning and sanitization schedules should be in
place.
Of special concern is the transfer of animals to the harvesting area (i.e., operating
room). You should ensure that animals are prepared in such a way as to exclude
potential surface contaminants, which may be carried from the animal facility.
2. 	Cleaning and Sanitizing Agents
You should demonstrate that agents used for cleaning and sanitizing work
surfaces and equipment as well as other surfaces in the harvesting and processing
areas (e.g., floors, walls), are effective against facility isolates, viruses, and other
potential adventitious agents. You should establish cleaning schedules which
maintain acceptable control in relation to the activities performed in the specified
area. We expect that validation studies, demonstrating the efficacy of the agents
used, will be performed as the trial progresses toward submission of a marketing
application
3. 	Environmental Monitoring
You should establish a program for monitoring the environment in the harvesting
and processing areas, based on the criticality of the manufacturing process
involved.
You should perform nonviable particulate monitoring to verify air cleanliness
classifications in the harvesting and processing areas (see section VIII.C.1. for
recommended air cleanliness classifications). This verification should include
laminar flow areas in the harvesting area and biological safety cabinets in the
processing area. After initial verification, you should perform nonviable
particulate monitoring at established intervals to demonstrate maintenance of the
assigned air cleanliness classification. [30]
You may monitor viable particulates, i.e., microbes, using a variety of techniques.
The use of settling plates during harvesting and processing activities, while not
quantitative, provides some assurance that the quality of the environment has not
been compromised. You should establish quantitative methods as clinical trials
progress toward submission of a marketing application. You should monitor
surfaces, including those of personnel performing production activities (e.g.,
gloved hands), using contact plates or swabs to demonstrate the continued
efficacy of the cleaning regimen, and maintenance of asepsis for personnel. We
recommend that personnel engaged directly in harvesting and processing
activities be monitored at the conclusion of each critical activity (e.g., surgery,
aseptic surgery). Additionally, you may randomly sample operators performing
cell expansion activities.

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4. 	Changeover Procedures
You should establish, follow, and document changeover procedures designed to
prevent contamination between harvests of xenotransplantation products. These
procedures should include clearance of all materials and waste from the operating
room or cell processing cabinet and cleaning/sanitization of surfaces. In addition,
you should address segregation procedures if multiple lots of xenogeneic cells or
tissues are processed at the same time. Adequate labeling of processing vessels
(e.g., tissue culture flasks) and dedication of equipment or portions of equipment
(e.g., shelves within incubators) are examples of such segregation procedures.
Centrifuges used for processing are of particular concern in terms of cross-
contamination. We recommend that only one lot of xenogeneic cells or tissues be
centrifuged at a time. You should demonstrate integrity of centrifuge tubes or
closed systems employed, when possible. You should adequately clean
centrifuges between each lot operation.
C. 	Validation and Qualification
As noted previously, validation and qualification efforts should be ongoing as clinical
trials progress. Minimally, we expect assurance that systems and equipment are
functioning as needed. You should submit validation protocols and data summaries to
FDA for review as part of the ongoing investigational file.
1. 	Air Handling Systems
HVAC systems should be designed to provide adequate air quality for harvesting
and processing of xenotransplantation products. You may use laminar flow units
above the operating table to provide high quality air during harvesting operations.
You may use biological safety cabinets to maintain aseptic conditions during
processing. We expect that this equipment will be capable of producing Class
100 conditions for the most critical of processes although we understand that
maintenance of these conditions may be difficult, during harvesting. Minimally,
the environment surrounding the Class 100 laminar flow units and/or biological
safety cabinets should be Class 100,000. Proceeding toward licensure, areas
surrounding critical Class 100 processes should meet Class 10,000 conditions.
Validation of these systems and units should include verification of air changes
and pressure differentials and should achieve the desired cleanliness level (see
section VIII.B.3.). Testing of the HEPA filters contained in the system should
address integrity and efficiency.
Routine environmental monitoring (see section VIII.B.3.), pressure differential
checks, and recertification of HEPA filters should demonstrate maintenance of the
desired conditions.

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2. 	Water
We expect that water used to formulate necessary reagents or for critical cleaning
purposes (i.e., equipment and surfaces in the harvesting and processing areas) will
meet the United States Pharmacopoeia USP<1231> monograph on Water for
Pharmaceutical Purposes [31] . If WFI is purchased, you should perform lot specific
testing and validate hold times for open containers. If WFI is generated at the
facility, you should properly validate and routinely monitor the system to ensure
continued quality.
3. 	Equipment
You should adequately calibrate and qualify equipment used for harvesting and/or
processing of xenogeneic cells and tissues. You should then routinely monitor
temperature-controlled equipment, such as refrigerators/freezers and incubators,
to assure proper conditions. Carbon dioxide supplied to incubators used for cell
expansion should be 0.2 micron filtered to minimize the potential for
contamination. If water baths are used, you should use maintenance procedures
for water quality. This may include the addition of agents to control
contamination.
4. 	Aseptic Processing
Generally, manipulation or expansion of xenogeneic cells or tissues is an entirely
aseptic process, i.e., there is no final sterilizing filtration of the product. In order
to validate this process, you should perform media fills (substitution of media for
product) to demonstrate that sterility may be maintained consistently. Assurance
of sterility of the final product is necessary from the very beginning of the clinical
studies [32] . You should adequately train and monitor personnel performing these
functions to assure consistent performance during normal production.
All product contact equipment should be sterile and free of pyrogens when
aseptically processing cells or tissues. You may use disposable labware where
possible. The sterility and depyrogenicity of the containers and closures used for
the final product are of particular importance. For equipment and components
that are to be sterilized, there should be evidence that the autoclave cycle(s) is
validated to provide an acceptable level of sterility assurance. Minimally, you
should establish and follow basic load configurations, and you should place
biological indicators within each load to verify lethality. As studies progress, we
expect that formal validation of all sterilizing/depyrogenating processes will be
performed.

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5. 	Process Validation
Ultimately, before licensure or product approval, you should validate all critical
processes used to manufacture the product. We have previously defined process
validation. [7, 32] We expect that process validation, when performed, will be
prospective and at full scale, with the exception of studies performed to
demonstrate viral clearance (removal/inactivation). Laboratory studies may also
help to establish appropriate operating and process parameters and may be used in
support of the formal study. We expect that information on the validation
protocol(s) and summaries of data resulting from its execution will be included in
the license application.
IX. 	PRECLINICAL CONSIDERATIONS FOR XENOTRANSPLANTATION
A. 	General Considerations
This section provides a general framework for the preclinical testing of
xenotransplantation products before use in clinical trials. You may also apply the general
principles as set forth in the document generated by the ICH on the safety of
biotechnology-derived pharmaceuticals to these products (see reference[33] ). In general,
studies to support the safety characterization of therapeutic agents should focus on the
intended alteration to the human pathophysiologic state (i.e., activity) as well as
unintended effects (i.e., toxicity) to the host system. Such studies serve to assess the
potential for clinical risks and constitute an important component of an FDA regulatory
application. Preclinical studies are particularly valuable for gaining insight into safety
issues which cannot be evaluated in human recipients for ethical or practical reasons.
Consequently, you should design strong preclinical safety programs and also consult the
FDA “Guidance for Industry: Preclinical Assessment of Investigational Cellular and
Gene Therapy Products.” [34]
Specific considerations in the design of preclinical studies that are intended to support the
safety of xenotransplantation products should include:
1. 	the animal source for the xenotransplantation product;
2. 	the tissue’s anatomic and physiologic similarity to its human homologue;
3. 	the determination of function of the xenotransplantation product;
4. 	the animal model system;
5. 	the integrity of the device components (if a device is used);
6. 	the dose levels (based on tissue mass as well as pharmacologic/metabolic
activity or release kinetics of bioactive molecules);

