[Federal Register: September 21, 1998 (Volume 63, Number 182)]
[Notices]
[Page 50244-50249]
From the Federal Register Online via GPO Access [wais.access.gpo.gov]
[DOCID:fr21se98-70]

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DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration
[Docket No. 97D-0159]


International Conference on Harmonisation; Guidance on Quality of
Biotechnological/Biological Products: Derivation and Characterization
of Cell Substrates Used for Production of Biotechnological/Biological
Products; Availability

AGENCY:  Food and Drug Administration, HHS.

ACTION:  Notice.

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SUMMARY:  The Food and Drug Administration (FDA) is publishing a
guidance entitled ``Q5D Quality of Biotechnological/Biological
Products: Derivation and Characterization of Cell Substrates Used for
Production of Biotechnological/Biological Products.'' The guidance was
prepared under the auspices of the International Conference on
Harmonisation of Technical Requirements for Registration of
Pharmaceuticals for Human Use (ICH). The document provides broad
guidance on appropriate standards for the derivation and
characterization of cell substrates used in the production of
biotechnological/biological products and recommends information in
these areas that should be presented in marketing applications.

DATES:  Effective September 21, 1998. Submit written comments at any
time.

ADDRESSES:  Submit written comments on the guidance to the Dockets
Management Branch (HFA-305), Food and Drug Administration, 5630 Fishers
Lane, rm. 1061, Rockville, MD 20852. Copies of the guidance are
available from the Drug Information Branch (HFD-210), Center for Drug
Evaluation and Research, Food and Drug Administration, 5600 Fishers
Lane, Rockville, MD 20857, 301-827-4573. Single copies of the guidance
may be obtained by mail from the Office of Communication, Training and
Manufacturers Assistance (HFM-40), Center for Biologics Evaluation and
Research (CBER), or by calling the CBER Voice Information System at 1-
800-835-4709 or 301-827-1800. Copies may be obtained from CBER's FAX
Information System at 1-888-CBER-FAX or 301-827-3844.

FOR FURTHER INFORMATION CONTACT:
    Regarding the guidance: Neil D. Goldman, Center for Biologics
Evaluation and Research (HFM-20), Food and Drug Administration, 1401
Rockville Pike, Rockville, MD 20852, 301-827-0377.
    Regarding ICH: Janet J. Showalter, Office of Health Affairs (HFY-
20), Food and Drug Administration, 5600 Fishers Lane, Rockville, MD
20857, 301-827-0864.

SUPPLEMENTARY INFORMATION:  In recent years, many important initiatives
have been undertaken by regulatory

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authorities and industry associations to promote international
harmonization of regulatory requirements. FDA has participated in many
meetings designed to enhance harmonization and is committed to seeking
scientifically based harmonized technical procedures for pharmaceutical
development. One of the goals of harmonization is to identify and then
reduce differences in technical requirements for drug development among
regulatory agencies.
    ICH was organized to provide an opportunity for tripartite
harmonization initiatives to be developed with input from both
regulatory and industry representatives. FDA also seeks input from
consumer representatives and others. ICH is concerned with
harmonization of technical requirements for the registration of
pharmaceutical products among three regions: The European Union, Japan,
and the United States. The six ICH sponsors are the European
Commission, the European Federation of Pharmaceutical Industries
Associations, the Japanese Ministry of Health and Welfare, the Japanese
Pharmaceutical Manufacturers Association, the Centers for Drug
Evaluation and Research and Biologics Evaluation and Research, FDA, and
the Pharmaceutical Research and Manufacturers of America. The ICH
Secretariat, which coordinates the preparation of documentation, is
provided by the International Federation of Pharmaceutical
Manufacturers Associations (IFPMA).
    The ICH Steering Committee includes representatives from each of
the ICH sponsors and the IFPMA, as well as observers from the World
Health Organization, the Canadian Health Protection Branch, and the
European Free Trade Area.
    In the Federal Register of May 2, 1997 (62 FR 24312), FDA published
a draft tripartite guideline entitled ``Quality of Biotechnological/
Biological Products: Derivation and Characterization of Cell Substrates
Used for Production of Biotechnological/Biological Products.'' The
notice gave interested persons an opportunity to submit comments by
June 16, 1997.
    After consideration of the comments received and revisions to the
guidance, a final draft of the guidance was submitted to the ICH
Steering Committee and endorsed by the three participating regulatory
agencies on July 17, 1997.
    In accordance with FDA's Good Guidance Practices (62 FR 8961,
February 27, 1997), this document has been designated a guidance,
rather than a guideline.
    The document provides broad guidance on appropriate standards for
the derivation of human and animal cell lines and microbial cells to be
used to prepare biotechnological/biological products and for the
preparation and characterization of cell banks to be used for
production. The guidance recommends information in these areas that
should be presented in marketing applications for these products.
    This guidance represents the agency's current thinking on standards
for the derivation and characterization of cell substrates used for
production of biotechnological/biological products. It does not create
or confer any rights for, or on, any person and does not operate to
bind FDA or the public. An alternative approach may be used if such
approach satisfies the requirements of the applicable statute,
regulations, or both.
    As with all of FDA's guidances, the public is encouraged to submit
written comments with new data or other new information pertinent to
this guidance. The comments in the docket will be periodically
reviewed, and, where appropriate, the guidance will be amended. The
public will be notified of any such amendments through a notice in the
Federal Register.
    Interested persons may, at any time, submit written comments on the
guidance to the Dockets Management Branch (address above). Two copies
of any comments are to be submitted, except that individuals may submit
one copy. Comments are to be identified with the docket number found in
brackets in the heading of this document. The guidance and received
comments may be seen in the office above between 9 a.m. and 4 p.m.,
Monday through Friday. An electronic version of this guidance is
available on the Internet at ``http://www.fda.gov/cder/guidance/
index.htm'' or ``http://www.fda.gov/cber/publications.htm''.
    The text of the guidance follows:

