50244 Federal Register/Vol. 63, No. 182/Monday, September 21, 1998/Notices
314.550 are reported and approved In the Federal Register of May 28, The estimated PRA reporting burden for
under OMB control number 09100376, 1998 (63 FR 29229), the agency 314.50 reflects an anticipated
which published in the Federal Register requested comments on the proposed reduction of 300 hours in burden.
at 62 FR 55408 and is not included in collection of information. No comments Based on the information provided by
the hour burden estimates in Table 1 of were received. the pharmaceutical industry for the
this document.) Elsewhere in this issue of the Federal number of hours per response, on FDAs
Respondents to this collection of Register, the agency has published a estimate of the reduction in reporting
information are all persons who submit Notice of Availability of a Draft resulting from the draft guidance for
an application or abbreviated Guidance for Industry on Submission of submitting abbreviated reports and
application or an amendment or Abbreviated Reports and Synopses in synopses for marketing applications,
supplement to FDA under part 314, to Support of Marketing Applications in and on FDAs prior experience with
obtain approval of a new drug or accordance with section 118 of the FDA respondents, the number of responses
antibiotic drug, and any person who Modernization Act. The goal of the draft per respondent, and the number of total
owns an approved application or guidance is to reduce the submission annual responses, FDA estimates the
abbreviated application. burden of applicants where appropriate. burden of this collection as follows:
TABLE 1.ESTIMATED ANNUAL REPORTING BURDEN1
No. of
21 CFR Section No. of Total Annual Hours per
Responses per Total Hours
[Form Number] Respondents Responses Response
Respondents
314.50 (b), (c), (d), (e), (f), and (k) 83 1.49 124 1,300 161,200
314.54 4 1.25 5 300 1,500
314.60 144 16.89 2,432 80 194,560
314.65 18 1.28 23 2 46
314.70 and 314.71 418 5.33 2,229 300 668,700
314.72 59 2.17 128 2 256
314.81(b)(1) [3331] 140 5 700 48 33,600
314.81(b)(2) [2252] 269 9.06 2,438 40 97,520
314.94(a) and (d) 117 3.96 464 480 222,720
314.96 315 12.43 3,915 80 313,200
314.97 152 19.74 3,000 80 240,000
314.98(c) [2252] 265 17.17 4,551 40 182,040
314.99(a) 46 13.04 600 2 1,200
314.110(a)(5) 55 1.13 62 8 496
314.120(a)(5) 26 1.12 29 8 232
314.420 450 1.11 500 8 4,000
Total 2,121,270
1There are no capital costs or operating and maintenance costs associated with this collection of information.
Dated: September 9, 1998. Biotechnological/Biological Products: Administration, 5600 Fishers Lane,
William K. Hubbard, Derivation and Characterization of Cell Rockville, MD 20857, 3018274573.
Associate Commissioner for Policy Substrates Used for Production of Single copies of the guidance may be
Coordination. Biotechnological/Biological Products. obtained by mail from the Office of
[FR Doc. 9825142 Filed 91898; 8:45 am] The guidance was prepared under the Communication, Training and
auspices of the International Conference Manufacturers Assistance (HFM40),
BILLING CODE 416001F
on Harmonisation of Technical Center for Biologics Evaluation and
Requirements for Registration of Research (CBER), or by calling the CBER
DEPARTMENT OF HEALTH AND Pharmaceuticals for Human Use (ICH). Voice Information System at 1800
HUMAN SERVICES The document provides broad guidance 8354709 or 3018271800. Copies may
on appropriate standards for the be obtained from CBERs FAX
Food and Drug Administration derivation and characterization of cell Information System at 1888CBER
substrates used in the production of FAX or 3018273844.
[Docket No. 97D0159]
biotechnological/biological products FORFURTHERINFORMATIONCONTACT:
International Conference on and recommends information in these Regarding the guidance: Neil D.
Harmonisation; Guidance on Quality of areas that should be presented in Goldman, Center for Biologics
Biotechnological/Biological Products: marketing applications. Evaluation and Research (HFM20),
Derivation and Characterization of Cell DATES: Effective September 21, 1998. Food and Drug Administration,
Substrates Used for Production of Submit written comments at any time. 1401 Rockville Pike, Rockville, MD
Biotechnological/Biological Products; ADDRESSES: Submit written comments 20852, 3018270377.