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7. 	the route of administration (site of implantation/injection, extracorporeal
or ex vivo use);
8. 	the study duration (as related to potential human exposure);
9. 	reactions between source animal and host immune systems;
10. 	interspecies extrapolation (i.e., cross-species activity of secreted
proteins/hormones at receptors); and
11. 	device biocompatibility.
Since a primary intent of the preclinical animal and in vitro studies is to identify potential
clinical risk factors, these evaluations should focus on maximizing the similarity between
animal and human testing strategies in test substance, route of administration, and dosing
regimen. Animal models of xenotransplantation should utilize a xenotransplantation
system evaluating the cell, tissue, or organ type being examined for use in humans and
should utilize clinically relevant immunosuppressive therapy. Rigorous preclinical
program design is needed to ensure comparability of preclinical to clinical study design
and is important for planning the clinical testing program, including selecting an
appropriate clinical indication, inclusion/exclusion criteria, recipient monitoring scheme,
dose, and concomitant therapies, as well as for advising potential recipients of risks
(informed consent).
B. 	Issues Related to Infectious Agents
Since the transfer of infectious agents that are pathogenic, latent, or even non-pathogenic
in their natural animal host may cause serious disease in an immunosuppressed patient,
you should consider the microbiologic burden carried by the xenotransplantation product
as well as the immune status of the recipient in preclinical study designs. Additionally,
designs of preclinical studies should incorporate:
1. 	careful veterinary monitoring of animals, taking note of any early signs of
infection; and
2. 	procedures needed to assign a cause of mortality (using appropriate
serologic or immunohistochemical identification of pathogens).
To prevent the spread of demonstrable or potential infectious agents, you should care for
animals with appropriate precautions, including isolation if necessary. Deaths from
infections in animal models may occur, due to immunosuppressive regimens that may be
intentionally more extreme than expected for use in humans in order to avoid rejection of
the xenogeneic live cells, tissues, or organs, and to obtain proof-of-concept data.
Therefore, data identifying cause of death (e.g., xenogeneic infectious agents or
activation of latent host infection) could assist in interpreting human risk, may be helpful

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in refining animal experimental models, and may identify pathogenic infectious agents in
the source animal. Animal models of xenotransplantation, while exploring these issues,
are limited by uncertainties in extrapolation of cross-species infectivity information; e.g.,
data indicating no infections in animal, even primate species, are not adequate to assure
that humans will not be susceptible to infections transmitted by the xenotransplantation
product.
Additional insight into refinements of animal immunosuppressant regimens may come
from evaluation of host resistance. You may evaluate host immunocompetence by
measuring resistance to infection by various pathogens, including those that may be
contained within the xenotransplantation product.
C. 	Xenotransplantation Product-Host Interactions
1. 	Immunologic Rejection
You should assess survival of the xenogeneic cells, tissues, or organs in animal
models, with attention given to:
a. 	identifying infiltration of immune or inflammatory cells into the
xenotransplantation product or alteration of such cells in other
relevant compartments, such as the blood and cerebrospinal fluid;
b. 	fibrotic encapsulation of the xenotransplantation product, e.g.,
resulting in impaired function or xenotransplantation product loss;
c. 	xenotransplantation product necrosis;
d. 	any evidence of graft versus host disease (GVHD);
e. 	in vivo function and durability of encapsulation or barriers
intended to diminish rejection or inflammatory responses;
f. 	any special concerns, regarding the site and nature of the
xenotransplantation product; and
g. 	if relevant to the particular xenotransplantation product, the
possibility that rejection of that product might predispose the
recipient to rejection of subsequent xenotransplantation products or
allotransplants.
2. 	Immunosuppression
Preclinical animal studies in which xenotransplantation is used in an
immunosuppressed host may raise questions, regarding the relevance of the model
to clinical pharmacology, toxicology, or immunology. Consideration of how both

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the host and source species handle the immunosuppressive drugs may be
necessary (for instance, where a nephrotoxic drug is metabolized by hepatic
enzymes), but intra-species differences in metabolism exist. Immunosuppressive
drugs often have very restricted therapeutic indices so that pharmacokinetics and
metabolism may markedly affect the activity and/or toxicity of the agents in the
host or xenotransplantation products. You should attempt to delineate toxicities
due to immunosuppressive drugs from toxicities due to the xenotransplantation
product.
You should consider and study, where appropriate, relative activity of
immunosuppression on the source species of the live xenogeneic cells, tissues, or
organs since immunosuppressive treatment that selectively suppresses immunity
in the host species may be permissive to GVHD. This might occur in cases where
immunologically active cells are contained, either intentionally or inadvertently,
within the xenotransplantation product.
3. 	Tumorigenicity in the Immunosuppressed Host
In addition, the tumorigenic potential of the xenotransplantation product, perhaps
due to altered cell growth regulation or to immunosuppression of the host, is an
important concern (refer to section IX.E.).
4. 	Cross-Species Compatibility of Bioactive Molecules
For xenotransplantation products for which you intend that the product synthesize
and provide bioactive molecules, such as cytokines or hormones, you should
provide to FDA, as part of the investigational application , data from preclinical
experiments that support that the molecules produced will be active in humans.
Experiments to address this issue should evaluate concentration-response issues.
You should perform the experiments in vitro and/or in appropriate preclinical
models in vivo.
Even when the xenotransplantation product is composed of a single cell type, the
product may secrete unintended molecules that could alter normal host
physiology. Moreover, host substances might affect product function. Therefore,
preclinical models should evaluate the overall health of the recipient (i.e., clinical
signs, gross pathology, and histopathology) as well as markers of activity of the
xenotransplantation product. You may use combination toxicity and activity
studies to evaluate both potential therapeutic and constitutive functions of the
xenotransplantation product. In some instances, the ability to biopsy
xenotransplantation products periodically is potentially an available tool for
evaluating the histopathologic status of the product and host immune response,
especially when evaluated in conjunction with clinical chemistries. You might
also perform control experiments to test the in vivo effects of live xenogeneic
cells, tissues, or organs taken from anatomic sites other than those used for
therapeutic procurement of the xenotransplantation product but lacking the