Q5D Quality of Biotechnological/Biological Products: Derivation and
Characterization of Cell Substrates Used for Production of
Biotechnological/Biological Products \1\
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    \1\ This guidance represents the agency's current thinking on
standards for the derivation and characterization of cell substrates
used for production of biotechnological/biological products. It does
not create or confer any rights for or on any person and does not
operate to bind FDA or the public. An alternative approach may be
used if such approach satisfies the requirements of the applicable
statute, regulations, or both.
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1.0 Introduction

1.1 Objective

    The objective of this guidance is to provide broad guidance on
appropriate standards for the derivation of human and animal cell
lines and microbial cells to be used to prepare biotechnological/
biological products defined in section 1.3, Scope, and for the
preparation and characterization of cell banks to be used for
production. The document, therefore, provides recommendations on the
information in these areas that should be presented in marketing
applications for these products.

1.2 Rationale

    Historically, some quality concerns for cell-derived biological
products have originated from the presence of adventitious
contaminants or from the properties of the cells used to prepare the
product. Recombinant DNA (rDNA)-derived products also carry quality
concerns regarding the expression construct contained in the cell
substrate. Thus, it is well established that the properties of the
cell substrate and events linked to the cell substrate can affect
resultant product quality and safety and, further, that effective
quality control of these products requires appropriate controls on
all aspects of handling the cell substrate.
    This document complements other guidances to provide a
comprehensive approach to quality issues arising from biological
aspects of processing products from metazoan and microbial cell
culture.

1.3 Scope

    This guidance covers cell substrates having a cell banking
system. In this document, ``cell substrate'' refers to microbial
cells or cell lines derived from human or animal sources that
possess the full potential for generation of the desired
biotechnological/biological products for human in vivo or ex vivo
use. Reagents for in vitro diagnostic use are outside the scope of
this document. Animal sources of cell lines include all those of
metazoan origin. Both continuous cell lines of indefinite in vitro
lifespan and diploid cells of finite in vitro lifespan are included.
Microbial sources include bacteria, fungi, yeast, and other
unicellular life forms.
    ``Biotechnological/biological products'' refers to any products
prepared from cells cultivated from cell banks with the exception of
microbial metabolites such as, for example, antibiotics, amino
acids, carbohydrates, and other low molecular weight substances.
Cell banks used to prepare gene therapy products or vaccines should
follow the recommendations presented in this document. Some
biological products, such as certain viral vaccines, are prepared in
primary cell cultures derived directly from animal tissues or
organs. Primary cells are not banked and therefore are not addressed
by this document. However, other considerations which may apply to
primary cells are discussed further in Appendix 1 of this document.

2.0 Guidelines

2.1.0 Source, History, and Generation of the Cell Substrate

2.1.1 Introduction

    It is important to provide supportive documentation which
describes the history of the cell substrate that is used in the
manufacture of a biotechnological/biological product, as well as any
parental cell line

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from which it was totally or partially derived. Events during the
research and development phases of the cell substrate may contribute
significantly to assessment of the risks associated with the use of
that particular cell substrate for production. The information
supplied in this regard is meant to facilitate an overall evaluation
that will ensure the quality and safety of the product.
    Careful records of the manipulation of the cell substrate should
be maintained throughout its development. Description of cell
history is only one tool of many used for cell substrate
characterization. In general, deficiencies in documented history may
not, by themselves, be an impediment to product approval, but
extensive deficiencies will result in increased reliance on other
methods to characterize the cell substrate.