Availability on the guidance to the Dockets Regarding ICH: Janet J. Showalter,
AGENCY: Food and Drug Administration, Management Branch (HFA305), Food Office of Health Affairs (HFY20),
HHS. and Drug Administration, 5630 Fishers Food and Drug Administration,
ACTION: Notice. Lane, rm. 1061, Rockville, MD 20852. 5600 Fishers Lane, Rockville, MD
Copies of the guidance are available 20857, 3018270864.
SUMMARY: The Food and Drug from the Drug Information Branch SUPPLEMENTARYINFORMATION: In recent
Administration (FDA) is publishing a (HFD210), Center for Drug Evaluation years, many important initiatives have
guidance entitled Q5D Quality of and Research, Food and Drug been undertaken by regulatory Federal Register/Vol. 63, No. 182/Monday, September 21, 1998/Notices 50245
authorities and industry associations to The document provides broad and microbial cells to be used to prepare
promote international harmonization of guidance on appropriate standards for biotechnological/biological products defined
regulatory requirements. FDA has the derivation of human and animal cell in section 1.3, Scope, and for the preparation
and characterization of cell banks to be used
participated in many meetings designed lines and microbial cells to be used to
for production. The document, therefore,
to enhance harmonization and is prepare biotechnological/biological
provides recommendations on the
committed to seeking scientifically products and for the preparation and
information in these areas that should be
based harmonized technical procedures characterization of cell banks to be used presented in marketing applications for these
for pharmaceutical development. One of for production. The guidance products.
the goals of harmonization is to identify recommends information in these areas 1.2 Rationale
and then reduce differences in technical that should be presented in marketing
Historically, some quality concerns for
requirements for drug development applications for these products.
cell-derived biological products have
among regulatory agencies. This guidance represents the agencys originated from the presence of adventitious
ICH was organized to provide an current thinking on standards for the contaminants or from the properties of the
opportunity for tripartite harmonization derivation and characterization of cell cells used to prepare the product.
initiatives to be developed with input substrates used for production of Recombinant DNA (rDNA)-derived products
from both regulatory and industry biotechnological/biological products. It also carry quality concerns regarding the
representatives. FDA also seeks input does not create or confer any rights for, expression construct contained in the cell
from consumer representatives and or on, any person and does not operate substrate. Thus, it is well established that the
others. ICH is concerned with to bind FDA or the public. An properties of the cell substrate and events
linked to the cell substrate can affect
alternative approach may be used if
harmonization of technical resultant product quality and safety and,
such approach satisfies the
requirements for the registration of further, that effective quality control of these
requirements of the applicable statute,
pharmaceutical products among three products requires appropriate controls on all
regions: The European Union, Japan, regulations, or both. aspects of handling the cell substrate.
and the United States. The six ICH As with all of FDAs guidances, the This document complements other
public is encouraged to submit written guidances to provide a comprehensive
sponsors are the European Commission,
comments with new data or other new approach to quality issues arising from
the European Federation of
information pertinent to this guidance. biological aspects of processing products
Pharmaceutical Industries Associations,
The comments in the docket will be from metazoan and microbial cell culture.
the Japanese Ministry of Health and
periodically reviewed, and, where 1.3 Scope
Welfare, the Japanese Pharmaceutical
Manufacturers Association, the Centers appropriate, the guidance will be This guidance covers cell substrates having
amended. The public will be notified of a cell banking system. In this document, cell
for Drug Evaluation and Research and
any such amendments through a notice substrate refers to microbial cells or cell
Biologics Evaluation and Research,
in the Federal Register. lines derived from human or animal sources
FDA, and the Pharmaceutical Research
Interested persons may, at any time, that possess the full potential for generation
and Manufacturers of America. The ICH submit written comments on the of the desired biotechnological/biological
Secretariat, which coordinates the guidance to the Dockets Management products for human in vivo or ex vivo use.