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therapeutic cell or tissue type and its anticipated pharmacologic activity. (Please
note that this advice also pertains to heterogeneous xenotransplantation products
as discussed in section IX.D.2.).
5. 	Migration of Xenogeneic Cells
Cells from xenotransplantation products may migrate within the host, thus
presenting clinical concerns regarding adverse reactions deriving from displaced,
bioactive cells or unexpected anatomical impediments. This may be especially
true for incompletely differentiated cells (see section IX.D.3.) and may be
evaluated in animals using histopathology, possibly coupled with enhancing
techniques, such as fluorescent dye loading and/or species-specific antibodies, or
more sensitive techniques, such as PCR.
D. 	Considerations for the Use of Heterogeneous Xenotransplantation Products
You should apply the following principles to the development of appropriate preclinical
testing of heterogeneous xenotransplantation products in order to assess potential adverse
effects. A xenotransplantation product may be considered heterogeneous if it is, for
example, a tissue or solid organ, possessing many varieties of cells; or a cellular implant,
containing extraneous tissues or cells which may be incompletely removed during tissue
dissection or present in short term cultures ex vivo.
1. 	Characterization of Constituent Cell Types in a Heterogeneous
Xenotransplantation Product
The procedures used in preclinical studies for the collection, isolation, and, where
relevant, activation or expansion of the xenotransplantation product, should
mimic the procedures intended for use in clinical trials, and you should
characterize cell types in the product being tested in an analogous fashion to the
proposed clinical xenotransplantation product. See section VI.B.2.c., regarding
recommendations for the evaluation of purity of heterogeneous
xenotransplantation products.
2. 	Secretion of Biologically Active Molecules by Xenotransplantation
Products
Uncharacterized cells or tissues present in the xenotransplantation product may
produce biologically active molecules with unintended activities. You should
perform experiments to identify released, bioactive substances of potential
biologic significance and appropriate to the particular xenotransplantation product
(e.g., neurotransmitters, hormones, cytokines), whether by intended or extraneous
cell types in the xenotransplantation product. For example, you may maintain or
culture in vitro samples of tissues being prepared for transplantation and test the

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supernatants for activities or relevant bioactive substances. You should consult
the ICH guidance document on safety preclinical evaluations in biotechnology for
additional guidance on these studies.[33]
In addition to assessment in vitro, you should evaluate heterogeneous
xenotransplantation products in appropriate animal models. (See section IX.C.4.
for additional discussion of this topic).
3. 	Differentiation in Heterogeneous Xenotransplantation Products
Xenotransplantation products derived from fetal animal sources, dedifferentiated
cells, or tissues or cells, expanded ex vivo, may comprise a heterogeneous
population with regard to cell maturity. The degree of heterogeneity may depend
on the cell or tissue type from which the xenotransplantation product is obtained,
the period of fetal development during which the tissue is procured, and/or the
time in culture. For such products, preclinical studies should compare the viable
cell types initially transplanted with those that exist subsequently in the
xenotransplantation product. This comparison may warrant preclinical studies
with sequential sacrifice groups or biopsies. Techniques, such as
immunohistochemical staining, trypan blue exclusion, bioassays, or PCR assays,
may be useful in identifying heterogeneous cell differentiation. You should
develop models to evaluate the effects of differentiation on the function of the
xenotransplantation product, using, for example, measurements of release or
secretion of biologically active molecules, including those that may not be
intended for efficacy of the xenotransplantation product but that it may produce.
Viable products may change over time as they respond to, adapt to, and
functionally integrate with the host environment. Therefore, you may monitor
cell viability, morphology, and functional endpoints (e.g., endocrine, behavioral,
or immunological) over time to guide development of clinical monitoring
regimens.
E. 	In Vitro and In Vivo Tumorigenicity Models for Xenotransplantation
Products Intended for Transplantation
Tumorigenicity is an important part of preclinical testing for certain xenotransplantation
products, such as those manipulated ex vivo. For further guidance, applicable to this
topic, see references.[3, 10, 33, 34]
Xenotransplantation products may be tumorigenic in a new species because of various
factors, such as transgenic manipulations, endogenous viruses, ex vivo culture, and
immunosuppression of the host. Therefore, for xenotransplantation products intended for
implantation, you should consider evaluation of tumorigenicity in vivo and in vitro.
1. 	Multiple models exist for testing tumorigenesis in vivo. The role of
immune challenge, immunosuppressive drugs, and exposure to certain
infectious agents comprise an important set of safety concerns that may be

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addressed preclinically. Preclinical experiments should include careful
evaluation of controls, background tumor growth rates, tumor incidence
and type, location, and time of appearance of tumors over an extended
period. These should make use of histopathologic evaluation as a primary
endpoint.
2. 	Colony formation in soft agar (clonogenic assays) and growth in organ
culture may be useful in vitro assays of the tumorigenic potential,
particularly for cell lines. These tests may provide information on stability
or abnormal characteristics of cell lines and may substitute for testing in
animals if you demonstrate that the tests have equivalent sensitivity in
your hands.
For xenotransplantation products consisting of cells that have been
expanded ex vivo, a change in cellular growth pattern, morphology, or
growth factor dependence may suggest transformation and a need for more
rigorous investigation.
F. 	Combinations of Xenotransplantation Products with Devices
A number of products for therapeutic use are combinations of xenotransplantation
products and device components, either for use as implants or extracorporeally. All of
the preceding recommendations in section IX. apply to such products. These products
also warrant further preclinical characterization for bioreactivity and biocompatibility of
the device components. Preclinical testing often will include characterization of the
device intended for human use, rather than a homologous product that has been made in
scale with a small laboratory species. This, in turn, may dictate that the device be studied
in an animal species with blood volume and size and, possibly, anatomic structures,
similar to those of humans.
Device elements may be reviewed jointly by staff in CBER and the Center for Devices
and Radiological Health (CDRH). Failure of device components (e.g., membranes and
filters) that serve to isolate animal tissue from the recipient is an important aspect of
safety assessment addressed by review staff in CDRH. Additional device toxicity issues,
also considered by staff at CDRH, are covered in the biocompatibility guidance published
by the International Organization for Standardization.
Implanted devices may be intended for permanent or extended residence in the human
body. Since these are considered chronic therapies, long-term risks (such as chronic
inflammation, carcinogenicity, consequences of re-implantation, and local/systemic
toxicities) of implanted xenotransplantation product/device combinations may necessitate
evaluation before product premarket approval and, to some extent, before initiation of
investigational studies in humans. Studies before initiation of clinical investigational
studies would usually last a minimum of 3 months.