2.1.2 Origin, Source, and History of Cells

    The source of cells (laboratory or culture collection) from
which the cell substrate was derived should be stated, and relevant
references from the scientific literature should be cited.
Information obtained directly from the source laboratory is
preferred. When this is not available, literature references may be
utilized.
    For human cell lines, it is relevant to describe the following
characteristics of the original donor: Tissue or organ of origin,
ethnic and geographical origin, age, sex, and general physiological
condition. If known, the state of health or medical history of the
donor should be reported along with the results of any tests of the
donor for pathogenic agents. Specifically for human diploid
fibroblasts, the age of the donor may influence the in vitro
lifespan of the cell line and this information should be provided if
available. For animal cell lines, relevant descriptions of the
source include species, strains, breeding conditions, tissue or
organ of origin, geographical origin, age and sex, the results of
tests for pathogenic agents, and general physiological condition of
the original donor.
    For microbes, manufacturers should describe the species, strain,
and known genotypic and phenotypic characteristics of the organism
from which the cell substrate was derived. Manufacturers should also
describe the pathogenicity, toxin production, and other biohazard
information, if any.
     The cultivation history of the cells should be documented. The
method originally used for the isolation of the cells should be
described as well as the procedures used in the culturing of the
cells in vitro and any procedures used to establish cell lines (for
example, use of any physical, chemical, or biological procedure, or
added nucleotide sequences). A description of any genetic
manipulation or selection should be provided. All available
information regarding the identification, characteristics, and
results of testing of these cells for endogenous and adventitious
agents should be provided.
    For continuous cell lines of metazoan origin, it is usually
adequate to quantitate culture duration by estimation of either
number of population doublings, or number of subcultivations at
defined dilution ratio, or time in days. For diploid cell lines
possessing finite in vitro lifespan, accurate estimation of the
number of population doublings during all stages of research,
development, and manufacturing is important. For microbial cells,
documentation of subcultivation frequency after cell substrate
generation is considered adequate.
    Regarding the generation of cell substrates, applicants should
provide a thorough discussion of procedures that would provide
exposure to infectious agents. Constituents of the culture medium
should be described, in particular, information regarding exposure
of the cells to materials of human or animal origin such as serum,
enzymes, hydrolysates, or other living cells. The description should
include the source, method of preparation and control, test results,
and quality assurance. Relevant literature on these points may be
referenced when available. This information will allow a detailed
analysis of potential entry routes for adventitious agents from
these sources, and would be part of the risk-benefit analysis of the
product.

2.1.3 Generation of the CelpaSubstrate

    A crucial step is the choice of a suitable parental cell line.
For recombinant products, a parental cell line is typically the
untransfected recipient cell line. The use of characterized parental
cell banks is suggested, but is not considered essential. A
characterized parental cell bank may be of benefit, especially when
multiple cell substrates are generated from the same parental cell
type, by providing a set of information on which the quality
assessment of the Master Cell Bank (MCB) can be based. For example,
the myeloma cell line may be banked as a parental cell line for
hybridomas.
    During the generation of the cell substrate, one or more
specific procedures may be utilized in the ultimate development of
the desired characteristics. These may include, for example, cell
fusion, transfection, selection, colony isolation, cloning, gene
amplification, and adaptation to specific culture conditions or
media. Information regarding the methodologies utilized in
developing the cell substrate can help to provide a clear
understanding of the history of the cell substrate. Some cell
substrates, such as human diploid fibroblasts, may not need
extensive manipulation or cloning prior to cell banking.
    For recombinant products, the cell substrate is the transfected
cell containing the desired sequences, which has been cloned from a
single cell progenitor. For further information on generation of
rDNA-modified cell substrates, consult other relevant (e.g.,
regional or international) guidances. For nonrecombinant products or
nonrecombinant vaccines, the cell substrate is the cell from the
parental cell line chosen for preparation of the MCB without further
modification. For products derived from hybridomas, the cell
substrate is the hybridoma cell line derived by fusion of the
parental myeloma cell line with other parental cells, e.g., immune
spleen cells.

2.2.0 Cell Banking

    One of the most important advantages of using serially
subcultivated cells to produce biotechnological/biological products
is the ability to have a characterized common starting source for
each production lot, i.e., the preserved bank of cells.
Manufacturers may prepare their own cell banks or may obtain them
from external sources. Manufacturers are responsible for ensuring
the quality of each cell bank and of the testing performed on each
bank.