preparation of documentation, is Reagents for in vitro diagnostic use are
Branch (address above). Two copies of
provided by the International outside the scope of this document. Animal
any comments are to be submitted,
Federation of Pharmaceutical sources of cell lines include all those of
except that individuals may submit one
Manufacturers Associations (IFPMA). metazoan origin. Both continuous cell lines
copy. Comments are to be identified of indefinite in vitro lifespan and diploid
The ICH Steering Committee includes
with the docket number found in cells of finite in vitro lifespan are included.
representatives from each of the ICH brackets in the heading of this Microbial sources include bacteria, fungi,
sponsors and the IFPMA, as well as document. The guidance and received yeast, and other unicellular life forms.
observers from the World Health comments may be seen in the office Biotechnological/biological products
Organization, the Canadian Health above between 9 a.m. and 4 p.m., refers to any products prepared from cells
Protection Branch, and the European Monday through Friday. An electronic cultivated from cell banks with the exception
Free Trade Area. version of this guidance is available on of microbial metabolites such as, for
In the Federal Register of May 2, 1997 the Internet at http://www.fda.gov/ example, antibiotics, amino acids,
carbohydrates, and other low molecular
(62 FR 24312), FDA published a draft cder/guidance/index.htm or http://
weight substances. Cell banks used to
tripartite guideline entitled Quality of www.fda.gov/cber/publications.htm. prepare gene therapy products or vaccines
Biotechnological/Biological Products: The text of the guidance follows: should follow the recommendations
Derivation and Characterization of Cell presented in this document. Some biological
Q5D Quality of Biotechnological/Biological
Substrates Used for Production of products, such as certain viral vaccines, are
Products: Derivation and Characterization of
Biotechnological/Biological Products. prepared in primary cell cultures derived
Cell Substrates Used for Production of
The notice gave interested persons an directly from animal tissues or organs.
Biotechnological/Biological Products1
opportunity to submit comments by Primary cells are not banked and therefore
June 16, 1997. 1.0 Introduction are not addressed by this document.
After consideration of the comments 1.1 Objective However, other considerations which may
apply to primary cells are discussed further
received and revisions to the guidance, The objective of this guidance is to provide
in Appendix 1 of this document.
a final draft of the guidance was broad guidance on appropriate standards for
submitted to the ICH Steering the derivation of human and animal cell lines 2.0 Guidelines
Committee and endorsed by the three 2.1.0 Source, History, and Generation of the
participating regulatory agencies on July 1This guidance represents the agencys current Cell Substrate
thinking on standards for the derivation and
17, 1997.
characterization of cell substrates used for 2.1.1 Introduction
In accordance with FDAs Good production of biotechnological/biological products.
It is important to provide supportive
Guidance Practices (62 FR 8961, It does not create or confer any rights for or on any
documentation which describes the history
February 27, 1997), this document has ppeurbsloicn.  aAnnd  adloteersn natoitv oe paeprparteo atcoh b minady F bDeA u soerd t hife such of the cell substrate that is used in the
been designated a guidance, rather than approach satisfies the requirements of the manufacture of a biotechnological/biological
a guideline. applicable statute, regulations, or both. product, as well as any parental cell line 50246 Federal Register/Vol. 63, No. 182/Monday, September 21, 1998/Notices
from which it was totally or partially derived. doublings during all stages of research, the preserved bank of cells. Manufacturers
Events during the research and development development, and manufacturing is may prepare their own cell banks or may
phases of the cell substrate may contribute important. For microbial cells, obtain them from external sources.
significantly to assessment of the risks documentation of subcultivation frequency Manufacturers are responsible for ensuring
associated with the use of that particular cell after cell substrate generation is considered the quality of each cell bank and of the
substrate for production. The information adequate. testing performed on each bank.