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Membranes with pores may partially isolate xenogeneic tissue housed in devices from
attack by host immune cells, but proteins and pathogens from the xenogeneic tissue may
still be released into the host, along with desired pharmacologically active molecules.
Such devices may reduce but may not eliminate a risk of xenogeneic infections. They
also may act as a stimulus, leading to local inflammation and fibrin deposition.
Adhesions and granulomas may form in host tissue, and deposits on implants may
interfere with activity and implanted cell viability. You should evaluate encapsulated
xenotransplantation products intended to be permeable to bioactive substances (such as
encapsulated islets) for preimplantation activity, and you should retrieve and assess the
products for activity, capsule integrity, and tissue viability after various periods of time in
the animals.
You should conduct extended animal studies (e.g., 12 to 24 months), using the clinical
route of administration (e.g., implant site) and clinical grade materials. You should
design studies to include groups that elucidate reactions to the biomaterials, alone, as well
as groups exposed to clinical and supraclinical doses of the complete product.
Toxicology studies for implanted biomaterials which have previously been utilized in
non-cellular devices may be relevant to safety determinations of the
xenotransplant/device combination products but cannot completely satisfy the need for
toxicity evaluation of the new product in its complete clinical form. You should be aware
that later changes in manufacture of the xenotransplantation product may necessitate new
toxicology studies.
For devices used for extracorporeal hemoperfusion, you should conduct studies to
evaluate the hemodynamic effects of establishing and discontinuing the extracorporeal
circuit, the products released from the tissues housed in the device (e.g., proteins that
could cause anaphylactic responses or stimulate unintended autoimmunity), the
deposition of blood cells (such as platelets) on device tubing or other components,
coagulation or complement activation, and removal of drugs from the recipient’s
circulation through filtration or device-localized cellular metabolism. Assessment of the
biologic activity of the combination product is often a component of preclinical safety
evaluations. For instance, studies should evaluate the duration and predictability of
cellular (e.g., cell cartridge) activity so that you may replace the biologic component of
the device at appropriate intervals to maintain life-supporting pharmacologic or metabolic
activity.
In summary, you should design animal studies of xenotransplantation product/device
combinations, as with other preclinical experiments, taking into consideration all aspects
of the clinical trial and proposed patient population, the need to study both desired and
undesired activities of the xenotransplantation product, and toxicities at the local and
systemic levels.

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X. 	CLINICAL ISSUES IN XENOTRANSPLANTATION
A. 	General Considerations
This section provides general principles rather than specific guidance. Since the
available basic knowledge and clinical experience with xenotransplantation are limited,
current issues may be resolved as new knowledge is acquired and new concerns may
emerge.
B. 	Clinical Protocol Review
Sponsors are responsible for ensuring reviews, as appropriate, by local review bodies,
including Institutional Review Boards (IRBs), Institutional Animal Care and Use
Committees (IACUCs), and Institutional Biosafety Committees (IBCs).[1]
In addition to the human subject protection issues traditionally addressed by local IRBs,
institutional review of xenotransplantation clinical trial protocols should also address:
1. 	the potential risks of infection for the contact populations (including health
care providers, family members, friends, and the community at large);
2. 	source animal husbandry (e.g., screening program, animal quarantine); and
3. 	adequacy of the proposal to address issues related to human and veterinary
infectious diseases (including virology, laboratory diagnostics,
epidemiology, and risk assessment).
C. 	Xenotransplantation Site
The PHS Guideline recommends that all clinical xenotransplantation procedures be
performed in transplantation centers with appropriate experience and expertise for
comparable allotransplantation procedures and with the capability to culture and to
identify viral agents, using in vitro and in vivo methods, either on-site or through active
and documented collaborations.[1]
D. 	Criteria for Patient Selection
Due to the potentially serious public health risks of possible zoonotic infections, you
should limit xenotransplantation to patients with serious or life-threatening diseases for
whom adequately safe and effective alternative therapies are not available, except when
very high assurance of safety can be demonstrated. You should limit candidates to those
patients who have potential for a clinically significant improvement with increased
quality of life, following the procedure. You should also consider the patient’s ability to
comply with public health measures as stated in the protocol, including long-term
monitoring.

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E. 	Risk/Benefit Analysis
We understand that the lack of other therapeutic options and the severity of disease may
raise the benefit-to-risk ratio for some individuals. However, consideration and
evaluation of risks and benefits of xenotransplantation should address both recipient and
public health concerns. You should consider the following in providing a benefit-to-risk
analysis. Infectious disease is among the potential risks both to the recipient and to the
public posed by the use of xenotransplantation products. You should describe and
examine the likelihood and uncertainties associated with any potential recipient and
public health consequences of each of the following events. Transmission of microbial
agents from xenotransplantation products could lead to systemic disease (for example,
infection or neoplasia) or failure of the xenotransplantation product in the recipient. In
addition, transmission of infectious agents could result in outbreaks of zoonotic disease,
silent transmission of latent viruses, or emergence of new strains of pathogens.
Experience has shown that widespread horizontal or vertical transmission of new
pathogens is possible before the pathogens are recognized (e.g., Human
Immunodeficiency Virus). You should describe and examine the possibilities and
uncertainties associated with any other types of adverse events that could have potential,
significant recipient or public health impact, arising from the xenotransplantation
product. Similarly, you should describe any immunological risks, including rejection of
the live xenogeneic cells, tissues, or organs, and, in some cases, GVHD.
F. 	Screening for Infectious Agents
Consult the PHS Guideline[1] for additional guidance and information on testing
recipients of xenotransplantation products.
1. 	Infectious Agents of Concern
Infectious agents of concern will differ among source animal species and among
cell or tissue types within each species. Therefore, you should individualize
clinical tests for the specific xenotransplantation product in question. The
categories of infectious agents of concern include bacteria (such as the
rickettsiae), fungi, mycoplasma, viruses, and the agent(s) causative for TSEs.
Tests should be available for agents known potentially to be present, including
those that are pathogenic in the source animal species and agents that are known
to infect human cells in vivo or in vitro. You should have the capability to test for
latent viruses or pathogens, and you should be prepared to develop and validate
clinical tests for new pathogens that may not be recognized at the time of
xenotransplantation. You should identify specific infectious agents for which
tests will be performed. (See section X.F.3. for additional information on testing.)

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2. 	Collection and Analysis of Clinical Samples
You should only perform xenotransplantation at clinical centers with available
state-of-the-art virology and microbiology laboratories that include a staff with
knowledge and experience in the isolation and identification of unusual
pathogens. In addition, there should be access to laboratory facilities where viral
cultures can be done in vivo, such as in embryonated eggs and suckling mice.
You should place specimens into viral transport medium at the bedside, store at 4
degrees Celsius, and inoculate into cell cultures as soon as possible and always
within 24 hours of collection. The sample(s) selected for culture will depend
upon clinical evaluation of the recipient. In your investigational application to
FDA (e.g., IND), you should describe tissue cell culture systems. These may
include primary monkey, primary human embryonic kidney, semicontinuous
human diploid, and continuous human heterodiploid cells. If isolation remains
difficult, then inoculation in vivo, e.g., into embryonated hen’s eggs and/or
suckling mice, may be necessary. In addition to culture, you may examine tissue
by EM. Immunohistopathology, immunofluorescent antibody,
radioimmunoassay, Enzyme-Linked Immunosorbent Assay, and PCR may be
helpful when appropriate antibodies and probes are available.
3. 	Testing and Scheduling of Testing of Recipients for Infectious Agents
In your investigational application to FDA, you should describe tests of clinical
specimens from recipients for specific agents of concern. The tests may include,
for example, serological and culture assays. You should also describe tests for
latent agents known to be in the source animal species (e.g., retroviruses,
herpesviruses). Assays should be able to distinguish between an infectious agent
derived from the source species and a related infectious agent present in humans
(i.e., porcine versus human Cytomegalovirus (CMV)). Data should be available
to demonstrate specificity, sensitivity, and reproducibility for all tests that are not
in widespread use or for newly-developed tests. In some cases, completion of
development of new tests, which have already demonstrated some level of utility,
specificity, and reproducibility, may proceed concurrently with the clinical trial.
You should also describe the schedule for screening of recipients for infections in
the application for investigational use.
a. 	Acute Infections
Recipients are at risk for the same infections that are common among
individuals who have received allografts. In general, these infections will
be related to the use of immunosuppression and will arise from the
recipient’s endogenous flora, reactivation of latent infections, and
environmental sources. The detection methods useful for these diseases
will not differ from the methods used to detect infections after
allotransplantation.