2.2.1 Cell Banking System

    The concept of a two-tiered cell bank, in which the MCB is used
to generate Working Cell Banks (WCB's), is generally accepted as the
most practical approach to providing a supply of cell substrate for
continued manufacture of the product. Manufacturers should describe
their strategy for providing a continued supply of cells from their
cell bank(s), including the anticipated utilization rate of the cell
bank(s) for production, the expected intervals between generation of
new cell bank(s), and the criteria for qualification of cell
bank(s).
    Generally, the MCB is made first, usually directly from an
initial clone or from a preliminary cell bank derived from an
initial clone. It is not considered necessary to prepare cell banks
from clones for certain types of cells (e.g., diploid cells, where
limited in vitro life span or other technical factors make cell
cloning impractical) or where the uncloned cell population is
already adequately homogeneous for the intended use.
    A WCB is derived from one or more containers of the MCB. It is
the WCB that is typically used to directly provide cells for the
manufacturing process. Additional WCB's are generated from the MCB
as needed. A newly prepared WCB should be appropriately qualified by
characterization and testing.
    It should be noted that the MCB and WCB may differ from each
other in certain respects, e.g., culture components and culture
conditions. Similarly, the culture conditions used to prepare the
MCB and WCB may differ from those used for the production process.
If changes in cell culture process do not affect product quality, it
is not considered necessary to reclone the cells or to rebank the
MCB or WCB. It is important that a characterized bank provides a
consistent product.
    A single-tiered banking system consisting only of the MCB but no
WCB's could be used in principle, for example, if relatively few
containers were needed each year to produce the desired product.
    In some microbial expression systems, a new transformation is
performed for each new cell substrate container lot, based upon
using aliquots of thoroughly tested host cell banks and plasmid
banks for each new transformation and on testing of each transformed
cell substrate bank. This transformed cell substrate bank is
considered the MCB, and it is used as the source of cell substrate
for production. Host cell banks, plasmid banks, and MCB's are
maintained by appropriate preservation methods. This alternative
system is considered adequate because the transformation of bacteria
and yeast is generally a very reproducible and easily performed
process, unlike the events needed for transfection of metazoan
cells. Manufacturers should provide information on the host cells,
rDNA molecules (such as plasmids), method of transformation and of

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cell banking, and the results of characterization studies.

2.2.2 Cell Banking Procedures

    It is important to prevent a contaminated cell substrate (or
bank) from being used in production and to avoid a loss of product
availability or development time resulting from the need to recreate
a cell bank found to be unusable due to contamination. It is
recognized that no cell bank testing regimen is able to detect all
potential contaminants; therefore, use of these preventive
principles during cell banking is important to provide reasonable
assurance of the absence of contamination and to provide a reliable
source of the cell substrate.
    Manufacturers should describe the type of banking system used,
the size of the cell bank(s), the container (vials, ampules, or
other appropriate vessels) and closure system used, the methods used
for preparation of the cell bank(s) including the cryoprotectants
and media used, and the conditions employed for cryopreservation and
storage.
    Manufacturers should describe the procedures used to avoid
microbial contamination and cross-contamination by other cell types
present in the laboratory and the procedures that allow the cell
bank containers to be traced. This should include a description of
the documentation system as well as that of a labeling system that
can withstand the process of preservation, storage, and recovery
from storage without loss of labeling information on the container.
    Manufacturers should describe their cell banking procedures.
Cells are generally prepared for banking by expanding cultures in a
progressively greater number or larger size of vessel until a pool
of cells can be obtained that is sufficient to generate enough
containers for the bank. To ensure the uniform composition of the
contents of each container, a single pool of cells for banking
should be prepared by combining the cells from all of the culture
vessels, if more than one vessel is used.
    Cells suspended in preservation medium are aliquoted from the
single pool into sterilized containers which are then sealed and
stored under appropriate conditions. For example, animal cells in
media containing a cryoprotectant are frozen in the sealed
containers under defined and controlled conditions and then
transferred to storage in the vapor or liquid phase of liquid
nitrogen or at equivalent ultra low temperatures. Other methods of
preservation and storage may be adequate depending on the organism
used, but they should be capable of maintaining a level of cell
viability upon reconstitution that is both consistent and adequate
for production use.
    To ensure continuous, uninterrupted production of
pharmaceuticals, manufacturers should carefully consider the steps
that can be taken to provide for protection from catastrophic events
that could render the cell bank unusable. Examples of these events
include fires, power outages, and human error. Manufacturers should
describe their plans for such precautions; for example, these may
include redundancy in the storage of bank containers in multiple
freezers, use of back-up power, use of automatic liquid nitrogen
fill systems for storage units, storage of a portion of the MCB and
WCB at remote sites, or regeneration of the MCB.
    The starting point of reference for estimates of in vitro cell
age during manufacturing should be the thawing of one or more
containers of the MCB. For diploid cell lines, in vitro lifespan
should be estimated in terms of population doubling levels. The
population doubling level at which senescence occurs should be
determined for diploid cells.