supplied in this regard is meant to facilitate Regarding the generation of cell substrates,
2.2.1 Cell Banking System
an overall evaluation that will ensure the applicants should provide a thorough
quality and safety of the product. discussion of procedures that would provide The concept of a two-tiered cell bank, in
Careful records of the manipulation of the exposure to infectious agents. Constituents of which the MCB is used to generate Working
cell substrate should be maintained the culture medium should be described, in Cell Banks (WCBs), is generally accepted as
throughout its development. Description of particular, information regarding exposure of the most practical approach to providing a
cell history is only one tool of many used for the cells to materials of human or animal supply of cell substrate for continued
cell substrate characterization. In general, origin such as serum, enzymes, hydrolysates, manufacture of the product. Manufacturers
deficiencies in documented history may not, or other living cells. The description should should describe their strategy for providing a
by themselves, be an impediment to product include the source, method of preparation continued supply of cells from their cell
approval, but extensive deficiencies will and control, test results, and quality bank(s), including the anticipated utilization
result in increased reliance on other methods assurance. Relevant literature on these points rate of the cell bank(s) for production, the
to characterize the cell substrate. may be referenced when available. This expected intervals between generation of new
2.1.2 Origin, Source, and History of Cells information will allow a detailed analysis of cell bank(s), and the criteria for qualification
potential entry routes for adventitious agents of cell bank(s).
The source of cells (laboratory or culture
from these sources, and would be part of the Generally, the MCB is made first, usually
collection) from which the cell substrate was
risk-benefit analysis of the product. directly from an initial clone or from a
derived should be stated, and relevant
references from the scientific literature 2.1.3 Generation of the CelpaSubstrate preliminary cell bank derived from an initial
clone. It is not considered necessary to
should be cited. Information obtained A crucial step is the choice of a suitable
prepare cell banks from clones for certain
directly from the source laboratory is parental cell line. For recombinant products,
types of cells (e.g., diploid cells, where
preferred. When this is not available, a parental cell line is typically the
limited in vitro life span or other technical
literature references may be utilized. untransfected recipient cell line. The use of
factors make cell cloning impractical) or
For human cell lines, it is relevant to characterized parental cell banks is
where the uncloned cell population is
describe the following characteristics of the suggested, but is not considered essential. A
already adequately homogeneous for the
original donor: Tissue or organ of origin, characterized parental cell bank may be of
intended use.
ethnic and geographical origin, age, sex, and benefit, especially when multiple cell
A WCB is derived from one or more
general physiological condition. If known, substrates are generated from the same
containers of the MCB. It is the WCB that is
the state of health or medical history of the parental cell type, by providing a set of
typically used to directly provide cells for the
donor should be reported along with the information on which the quality assessment
manufacturing process. Additional WCBs are
results of any tests of the donor for of the Master Cell Bank (MCB) can be based.
generated from the MCB as needed. A newly
pathogenic agents. Specifically for human For example, the myeloma cell line may be
prepared WCB should be appropriately
diploid fibroblasts, the age of the donor may banked as a parental cell line for hybridomas.
qualified by characterization and testing.
influence the in vitro lifespan of the cell line During the generation of the cell substrate,
It should be noted that the MCB and WCB
and this information should be provided if one or more specific procedures may be
may differ from each other in certain
available. For animal cell lines, relevant utilized in the ultimate development of the
respects, e.g., culture components and
descriptions of the source include species, desired characteristics. These may include,
culture conditions. Similarly, the culture
strains, breeding conditions, tissue or organ for example, cell fusion, transfection,
conditions used to prepare the MCB and
of origin, geographical origin, age and sex, selection, colony isolation, cloning, gene
WCB may differ from those used for the
the results of tests for pathogenic agents, and amplification, and adaptation to specific
general physiological condition of the culture conditions or media. Information production process. If changes in cell culture
original donor. regarding the methodologies utilized in process do not affect product quality, it is not
For microbes, manufacturers should developing the cell substrate can help to considered necessary to reclone the cells or
describe the species, strain, and known provide a clear understanding of the history to rebank the MCB or WCB. It is important
genotypic and phenotypic characteristics of of the cell substrate. Some cell substrates, that a characterized bank provides a
the organism from which the cell substrate such as human diploid fibroblasts, may not consistent product.
was derived. Manufacturers should also need extensive manipulation or cloning prior A single-tiered banking system consisting
describe the pathogenicity, toxin production, to cell banking. only of the MCB but no WCBs could be used
and other biohazard information, if any. For recombinant products, the cell in principle, for example, if relatively few
The cultivation history of the cells should substrate is the transfected cell containing containers were needed each year to produce
be documented. The method originally used the desired sequences, which has been the desired product.