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In addition to being at risk for these infections, a recipient may be at risk
for infection by agents contained in the xenotransplantation product.
Little clinical experience exists with xenogeneic infectious agents’
infecting humans from xenotransplantation products. We anticipate that a
recipient will be at greatest risk for infection during the first few months
after the procedure. However, there may be significant delay in the
clinical manifestations of infection in some cases. The timing of
occurrence of infectious episodes may vary, depending on
immunosuppression. It is important that you collect relevant data, during
the clinical trial and for the lifetime of the recipient, and that investigation
of acute infectious episodes includes appropriate tests. It is difficult to
predict the diagnostic symptoms and signs of such infections in the
immunosuppressed patient. When the source of a recipient’s post-
transplant illness remains obscure, you should perform testing on
appropriate fluid and tissue samples. Such testing should include the use
of serology as well as various cell and microbial culture systems and in
vivo systems. Culturing may detect infections that serologic testing has
missed (for example, when immunosuppressed transplant recipients are
unable to mount the usual immunological response to a pathogen).
Patient care workers who work with acutely ill recipients should follow
recommended procedures for handling and disinfection/sterilization of
medical instruments and disposal of infectious waste[35] . When there is a
suspicion of a possible xenogeneic infection, you should notify FDA
promptly if a non-xenogeneic causative infectious agent is not readily
identified and notify FDA immediately if a potentially xenogeneic,
causative infectious agent is identified.
b. 	Chronic Infections
An immunosuppressed recipient will be at risk for infection by the
pathogens most commonly associated with allotransplantation. In
addition, you should consider pathogens potentially derived from the
source animal. With adequate preclinical and xenotransplantation product
testing before the procedure, the most likely chronic pathogens from the
animal may be endogenous or exogenous viruses although parasites, such
as Toxoplasma, should be considered.
c. 	Routine Screening for Clinically Inapparent Infections and
Seroconversions
In addition to diagnostic testing when a recipient appears ill, it is
important to establish ongoing recipient screening programs. Sponsors
should describe and validate their screening programs, taking into

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consideration the source animal species and type of cell, tissue, or organ
used.
i. 	Passive Screening Program
In passive screening programs, appropriate clinical samples, such
as blood, plasma, urine, etc., are obtained periodically and archived
for possible future testing. In the event of a diagnosed infection or
the onset of symptoms that may represent infection in one
recipient, these samples are then available for retrospective
screening of persons, regardless of the presence or absence of
symptoms, who shared a common or similar exposure to a
xenotransplantation product. We recommend that a passive
screening program be accomplished through an established
schedule for routine sample collection and storage of samples from
asymptomatic recipients. Such a passive screening program would
be in addition to the collection and archiving of biologic specimens
designated for PHS use as described in the PHS Guideline[1] .
However, the time points identified by the PHS Guideline as
appropriate for archiving specimens designated for PHS use also
provide guidance on the minimal frequency with which specimens
should be obtained and stored as part of a passive screening
program. These time points include:
• 	before xenotransplantation (two samples, one month apart);
• 	at the time of transplantation;
• 	in the immediate post-transplant period;
• 	at one month and six months after transplant;
• 	annually, for the first two years; and
• 	every five years, subsequently.
In certain cases, more frequent acquisition of samples may be
appropriate. The sponsor should consider the animal source and
type of product in proposing the schedule and tests to be used in
the passive screening program.
See section X.H. for recommendations regarding the number, size,
use, and duration of storage of collected samples.

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ii. 	Active Screening
In addition to a passive screening program, you should consider an
active screening program. Possible mechanisms of active
screening range from testing of samples immediately after
collection from recipients to periodic performance of specific
additional laboratory tests on a subset of the samples collected in
the passive screening program. In addition, you should consider
implementation of centralized review of routinely collected clinical
data to detect trends suggestive of emerging diseases. Section
4.1.1.2 of the PHS Guideline[1] suggests an active screening
program for agents known to be in the xenotransplantation product
with testing of samples acquired at 2, 4, and 6 weeks after the
patient receives the xenotransplantation product.
A significant advantage of such a program is screening
prospectively for evidence of infection in the absence of symptoms
provides for a prospective understanding of the patterns of
infection and disease that may be occurring in recipients. Active
screening could allow potential detection of a novel infection in the
asymptomatic recipient and enable implementation of infection
control practices to contain it before secondary human to human
transmission or widespread dissemination in the general public,
even in the absence of manifestation of associated disease (which
may be absent altogether or simply delayed in onset). If a
xenotransplantation product known to harbor an infectious agent is
used for xenotransplantation, you should implement active
screening for that infectious agent. For example, you should assess
all recipients of xenotransplantation products involving the use of
porcine cells, tissues, or organs for evidence of infection by
PERV.[29] Recipient screening for PERV should include analysis
by multiple methods. Ideally, you should use all of the following
detection methods:
• 	PCR of recipient’s PBMC for PERV DNA sequence,
• 	serologic analysis for PERV-specific antibodies, and
• 	assays capable of detecting plasma virions, such as RT-
PCR for detection of viral RNA or highly sensitive
methods for detection of RT activity. [29]
You should collect a sufficient quantity of each sample in the
active screening program to permit archiving for future use, should
the need arise. See section X.H. for recommendations regarding
the number, size, use, and duration of storage of collected samples.

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d. 	Identification of Xenogeneic Retroviruses in Recipients
One particular concern is the potential transmission of xenogeneic
retroviruses, such as PERV in the case of recipients of porcine products.
If you are a sponsor of porcine xenotransplantation product clinical trials,
you should develop a plan to address the possibility that a recipient tests
positive for the presence of PERV or other similar xenogeneic infectious
agents. The plan should include the following:
i. 	Strategies to identify the source of a positive signal in the
screening test (e.g., infection versus false positive). For
example, in the case of porcine xenotransplantation
products, we recommend PCR of DNA isolated from
recipient PBMC for detection of PERV genetic sequences.
However, if a positive result is obtained from this analysis,
one possible explanation would be the presence of porcine
cells. Therefore, you should perform additional DNA PCR
for a repetitive porcine genetic element to determine
whether the source of the positive result may be from
microchimerism for pig cells rather than from human cells
infected with a pig retrovirus. If this analysis suggests the
latter possibility, additional analysis should include an
attempt to isolate the virus from relevant recipient
specimens in an appropriate co-culture assay;
ii. 	Determination of infectivity of the agent using appropriate
assays (e.g., co-cultivation) and additional characterization
of the agent as necessary;
iii. 	A plan to notify FDA and relevant sponsors and
investigators and appropriate IRBs;
iv. 	A contingency plan to modify the clinical trial (including
suspension or termination of enrollment);
v. 	Provisions for acute and follow-up medical care and
counseling of the patients in the study; and
vi. 	Additional actions, if required, for the safety of the
recipient and intimate contacts and to address possible
public health risks.