2.3.0 General Principles of Characterization and Testing of Cell Banks

    The characterization and testing of banked cell substrates is a
critical component of the control of biotechnological and biological
products. Characterization of the MCB allows the manufacturer to
assess this source with regard to presence of cells from other
lines, adventitious agents, endogenous agents and molecular
contaminants (e.g., toxins or antibiotics from the host organism).
The objective of this testing is to confirm the identity, purity,
and suitability of the cell substrate for manufacturing use. In some
cases, additional testing such as tumorigenicity or karyology may be
useful. The testing program chosen for a given cell substrate will
vary according to the biological properties of the cells (for
example, growth requirements), its cultivation history (including
use of human-derived and animal-derived biological reagents), and
available testing procedures. The extent of characterization of a
cell substrate may influence the type or level of routine testing
needed at later stages of manufacturing. Manufacturers should
perform tests for identity and purity once for each MCB and tests of
stability during cell cultivation once for each product to be
registered. In addition, tests of purity and limited tests of
identity should be performed once on each WCB. Also, applicants
should consult the ICH guidance on viral safety. Relevant tests
among those described below should be performed and described in the
marketing application, along with the results of the testing.
    For cell lines containing exogenously assembled expression
constructs, the relevant ICH guidance on rDNA expression constructs
should be consulted for guidance on the characterization of
nucleotide and amino acid sequences. It may also be useful to
examine, by similar methods, the coding sequences in some
nonrecombinant DNA-derived cell lines where the gene sequences have
been characterized and are well understood. However, it is not
considered necessary to carry out investigations of the sequences
encoding complex natural products, for example, families of related
gene products, microbial vaccine antigens, or monoclonal antibodies
from hybridomas.
    Manufacturers are also encouraged to employ ``state-of-the-art''
methods and technological improvements in cell substrate
characterization and testing as they become available, as long as
the specificity, sensitivity, and precision of the newer methods are
at least equivalent to those of existing methods.
    The manufacturer may choose to characterize the WCB instead of
the MCB, if justified.

2.3.1.0 Tests of Identity

    Appropriate tests should be performed to determine that the
banked cell is what it is represented to be. Either phenotypic or
genotypic characteristics may be used in identity testing. It is not
considered necessary to do all the possible tests. Tests of identity
are generally performed on the MCB. In addition, limited identity
testing is generally performed on each WCB.

2.3.1.1 Metazoan Cells

    For human or animal cells that grow attached to a substratum,
morphological analysis may be a useful tool in conjunction with
other tests. In most cases, isoenzyme analysis is sufficient to
confirm the species of origin for cell lines derived from human or
animal sources; other tests may be appropriate depending on the
history of the cell line. Other technologies may be substituted to
confirm species of origin, including, for example, banding
cytogenetics or use of species-specific antisera. An alternative
strategy would be to demonstrate the presence of unique markers, for
example, by using banding cytogenetics to detect a unique marker
chromosome, or DNA analysis to detect a genomic polymorphism pattern
(for example, restriction fragment length polymorphism, variable
number of tandem repeats, or genomic dinucleotide repeats). Either
confirmation of species of origin or presence of known unique cell
line markers is considered an adequate test of identity. Expression
of the desired product may represent a complementary approach to
confirmation of identity.

2.3.1.2 Microbial Cells

    For most microbial cells, analysis of growth on selective media
is usually adequate to confirm host cell identity at the species
level for the host cell bank and the transformed cell bank. For E.
coli, where a variety of strains may be used, biological
characterization methods such as phage typing should be considered
as supplementary tests of identity. For plasmid banks, identity
assessment can be accomplished as described by the ICH document on
analysis of the expression construct. Expression of the desired
product is also considered adequate to confirm the identity of the
microbial expression system.

2.3.2.0 Tests of Purity

    A critical aspect of cell development and banking is the
assessment that the MCB and WCB are biologically pure, i.e., are
free from adventitious microbial agents and adventitious cellular
contaminants. The impact of selective agents and antibiotics on the
detection of adventitious microbial contaminants should be
considered when planning and performing these tests.