for the isolation of the cells should be cloned from a single cell progenitor. For In some microbial expression systems, a
described as well as the procedures used in further information on generation of rDNA- new transformation is performed for each
the culturing of the cells in vitro and any modified cell substrates, consult other new cell substrate container lot, based upon
procedures used to establish cell lines (for relevant (e.g., regional or international) using aliquots of thoroughly tested host cell
example, use of any physical, chemical, or guidances. For nonrecombinant products or banks and plasmid banks for each new
biological procedure, or added nucleotide nonrecombinant vaccines, the cell substrate transformation and on testing of each
sequences). A description of any genetic is the cell from the parental cell line chosen transformed cell substrate bank. This
manipulation or selection should be for preparation of the MCB without further transformed cell substrate bank is considered
provided. All available information regarding modification. For products derived from the MCB, and it is used as the source of cell
the identification, characteristics, and results hybridomas, the cell substrate is the substrate for production. Host cell banks,
of testing of these cells for endogenous and hybridoma cell line derived by fusion of the plasmid banks, and MCBs are maintained by
adventitious agents should be provided. parental myeloma cell line with other appropriate preservation methods. This
For continuous cell lines of metazoan parental cells, e.g., immune spleen cells. alternative system is considered adequate
origin, it is usually adequate to quantitate because the transformation of bacteria and
2.2.0 Cell Banking
culture duration by estimation of either yeast is generally a very reproducible and
number of population doublings, or number One of the most important advantages of easily performed process, unlike the events
of subcultivations at defined dilution ratio, or using serially subcultivated cells to produce needed for transfection of metazoan cells.
time in days. For diploid cell lines biotechnological/biological products is the Manufacturers should provide information
possessing finite in vitro lifespan, accurate ability to have a characterized common on the host cells, rDNA molecules (such as
estimation of the number of population starting source for each production lot, i.e., plasmids), method of transformation and of Federal Register/Vol. 63, No. 182/Monday, September 21, 1998/Notices 50247
cell banking, and the results of for storage units, storage of a portion of the The manufacturer may choose to
characterization studies. MCB and WCB at remote sites, or characterize the WCB instead of the MCB, if
2.2.2 Cell Banking Procedures regeneration of the MCB. justified.
It is important to prevent a contaminated The starting point of reference for estimates 2.3.1.0 Tests of Identity
of in vitro cell age during manufacturing
cell substrate (or bank) from being used in Appropriate tests should be performed to
should be the thawing of one or more
production and to avoid a loss of product determine that the banked cell is what it is
containers of the MCB. For diploid cell lines,
availability or development time resulting represented to be. Either phenotypic or
in vitro lifespan should be estimated in terms
from the need to recreate a cell bank found genotypic characteristics may be used in
of population doubling levels. The
to be unusable due to contamination. It is identity testing. It is not considered
recognized that no cell bank testing regimen population doubling level at which necessary to do all the possible tests. Tests
is able to detect all potential contaminants; senescence occurs should be determined for of identity are generally performed on the
therefore, use of these preventive principles diploid cells. MCB. In addition, limited identity testing is
during cell banking is important to provide 2.3.0 General Principles of Characterization generally performed on each WCB.
reasonable assurance of the absence of and Testing of Cell Banks 2.3.1.1 Metazoan Cells
contamination and to provide a reliable
source of the cell substrate. The characterization and testing of banked For human or animal cells that grow
Manufacturers should describe the type of cell substrates is a critical component of the attached to a substratum, morphological
banking system used, the size of the cell control of biotechnological and biological analysis may be a useful tool in conjunction
bank(s), the container (vials, ampules, or products. Characterization of the MCB allows with other tests. In most cases, isoenzyme
other appropriate vessels) and closure system the manufacturer to assess this source with analysis is sufficient to confirm the species
used, the methods used for preparation of the regard to presence of cells from other lines, of origin for cell lines derived from human
cell bank(s) including the cryoprotectants adventitious agents, endogenous agents and or animal sources; other tests may be
and media used, and the conditions molecular contaminants (e.g., toxins or appropriate depending on the history of the
employed for cryopreservation and storage. antibiotics from the host organism). The cell line. Other technologies may be
Manufacturers should describe the objective of this testing is to confirm the substituted to confirm species of origin,
procedures used to avoid microbial identity, purity, and suitability of the cell including, for example, banding cytogenetics
contamination and cross-contamination by substrate for manufacturing use. In some or use of species-specific antisera. An
other cell types present in the laboratory and cases, additional testing such as alternative strategy would be to demonstrate
the procedures that allow the cell bank tumorigenicity or karyology may be useful. the presence of unique markers, for example,
containers to be traced. This should include The testing program chosen for a given cell by using banding cytogenetics to detect a
a description of the documentation system as substrate will vary according to the biological unique marker chromosome, or DNA analysis
well as that of a labeling system that can properties of the cells (for example, growth to detect a genomic polymorphism pattern
withstand the process of preservation, requirements), its cultivation history (for example, restriction fragment length
storage, and recovery from storage without (including use of human-derived and animal- polymorphism, variable number of tandem
loss of labeling information on the container. derived biological reagents), and available repeats, or genomic dinucleotide repeats).