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e. 	Post-mortem Detection of Agents and Archiving of Autopsy
Samples
You should request a complete postmortem examination, including
histopathology and cultures of all recipients. At post-mortem, you should
fix and embed samples of body tissue for examination by light and
electron microscopy. You should obtain samples from the
xenotransplantation product and, as appropriate, all major organs related
to the product or to clinical syndromes that resulted in the recipient’s
death, were deemed to have been serious, or were of unexplained etiology.
You should archive tissue and fluid samples at -70 degrees Celsius or
lower, as appropriate for preserving the sample, for 50 years beyond the
recipient’s death as discussed in section X.H.1.
4. 	Infections in Recipient Contacts
We recommend that you develop a program to educate and monitor health care
providers and to monitor other intimate contacts of recipients (e.g., persons with
whom recipients repeatedly engage in activities that could result in intimate
exchange of body fluids). In these groups, passive screening (see section
X.F.3.c.i.) may be appropriate. You should obtain baseline samples of plasma and
archive them at -70 degrees Celsius, and you should obtain leukocytes and
archive them in liquid nitrogen for health care personnel, for example, when they
join the clinical teams. You should also ensure that such contacts are advised and
counseled, regarding potential risks.
G. 	Patient Follow-up
The sponsor should propose and submit a plan for clinical follow-up of recipients in a
xenotransplantation protocol in the FDA application, requesting investigational use. This
plan should take into account the timetable for collection and storage of specimens for the
passive screening program and should extend for the life of the recipient (see section
X.F.3.c.i.). The frequency of follow-up will decrease with time, post-procedure. It is
reasonable to plan for a tapering frequency of clinical monitoring and follow-up, with the
flexibility to increase the frequency for individual recipients or trial participants as a
whole, if events occur to make this appropriate.
H. 	Archiving of Patient Plasma and Tissue Specimens
1. 	Before patients are treated, you should create protocols or SOPs for
archiving all samples of patient tissue and fluids, including samples
archived as part of recipient screening, post-mortem samples, and samples
for PHS use.
a. 	You should follow appropriate biosafety precautions in collection
of clinical samples from recipients. You should follow standard

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precautions in obtaining blood from recipients. [35] The PHS
Guideline[1] recommends the use of at least a BioSafety Level
(BSL)-2 containment facility with BSL-3 practices for any
manipulation of clinical samples.
b. 	For the schedule for archiving biological specimens recommended
by PHS, see reference 1 and section X.F.3.c.i. of this document.
The specific protocol or the recipient’s medical course may
indicate more frequent archiving.
c. 	Plans should exist to maintain all archived samples, according to
the procedures recommended in the PHS Guideline[1] , including
those obtained from patients during acute infectious episodes and
from health care workers.
d. 	You should store patient blood and plasma samples in volumes and
quantities, according to the recommendations for animal plasma
and blood cell samples (see section V.E.3.b.).
In addition to recipient samples collected during screening programs or
post mortem, when xenotransplantation recipient tissues are collected for
any medical use, such as a biopsy for diagnostic purposes, you should also
archive samples of such tissues. You should store samples at -70 degrees
Celsius or lower as appropriate for preserving the sample.
2. 	Archive Samples
a. 	You should follow the recommendations in the PHS Guideline[1]
and also section X.F.3.c.i. of this document, regarding archiving of
plasma, blood, and other specimens. You should collect, archive,
and reserve samples for use by PHS in the event that the need for a
PHS-led investigation arises. The PHS Guideline[1] recommends
that biologic specimens for PHS use be maintained for 50 years
beyond the date of xenotransplantation, based on the latency
periods of known human pathogenic persistent viruses and the
precedents established by the U.S. Occupational Safety and Health
Administration with respect to recordkeeping requirements.
b. 	In addition to the designated PHS samples, the sponsor should
archive separate samples of patient plasma, blood cells,
xenotransplantation product, or other tissues for clinical follow-up
and for storage as part of a passive screening program, as detailed
above. (See section X.H.1.).

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c. 	You should not use samples archived for use by PHS (see section
X.H.2.a.) or for monitoring of the recipient through a passive
screening program (see section X.H.2.b.) for other purposes, such
as research.
I. 	Health Records and Data Management
1. 	You should ensure that the recipient’s medical record contains information
on the recipient’s health and all xenotransplantation-related information,
including procedures, a description of the xenotransplantation product, and
any xenotransplantation product-related adverse events. In addition, you
should develop an appropriate tracking system for all recipients of their
xenotransplantation products and use this tracking information to facilitate
notification in the case of a serious adverse event related to a
xenotransplantation product. You should collect information when events
occur, such as a xenotransplantation procedure or an adverse event, and at
the time of clinical follow-up examinations.
Reporting forms should be uniform and include information relevant to the
recipient. We recommend that the information to be collected and tracked
include, at a minimum, the following:
a. 	Facility information - Sponsors should record information
regarding their animal facilities, manufacturing facilities, and
clinical centers associated with each source animal,
xenotransplantation product, and recipient.
b. 	Recipient information - You should identify recipients by code
number or other identifier to link the recipient to relevant
information in the tracking system.
c. 	Procedure information - You should record information about each
xenotransplantation procedure. This information should include
but is not limited to:
i. 	recipient identifiers;
ii. 	the date of the procedure;
iii. 	the clinical center where the procedure was performed;
iv. 	the physician or investigator who performed the procedure;
v. 	the clinical indication for the xenotransplantation
procedure;

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vi. 	medications and therapies administered at the time of the
procedure;
vii. 	a description of the xenotransplantation product(s);
viii. 	identification of the animal source(s);
ix. 	animal facilities for each animal source;
x. 	xenotransplantation product manufacturing facilities; and
xi. 	other pertinent clinical information.
d. 	Adverse Event Reports - A sponsor must record adverse events and
report the events to FDA, pursuant to existing regulation
(21 CFR 312.32). Sponsors should keep records of each event.
e. 	Recipient clinical follow-up examinations - You should
periodically collect clinical status information for recipients of
xenotransplantation products (see section X.F.). This information
should include but is not limited to:
i. 	the date of the clinical follow-up examination;
ii. 	the location of the clinical follow-up examination;
iii. 	the status of the xenotransplantation product in the
recipient;
iv. 	any new significant co-morbidities;
v. 	any hospitalizations since the recipient’s last clinical
follow-up examination.
f. 	Animal Health Events - Animal facilities should record animal
health events. These events include but are not limited to:
i. 	breaks in environmental barriers of the secured animal
facility;
ii. 	disease outbreaks; and
iii. 	sudden, unexplained or unexpected animal deaths.