2.3.2.1 Metazoan Cells

    Tests for the presence of bioburden (bacteria and fungi) should
be performed on individual containers (1 percent of the total number
but not less than two containers) of the MCB and WCB. In all other
aspects, the current methodologies described in either the

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European Pharmacopoeia (Ph. Eur.), the Japanese Pharmacopoeia (JP),
or the U.S. Pharmacopoeia (U.S.P.) for testing microbial limits or
microbial sterility may be considered adequate.
    Tests for the presence of mycoplasma should be performed on the
MCB and WCB. Current procedures considered adequate include both the
agar and broth media procedures as well as the indicator cell
culture procedure. Current methods for mycoplasma testing are
described in Ph. Eur., JP, and ``Points to Consider in the
Characterization of Cell Lines Used to Produce Biologicals'' (FDA,
CBER, 1993). Testing cells derived from a single container is
generally considered adequate. For nonmammalian animal cell lines,
alternative controls and/or assay conditions may be appropriate;
manufacturers should consult with the national/regional regulatory
authority for appropriate methodology.
    If future efforts to harmonize bioburden and mycoplasma assays
are fruitful, then the scientifically appropriate harmonized assay
should be used.
    Virus testing of cell substrates should be designed to detect a
wide spectrum of viruses by using appropriate screening tests and
relevant specific tests, based on the cultivation history of the
cell line, to detect possible contaminating viruses. Applicants
should consult the ICH guidance on viral safety. For product classes
not covered by the viral safety guidance, the current World Health
Organization (WHO) documents for use of animal cells may be
consulted.
    The purity of cell substrates can be compromised through
contamination by cell lines of the same or different species of
origin. The choice of tests to be performed depends upon whether
opportunities have existed for cross-contamination by other cell
lines. In some cases, it may be necessary to maintain growing
cultures of different cell lines in the same laboratory. During
procedures in cell banking where open manipulations are performed,
care should be taken to ensure that simultaneous open manipulations
of other cell lines are avoided to prevent cross-contamination.
Whenever another cell line was present in the cell banking room at
the same time that open cell banking procedures were being performed
(such as cell expansion, pooling, or aliquoting of the chosen cell
line), the cell banks should be tested for the presence of cells
from (or products derived from) the second cell line. In general,
the methods described in section 2.3.1.0 to assess cell identity are
also considered adequate tests to detect cross-contamination by
other cell lines. Additional assurance of lack of cross-
contamination can be provided by successful preparation of the
intended product from the cell substrate.

2.3.2.2 Microbial Cells

    The design and performance of specific tests for adventitious
microbial agents and adventitious cellular contaminants in microbial
cell banks should take into account the properties of the banked
cell, the likely contaminants based upon scientific literature,
source, methods and materials used for cultivation, and other
organisms present in the banking laboratory. For example, visual
examination of the characteristics of well-isolated colonies is
suggested, using several microbiological media, of which some do and
some do not support growth of the cell substrate. However, it is not
intended that manufacturers necessarily characterize resistant
mutants of the cell substrate arising from such studies, or other
artifacts of such assays. Rather, the purpose of such assays is to
detect existing contaminants.