Manufacturers should describe their cell testing procedures. The extent of Either confirmation of species of origin or
banking procedures. Cells are generally characterization of a cell substrate may presence of known unique cell line markers
prepared for banking by expanding cultures influence the type or level of routine testing is considered an adequate test of identity.
in a progressively greater number or larger needed at later stages of manufacturing. Expression of the desired product may
size of vessel until a pool of cells can be Manufacturers should perform tests for represent a complementary approach to
obtained that is sufficient to generate enough identity and purity once for each MCB and confirmation of identity.
containers for the bank. To ensure the tests of stability during cell cultivation once 2.3.1.2 Microbial Cells
uniform composition of the contents of each for each product to be registered. In addition, For most microbial cells, analysis of
container, a single pool of cells for banking tests of purity and limited tests of identity growth on selective media is usually
should be prepared by combining the cells should be performed once on each WCB. adequate to confirm host cell identity at the
from all of the culture vessels, if more than
Also, applicants should consult the ICH species level for the host cell bank and the
one vessel is used.
guidance on viral safety. Relevant tests transformed cell bank. For E. coli, where a
Cells suspended in preservation medium
among those described below should be variety of strains may be used, biological
are aliquoted from the single pool into
performed and described in the marketing characterization methods such as phage
sterilized containers which are then sealed
application, along with the results of the typing should be considered as
and stored under appropriate conditions. For
testing. supplementary tests of identity. For plasmid
example, animal cells in media containing a
For cell lines containing exogenously banks, identity assessment can be
cryoprotectant are frozen in the sealed
assembled expression constructs, the relevant accomplished as described by the ICH
containers under defined and controlled
ICH guidance on rDNA expression constructs document on analysis of the expression
conditions and then transferred to storage in
should be consulted for guidance on the construct. Expression of the desired product
the vapor or liquid phase of liquid nitrogen
characterization of nucleotide and amino is also considered adequate to confirm the
or at equivalent ultra low temperatures.
acid sequences. It may also be useful to identity of the microbial expression system.
Other methods of preservation and storage
may be adequate depending on the organism examine, by similar methods, the coding 2.3.2.0 Tests of Purity
sequences in some nonrecombinant DNA-
used, but they should be capable of A critical aspect of cell development and
maintaining a level of cell viability upon derived cell lines where the gene sequences banking is the assessment that the MCB and
reconstitution that is both consistent and have been characterized and are well WCB are biologically pure, i.e., are free from
adequate for production use. understood. However, it is not considered adventitious microbial agents and
To ensure continuous, uninterrupted necessary to carry out investigations of the adventitious cellular contaminants. The
production of pharmaceuticals, sequences encoding complex natural impact of selective agents and antibiotics on
manufacturers should carefully consider the products, for example, families of related the detection of adventitious microbial
steps that can be taken to provide for gene products, microbial vaccine antigens, or contaminants should be considered when
protection from catastrophic events that monoclonal antibodies from hybridomas. planning and performing these tests.