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The animal facility should report animal health events to the IND
sponsor. The sponsor should include this information in its
tracking system for recipients and in reports to the FDA.
g. 	Recipient Death Reports - Sponsors should maintain death reports
on recipients. This information should include recipient
identifying information, the date of death, and the cause of death.
You should record death certificate and autopsy information, if
available. You should also report deaths to FDA.
2. 	You should maintain health records for at least 50 years beyond the date
of transplantation.
3. 	The sponsor should make provisions for all records and samples
(including post-mortem samples) to be maintained for the requested period
of time in the event that the establishment ceases operation.
J. 	Informed Consent
1. 	General Comments
The informed consent document must include the standard contents (see
21 CFR Part 50).
2. 	Specific Issues
Within the general outline of the informed consent document, you should address
certain specific issues regarding recipients.
a. 	Participation in the Study
i. 	Since the zoonotic, opportunistic, and xenogeneic
infectious risks to the recipient may extend to the
recipient’s family or intimate contacts, the patient should
consent to inform his or her current and future intimate
contacts of their potential risks from the source animal
species and of their deferral from blood donation. Intimate
contacts of xenotransplantation product recipients include
persons who have engaged repeatedly in activities that
could result in intimate exchange of body fluids, including
blood or saliva, with a xenotransplantation product
recipient. Examples of intimate contacts include but are
not limited to sexual partners, household members who
share razors or toothbrushes, and health care workers or
laboratory personnel with repeated percutaneous, mucosal
or other direct exposures. Mere sharing of housing or

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casual contact, such as hugging or kissing without the
exchange of saliva, would not be interpreted as intimate
contact. You should offer the recipient assistance with this
education process, if desired. This discussion should
include the recipient’s potential to transmit zoonotic or
opportunistic infections if such an infection were to occur
and the possibly increased risk of such transmission to
individuals who may be at increased risk for zoonotic or
opportunistic pathogens, such as infants, pregnant women,
the elderly, and chronically ill or immunosuppressed
individuals.
ii. 	Pending further information, clarifying risks, and further
public consultation and discussion, xenotransplantation
product recipients and their intimate contacts (see section
X.J.2.a.i.) should be deferred from donation of Whole
Blood; blood components, including Source Plasma and
Source Leukocytes; tissues; breast milk; ova; sperm; or any
other body parts for use in humans. For more detailed
information, please consult the FDA “Guidance for
Industry: Eligibility Determination for Donors of Human
Cells, Tissues, and Cellular and Tissue-Based Products
(HCT/Ps).”[8]
iii. 	You should counsel the recipient, regarding other
behavioral modifications. You should provide, as
appropriate, advice on the use of barriers to transmission of
infectious agents during sexual activity and the use of
appropriate precautions for nonsexual contacts.
iv. 	The informed consent document should contain
information about the proposed life-long surveillance for
all recipients and the need for clinical and laboratory
monitoring throughout. You should explain, to the extent
possible, the schedule for such clinical and laboratory
monitoring.
v. 	The document should address the need for archiving
plasma and tissue specimens from the source animal and
the recipient for analysis in the case of xenogeneic disease
concerns. The document should explain that such
specimens may be tested in the future by the sponsor or
PHS agencies as needed to evaluate concerns regarding
xenogeneic infections.

Contains Nonbinding Recommendations
56
vi. 	The document should inform the recipient of the
responsibility to inform the investigator or his/her designee
of any change in address or telephone number for the
purpose of enabling life-long health surveillance.
vii. 	The document should inform the recipient of the long term
need for access by the appropriate public health agencies
(e.g., FDA, CDC) to the recipient’s medical records. To
the extent permitted by applicable laws and/or regulations,
you should maintain the confidentiality of medical records.
viii. 	You should include a request for autopsy in the informed
consent document signed by the intended recipient or
his/her appropriate representative.
b. 	Risks to the Recipient and his/her Intimate Contacts
i. 	The informed consent document should address the specific
and known risks of all protocol-related activities not
directly associated with source animal issues as well as the
known and unknown zoonoses that may be associated with
the source species. It should mention the uncertainty of the
risks of infection or its transmission and of the risk of
tumorigenesis. It should mention the possibility of a long
latency period before detection of possible adverse effects.
It should specify the need for and risks from prophylactic
antimicrobial, antiviral, or other chemo- or immunotherapy.
It should provide in an attachment for the recipient and the
recipient’s family the reasoning behind the use of any
prophylactic treatments.
ii. 	In addition, it should describe the possible need for
confinement, reverse isolation, or other specialized medical
housing, including the estimated duration of such
confinement. It should describe any specialized dietary,
travel, or other precautions in as much detail as possible.
iii. 	It should include any known time course for the risks of
disease development and transmission. It should discuss
infectious diseases with protracted incubation periods,
including TSEs and other unusual pathogens.
iv. 	In the specific case of xenotransplantation products from
porcine sources, the informed consent document should
include the following information:

Contains Nonbinding Recommendations
57
• 	PERV can be transmitted from pig cells to human
cells in culture, and this virus can be transmitted
from a human cell line to other human cell lines in
culture.
• 	The clinical significance, if any, of this observation
is unknown and is an area of active research;
however, it is known that infection by certain type
C retroviruses, similar in structure to PERV, can
cause neurological disorders and diseases, such as
lymphomas and other malignancies, in certain
animal models.
c. 	Potential Benefits
It should be clear in the informed consent whether xenotransplantation is
being studied as a first-line, second-line, or salvage therapy of the
condition for which it is being proposed for the individual recipient. It
should clearly convey the specific desired benefits, e.g., limited
prolongation of survival, improved specific organ function,
xenotransplantation product support until allograft becomes available, or
experimental use without known or anticipated benefit.
d. 	Alternative Treatments
The informed consent document should also explain in detail the
anticipated therapeutic options available to participants in the event of
failure of the xenotransplantation product.
e. 	Possible Consequences and Subsequent Treatment Options
The informed consent document, to the extent possible, should explain the
consequences to the patient, should the product fail or undergo irreversible
rejection, including clear and unambiguous statements about the options
that may not be possible after rejection of the xenotransplantation product,
e.g., allotransplantation.
f. 	Confidentiality Issues
The informed consent should inform the patient that all data, including
data collected during the follow-up period, could be made available to
PHS agencies.

Contains Nonbinding Recommendations
58
K. 	Responsibility of the Sponsor in Informing the Patient of New Scientific
Information
You should commit to providing recipients with updated information as soon as possible,
in the event that new data on risks, benefits, or the need for additional treatments relevant
to the recipient’s clinical course become available or necessary. You should be willing to
make a long-term commitment to provide information to the recipient’s families, in the
event that a recipient has died and new safety information of relevance to their potential
exposures becomes known. If you are the sponsor, you should ensure that the
investigators are also willing to commit to providing new information to recipients and
their families.