2.3.3 Cell Substrate Stability

    Another dimension to cell characterization is appropriateness
for intended use in production. There are two concerns for cell
substrate stability: Consistent production of the intended product
and retention of production capacity during storage under defined
conditions.
    For the evaluation of stability during cultivation for
production, at least two time points should be examined, one using
cells that have received a minimal number of subcultivations, and
another using cells at or beyond the limit of in vitro cell age for
production use described in the marketing application. The limit of
in vitro cell age for production use should be based on data derived
from production cells expanded under pilot plant scale or commercial
scale conditions to the proposed limit of in vitro cell age for
production use or beyond. Generally, the production cells are
obtained by expansion of cells from the WCB; cells from the MCB
could be used with appropriate justification. This demonstration of
cell substrate stability is commonly performed once for each product
marketing application.
    Evaluation of the cell substrate with respect to the consistent
production of the intended product of interest should be the primary
subject of concern. The type of testing and test article(s) used for
such assessments will depend on the nature of the cell substrate,
the cultivation methods, and the product. For cell lines containing
recombinant DNA expression constructs, consistency of the coding
sequence of the expression construct should be verified in cells
cultivated to the limit of in vitro cell age for production use or
beyond by either nucleic acid testing or product analysis, as
described in the relevant ICH guidance. For nonrecombinant cell
lines in which the coding sequence for the desired product has
already been analyzed at the MCB or WCB level, invariability of the
protein coding sequence during production should be verified in the
production cells cultivated to the proposed limit of in vitro cell
age for production use or beyond by either nucleic acid testing or
analysis of the purified protein product.
    Where the product cannot be analyzed as described above, other
specific traits, which may include, for example, morphological
characteristics, growth characteristics, biochemical markers,
immunological markers, productivity of the desired product, or other
relevant genotypic or phenotypic markers, may be useful for the
assessment of cell substrate stability. In some cases, where direct
comparison of the characteristics of the MCB with those of the
production cells at or beyond the limit of in vitro cell age is
difficult or impossible, one may compare the characteristics of
cells at the initial stages of cultivation or production to those of
cells at or beyond the limit of in vitro cell age for production use
in order to assess cell stability during production. Indices such
as, for example, oxygen or glucose consumption rates, ammonia or
lactate production rates may be useful for such testing. Increases
in the defined limit of in vitro cell age for production use should
be supported by data from cells which have been expanded to the
proposed new limit of in vitro cell age. For diploid cell lines,
data should be presented that establish the finite in vitro lifespan
of the cells from the WCB under conditions representative of those
employed for manufacturing use.
    Evidence for banked cell stability under defined storage
conditions will usually be generated during production of clinical
trial material from the banked cells. Data from the determination of
cell viability when the preserved cells are reconstituted for
production of clinical trial supplies will verify that the revived
cells have survived the preservation process. Data from the
preparation of clinical materials will demonstrate that the revived
cells can be used to prepare the desired product. Available data
should be clearly documented in the application dossiers, plus a
proposal for monitoring of banked cell stability should be provided.
The proposed monitoring can be performed at the time that one or
more containers of the cryopreserved bank is thawed for production
use, when the product or production consistency is monitored in a
relevant way, or when one or more containers of the cryopreserved
MCB is thawed for preparation of a new WCB (and the new WCB is
properly qualified), as appropriate. In the case when production
does not take place for a long period of time, viability testing on
the cell bank used as a source of the production substrate should be
performed at an interval described in the marketing application. If
the viability of the cell substrate is not significantly decreased,
generally no further testing of the MCB or WCB is considered
necessary.

2.3.4 Tests for Karyology and Tumorigenicity

    Utilization of karyology and tumorigenicity testing for
evaluating the safety of a diploid cell line or characterizing a new
cell line may be useful depending on the cells, the nature of the
product, and the manufacturing process. Extensive analysis to
determine the relative abundance of aneuploid cells has not been
found to be useful. Karyology need not be determined for rodent cell
lines or new cell lines known to be nondiploid. However, cytogenetic
analysis may be an adequate method to assess cell substrate identity
or purity as described in sections 2.3.1.0 and 2.3.2.0. Repetition
of tumorigenicity testing for cells with already documented evidence
of tumorigenicity is not considered necessary.
    For products that are highly purified and that contain no cells,
karyology and tumorigenicity testing are generally not considered
necessary, provided that appropriate limits for residual host cell
DNA are shown to be consistently met by either

[[Page 50249]]

process validation studies or by lot release testing.
    In general, products for which the presence of live cells cannot
be excluded or which have little downstream purification (for
example, some conventional live virus vaccines) will need such
characterization of the cell substrate. The utility of
tumorigenicity testing and chromosomal analysis for new cell
substrates for unpurified products should be evaluated on a case-by-
case basis. Use of cell lines known to be tumorigenic or to possess
abnormal karyology should be evaluated in terms of risk-benefit for
each product application when the product contains cells or when not
highly purified.
    Products that are manufactured in genetically unmodified MRC-5
or WI-38 cells do not need characterization of these cell substrates
by karyology or tumorigenicity since extensive characterization has
already been performed and published for these cell lines. However,
for each MRC-5 and WI-38 WCB generated, manufacturers should
confirm, once, that the cells grown in the manner to be used in
production are diploid and have the expected lifespan.
    For new or previously uncharacterized diploid cell substrates,
confirmation of diploid karyology should be presented and
tumorigenic potential should be established, using cells from the
MCB.

