[Federal Register Volume 62, Number 85 (Friday, May 2, 1997)]
[Notices]
[Pages 24312-24317]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 97-11441]


      
      

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Part IX





Department of Health and Human Services





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Food and Drug Administration



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

  Federal Register / Vol. 62, No. 85 / Friday, May 2, 1997 / Notices  

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DEPARTMENT OF HEALTH AND HUMAN SERVICES

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


International Conference on Harmonisation; Draft Guideline 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.

-----------------------------------------------------------------------

SUMMARY: The Food and Drug Administration (FDA) is publishing a draft 
guideline entitled ``Quality of Biotechnological/Biological Products: 
Derivation and Characterization of Cell Substrates Used for Production 
of Biotechnological/Biological Products.'' The draft guideline was 
prepared under the auspices of the International Conference on 
Harmonisation of Technical Requirements for Registration of 
Pharmaceuticals for Human Use (ICH). The draft guideline provides 
guidance on appropriate standards for the derivation and 
characterization of cell substrates used in the production of 
biotechnological/biological products, and provides recommendations on 
the information in these areas that should be presented in marketing 
applications.
DATES: Written comments by June 16, 1997.

ADDRESSES: Submit written comments on the draft guideline to the 
Dockets Management Branch (HFA-305), Food and Drug Administration, 
12420 Parklawn Dr., rm. 1-23, Rockville, MD 20857. Copies of the draft 
guideline 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 draft guideline may be obtained by mail from the Office of 
Communication, Training and Manufacturers Assistance (HFM-40), Center 
for Biologics Evaluation and Research (CBER), 1401 Rockville Pike, 
Rockville, MD 20852-1448 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 guideline: Ruth H. Wolff, Center for Biologics 
Evaluation and Research (HFM-594), Food and Drug Administration, 1401 
Rockville Pike, Rockville, MD 20852, 301-827-5103.
    Regarding the 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 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.
    On January 10, 1997, the ICH Steering Committee agreed that a draft 
guideline entitled ``Quality of Biotechnological/Biological Products: 
Derivation and Characterization of Cell Substrates Used for Production 
of Biotechnological/Biological Products'' should be made available for 
public comment. The draft guideline is the product of the Quality 
Expert Working Group of the ICH. Comments about this draft will be 
considered by FDA and the Quality Expert Working Group.
    The draft guideline provides 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 draft guideline recommends information in these areas 
that should be presented in marketing applications for 
biotechnological/biological products.
    This guidance document 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.
    Interested persons may, on or before June 16, 1997, submit to the 
Dockets Management Branch (address above) written comments on the draft 
guideline. 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 
draft guideline 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 guideline is available via Internet using the World Wide Web 
(WWW) (http://www.fda.gov/cder/guidance.htm). To connect to CBER's WWW 
site, type http://www.fda.gov/cber/cberftp.html.
    The text of the draft guideline follows:

Quality of Biotechnological/Biological Products: Derivation and 
Characterization of Cell Substrates Used for Production of 
Biotechnological/Biological Products

1.0 Introduction

1.1 Objective

    The objective of this guideline 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

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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 guidelines 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 guideline 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 product 
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 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 which 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 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 Source

    The source of cells (laboratory or culture collection) from 
which the cell substrate was derived should be stated, the materials 
and methods used should be described, 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.

2.1.3 Cell History

    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 adequate.
    Regarding the generation of cell substrates, applicants should 
provide a thorough discussion of procedures which 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 will be part of the risk-benefit analysis of the 
product.

2.1.4 Generation of the Cell Substrate

    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 database of information on which the quality 
assessment of the Master Cell Bank (MCB) can be built. 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) guidelines. 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.

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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, an 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 which 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 a 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 an MCB, and it is used as the source of cell substrate 
for production. Host, plasmid, 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 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 and the 
cell bank containers to be traced. This should include a description 
of the labeling system which withstands 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 which 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 ultralow 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 establishment 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 once as part of process validation for each product to 
be registered. In addition, tests of purity and limited tests of 
identity should be performed once on each WCB. 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 guideline 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

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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 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 adventious 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, any of the current methodologies described in the European 
Pharmacopoeia (EP), the Japanese Pharmacopoeia (JP), or the U.S. 
Pharmacopeia (U.S.P.) for testing microbial limits or microbial 
sterility are considered adequate.
    Tests for the presence of both cultivatable and nonagar 
cultivatable 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. Suggested current methods for mycoplasma testing are 
described in either EP, JP, or ``Points to Consider in the 
Characterization of Cell Lines Used to Produce Biologicals'' (FDA, 
CBER, 1993). 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. Testing cells derived from a single 
container is generally considered adequate. 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 guideline on viral safety. For product 
classes not covered by the viral safety guideline, 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 is present in the cell banking room at 
the same time that open cell banking procedures are 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 is 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 which 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 for 
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 need only be 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 in vitro cell age limit for production use or 
beyond by either nucleic acid testing or product analysis, as 
described in the relevant ICH guideline. 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 in vitro age limit 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 the in vitro cell age limit is difficult or 
impossible, one may compare the characteristics of cells at the 
initial stages of cultivation or production to those of cells at the 
in vitro cell age limit for production use in order to assess cell 
stability during production. Indices such as, for example, oxygen or 
glucose consumption rates,

[[Page 24316]]

ammonia or lactate production rates may be useful for such testing. 
Increases in the defined limit for in vitro cell age for production 
use should be supported by data from cells that have been expanded 
to the proposed new in vitro cell age limit. For diploid cell lines, 
data should be presented that established 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 met consistently either by 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 is 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. Methods for karyological and tumorigenicity analyses may be 
found in the current WHO document on animal cells.

3. 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 which 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 with 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) to 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 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

Annex to Quality of Biotechnological/Biological Products: Derivation 
and Characterization of Cell Substrates Used for Production of 
Biotechnological/Biological Products

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 assure 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 guidelines.
    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.

[[Page 24317]]

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 guidelines.
    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: April 25, 1997.
William K. Hubbard,
Associate Commissioner for Policy Coordination.
[FR Doc. 97-11441 Filed 5-1-97; 8:45 am]
BILLING CODE 4160-01-F