Contains Nonbinding Recommendations
59
XI. 	REFERENCES
1. 	PHS Guideline on Infectious Disease Issues in Xenotransplantation; January 2001.
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformatio
n/Guidances/Xenotransplantation/ucm074727.htm
2. 	Second WHO Global Consultation on Regulatory Requirements for Xenotransplantation
Clinical Trials; October 2011.
http://www.who.int/transplantation/xeno/report2nd_global_consultation_xtx.pdf
3. 	Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry,
Manufacturing, and Control (CMC) Information for Human Somatic Cell Therapy
Investigational New Drug Applications (INDs); April 2008.
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformatio
n/Guidances/Xenotransplantation/ucm074131.htm
4. 	Guidance for Industry: Regulation of Genetically Engineered Animals Containing
Heritable Recombinant DNA Constructs; #187; June 2015.
http://www.fda.gov/downloads/AnimalVeterinary/GuidanceComplianceEnforcement/Gui
danceforIndustry/ucm113903.pdf
5. 	Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products; January
2011.
http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulator
yInformation/Guidances/CellularandGeneTherapy/UCM243392.pdf
6. 	Guidance for Industry: Pyrogen and Endotoxins Testing: Questions and Answers; June
2012.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guid
ances/UCM310098.pdf
7. 	Guidance for Industry: Process Validation: General Principles and Practices; January
2011.
http://www.fda.gov/downloads/Drugs/.../Guidances/UCM070336.pdf
8. 	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/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulator
yInformation/Guidances/Tissue/ucm091345.pdf
9. 	Medical Devices Containing Materials Derived From Animal Sources (Except for In
Vitro Diagnostic Devices) - Draft Guidance for Industry and Food and Drug
Administration Staff; January 23, 2014. This document, when finalized, will supersede
“Medical Devices Containing Materials Derived From Animal Sources (Except for In
Vitro Diagnostic Devices)” issued November 6, 1998.
http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/Guidanc
eDocuments/UCM381491.pdf
10. 	Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy;
March 1998.
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformatio
n/Guidances/CellularandGeneTherapy/ucm072987.htm
11. 	Guidance for Industry: Public Health Issues Posed by the Use of Nonhuman Primate
Xenografts in Humans; April 1999.

Contains Nonbinding Recommendations
60
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformatio
n/Guidances/Xenotransplantation/ucm074730.htm
12. 	WHO Tables on Tissue Infectivity Distribution in Transmissible Spongiform
Encephalopathies; 2010.
http://www.who.int/bloodproducts/tablestissueinfectivity.pdf
13. 	World Organisation for Animal Health (OIE). List of Bovine Spongiform
Encephalopathy Risk Status of Member Countries. Negligible BSE risk.
http://www.oie.int/animal-health-in-the-world/official-disease-status/bse/list-of-bse-risk-
status
14. 	Final rule: Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine
Products (December 4, 2013; 78 FR 72980).
https://www.federalregister.gov/documents/2013/12/04/2013-28228/bovine-spongiform-
encephalopathy-importation-of-bovines-and-bovine-products
15. 	International Organization for Standardization, ISO 9001:2015 Quality Management
Systems -- Requirements; September 2015.
http://www.iso.org/iso/home/store/catalogue_ics/catalogue_detail_ics.htm?csnumber=62
085
16. 	USDA APHIS - BSE Diagnostics.
https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/animal-disease-
information/cattle-disease-information/sa_bse/ct_diagnostics
17. 	Final rule: Substances Prohibited From Use in Animal Food or Feed (April 25, 2008; 73
FR 22720).
18. 	Occupational Health and Safety in the Care and Use of Research Animals. The National
Academies Press; 1997.
http://www.nap.edu/catalog/4988/occupational-health-and-safety-in-the-care-and-use-of-
research-animals
19. 	Points to Consider in the Characterization of Cell Lines Used to Produce Biological
Products; June 1984.
http://www.fda.gov/medicaldevices/deviceregulationandguidance/guidancedocuments/uc
m081602.htm
20. 	ICH Guideline: Derivation and Characterisation of Cell Substrates Used for Production
of Biotechnological/Biological Products Q5D; July 1997.
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q5D/S
tep4/Q5D_Guideline.pdf
21. 	ICH Guideline: Specifications: Test Procedures and Acceptance Criteria for New Drug
Substances and New Drug Products: Chemical Substances Q6A; October 1999.
http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html
22. 	ICH Guideline: Specifications: Test Procedures and Acceptance Criteria for
Biotechnological/Biological Products Q6B; March 1999.
http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html
23. 	ICH Guideline: Viral Safety Evaluation of Biotechnology Products Derived From Cell
Lines of Human or Animal Origin Q5A(R1); September 1999.
http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html
24. 	Michaels MG, Simmons RL. Xenotransplant-Associated Zoonoses. Transplantation.
1994;57:1-7.

Contains Nonbinding Recommendations
61
25. 	Wilson CA, Wong S, Muller J, Davidson CE, Rose TM, Burd P. Type C Retrovirus
Released from Porcine Primary Peripheral Blood Mononuclear Cells Infects Human
Cells. Journal of Virology. 1998;72(4):3082-3087.
http://jvi.asm.org/content/72/4/3082.full
26. 	Martin U, Kiessig V, Blusch JH, Haverich A, von der Helm K, Herden T, Steinhoff G.
Expression of Pig Endogenous Retrovirus by Primary Porcine Endothelial Cells and
Infection of Human Cells. The Lancet. 1998;352:692-694.
http://www.thelancet.com/pdfs/journals/lancet/PIIS014067369807144X.pdf
27. 	Coffin J. Endogenous Viruses. In: Weiss R, Teich N, Varmus H, Coffin J, editors. RNA
Tumor Viruses: Molecular Biology of Tumor Viruses. 2nd ed. Cold Spring Harbor (NY):
Cold Spring Harbor Laboratory Press; 1984. Chapter 10; 1109-1203.
28. 	McIntosh K. Diagnostic Virology. In: Fields BN, Knipe DM, Chenock, RM, Hirsch MS,
Melnick JL, Monath TP, Roizman B, editors. Virology. 2nd ed. New York (NY): Raven
Press; 1990
29. 	Mattiuzzo G, Takeuchi Y, Scobie L. Potential Zoonotic Infection of Porcine Endogenous
Retrovirus in Xenotransplantation. Methods in Molecular Biology. 2012;885:263-279.
http://www.ncbi.nlm.nih.gov/pubmed/22566002
30. 	International Organization for Standardization, ISO 14644-5:2004 Cleanrooms and
Associated Controlled Environments -- Part 5: Operations;2013.
http://www.iso.org/iso/home/store/catalogue_tc/catalogue_detail.htm?csnumber=33445
31. 	United States of Pharmacopoeia USP<1231> Water for Pharmaceutical Purposes.
http://pharmacopeia.cn/v29240/usp29nf24s0_c1231.html
32. 	Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing - Current
Good Manufacturing Practice; September 2004.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guid
ances/UCM070342.pdf
33. 	ICH Guideline: Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals
S6(R1); June 2011.
http://www.ich.org/products/guidelines/safety/article/safety-guidelines.html
34. 	Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene
Therapy Products; November 2013.
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformatio
n/Guidances/CellularandGeneTherapy/ucm376136.htm
35. 	Centers for Disease Control and Prevention 2007 Guideline for Isolation Precautions:
Preventing Transmission of Infectious Agents in Healthcare Settings; 2007.
http://www.cdc.gov/hicpac/2007IP/2007isolationPrecautions.html