3.0 Glossary

    Cell bank--A cell bank is a collection of appropriate
containers, whose contents are of uniform composition, stored under
defined conditions. Each container represents an aliquot of a single
pool of cells.
    Cell line--Type of cell population that originates by serial
subculture of a primary cell population, which can be banked.
     Continuous cell line--A cell line having an infinite capacity
for growth. Often referred to as ``immortal'' and previously
referred to as ``established.''
    Diploid cell line--A cell line having a finite in vitro lifespan
in which the chromosomes are paired (euploid) and are structurally
identical to those of the species from which they were derived.
    Host cells--See parental cells.
    In vitro cell age--Measure of time between thaw of the MCB
vial(s) and harvest of the production vessel measured by elapsed
chronological time, by population doubling level of the cells, or by
passage level of the cells when subcultivated by a defined procedure
for dilution of the culture.
    Metazoan--Organism of multicellular animal nature.
    MCB (Master Cell Bank)--An aliquot of a single pool of cells
which generally has been prepared from the selected cell clone under
defined conditions, dispensed into multiple containers, and stored
under defined conditions. The MCB is used to derive all working cell
banks. The testing performed on a new MCB (from a previous initial
cell clone, MCB, or WCB) should be the same as for the original MCB
unless justified.
    Parental cells--Cells to be manipulated to give rise to a cell
substrate or an intermediate cell line. For microbial expression
systems, it is typical to also describe the parental cells as the
host cells. For hybridomas, it is typical to also describe the
parental cells as the cells to be fused.
    WCB (Working Cell Bank)--The Working Cell Bank is prepared from
aliquots of a homogeneous suspension of cells obtained from
culturing the MCB under defined culture conditions.

Appendix 1

Primary Cell Substrates

I. Introduction

    The principles contained in this document apply in general to
biotechnological/biological products prepared from characterized
banked cells. However, a number of biological products, in
particular certain viral vaccines, are prepared using primary cells.
    Because primary cell cultures are used within the first passage
after establishment from the tissue of origin, it is not possible to
carry out extensive characterization of the cells prior to their use
as is done for banked cell substrates. In addition, biological
products produced using primary cell substrates often do not undergo
extensive processing (e.g., purification). Despite these
differences, the approach taken to ensure the suitability and safety
of primary cell substrates for production of biologicals is
analogous, in many respects, to that outlined in this document and
in other guidances.
    This annex outlines cell substrate-related information that
should be included in marketing applications for biological products
prepared using primary cells. This information falls into three
general categories: (1) Information concerning the source tissue (or
organ) and other animal-derived raw materials used for the
establishment of primary cell substrates, (2) information concerning
the preparation of primary cell substrates, and (3) testing
performed on primary cell substrates to ensure the safety of the
product.

II. Source Tissue and Other Raw Materials

    Information should be provided about the animals used as a
source of tissue for the preparation of primary cell substrates.
Tissue should be derived from healthy animals subjected to
veterinary and laboratory monitoring to certify the absence of
pathogenic agents. Whenever possible, donor animals should be
obtained from closed, specific pathogen-free (when available)
colonies or flocks. Animals used as tissue donors should not have
been used previously for experimental studies. Animals should be
adequately quarantined for an appropriate period of time prior to
use for the preparation of cells. In some countries, animals may
need to be quarantined in the country where the primary cells are
prepared. Manufacturers should consult with national/regional
authorities for specific requirements.
    Information on materials and components used for the preparation
of primary cell substrates should be provided, including the
identity and source of all reagents of human or animal origin. A
description of testing performed on components of animal origin to
certify the absence of detectable contaminants and adventitious
agents should be included.

III. Preparation of Primary Cell Substrates

    Methods used for isolation of cells from tissue, establishment
of primary cell cultures, and maintenance of cultures should be
described.

IV. Testing of Primary Cell Substrates

    Tests performed on primary cell substrates to qualify them for
use in production should be described. As noted, the nature of
primary cell substrates precludes extensive testing and
characterization prior to use. Testing to demonstrate the absence of
adventitious agents in these substrates is therefore conducted
concurrently and may include: Observation of production or
uninfected control cultures before, during, and beyond the period of
production; inoculation of culture fluids from production and
uninfected control cultures into various susceptible indicator cell
cultures capable of detecting a wide range of relevant viruses,
followed by examination for cytopathic changes and testing for the
presence of hemadsorbing viruses; and other tests for specific
agents (such as relevant retroviruses) as necessary. Additional
information concerning specific viral tests may be found in the
relevant national/regional/international guidances.
    Appropriate testing regimens and test methods for cells used in
the production of specific products will vary depending on the donor
species used as a source of tissue, adventitious agents potentially
present, the nature of the product, its intended clinical use,
aspects of the manufacturing process, and the extent of testing
performed on the final product. Applicants should explain and
justify the approach taken with respect to their specific product.

    Dated: August 28, 1998.
 William B. Schultz,
 Deputy Commissioner for Policy.
[FR Doc. 98-25108 Filed 9-18-98; 8:45 am]
BILLING CODE 4160-01-F