could render the cell bank unusable. Manufacturers are also encouraged to
Examples of these events include fires, power employ state-of-the-art methods and 2.3.2.1 Metazoan Cells
outages, and human error. Manufacturers technological improvements in cell substrate Tests for the presence of bioburden
should describe their plans for such characterization and testing as they become (bacteria and fungi) should be performed on
precautions; for example, these may include available, as long as the specificity, individual containers (1 percent of the total
redundancy in the storage of bank containers sensitivity, and precision of the newer number but not less than two containers) of
in multiple freezers, use of back-up power, methods are at least equivalent to those of the MCB and WCB. In all other aspects, the
use of automatic liquid nitrogen fill systems existing methods. current methodologies described in either the 50248 Federal Register/Vol. 63, No. 182/Monday, September 21, 1998/Notices
European Pharmacopoeia (Ph. Eur.), the example, visual examination of the difficult or impossible, one may compare the
Japanese Pharmacopoeia (JP), or the U.S. characteristics of well-isolated colonies is characteristics of cells at the initial stages of
Pharmacopoeia (U.S.P.) for testing microbial suggested, using several microbiological cultivation or production to those of cells at
limits or microbial sterility may be media, of which some do and some do not or beyond the limit of in vitro cell age for
considered adequate. support growth of the cell substrate. production use in order to assess cell
Tests for the presence of mycoplasma However, it is not intended that stability during production. Indices such as,
should be performed on the MCB and WCB. manufacturers necessarily characterize for example, oxygen or glucose consumption
Current procedures considered adequate resistant mutants of the cell substrate arising rates, ammonia or lactate production rates
include both the agar and broth media from such studies, or other artifacts of such may be useful for such testing. Increases in
procedures as well as the indicator cell assays. Rather, the purpose of such assays is the defined limit of in vitro cell age for
culture procedure. Current methods for to detect existing contaminants. production use should be supported by data
mycoplasma testing are described in Ph. Eur., 2.3.3 Cell Substrate Stability from cells which have been expanded to the
JP, and Points to Consider in the proposed new limit of in vitro cell age. For
Characterization of Cell Lines Used to Another dimension to cell characterization diploid cell lines, data should be presented
Produce Biologicals (FDA, CBER, 1993). is appropriateness for intended use in that establish the finite in vitro lifespan of
Testing cells derived from a single container production. There are two concerns for cell
is generally considered adequate. For substrate stability: Consistent production of the cells from the WCB under conditions
nonmammalian animal cell lines, alternative the intended product and retention of representative of those employed for
controls and/or assay conditions may be production capacity during storage under manufacturing use.
appropriate; manufacturers should consult defined conditions. Evidence for banked cell stability under
with the national/regional regulatory For the evaluation of stability during defined storage conditions will usually be
authority for appropriate methodology. cultivation for production, at least two time generated during production of clinical trial
If future efforts to harmonize bioburden points should be examined, one using cells material from the banked cells. Data from the
and mycoplasma assays are fruitful, then the that have received a minimal number of determination of cell viability when the
scientifically appropriate harmonized assay subcultivations, and another using cells at or preserved cells are reconstituted for
should be used. beyond the limit of in vitro cell age for production of clinical trial supplies will
Virus testing of cell substrates should be production use described in the marketing verify that the revived cells have survived the
designed to detect a wide spectrum of viruses application. The limit of in vitro cell age for preservation process. Data from the
by using appropriate screening tests and production use should be based on data preparation of clinical materials will
relevant specific tests, based on the derived from production cells expanded demonstrate that the revived cells can be
cultivation history of the cell line, to detect under pilot plant scale or commercial scale used to prepare the desired product.
possible contaminating viruses. Applicants conditions to the proposed limit of in vitro Available data should be clearly documented
should consult the ICH guidance on viral cell age for production use or beyond. in the application dossiers, plus a proposal
safety. For product classes not covered by the Generally, the production cells are obtained for monitoring of banked cell stability should
viral safety guidance, the current World by expansion of cells from the WCB; cells be provided. The proposed monitoring can be
Health Organization (WHO) documents for from the MCB could be used with performed at the time that one or more
use of animal cells may be consulted. appropriate justification. This demonstration containers of the cryopreserved bank is
The purity of cell substrates can be of cell substrate stability is commonly thawed for production use, when the product
compromised through contamination by cell performed once for each product marketing or production consistency is monitored in a
lines of the same or different species of application. relevant way, or when one or more
origin. The choice of tests to be performed Evaluation of the cell substrate with containers of the cryopreserved MCB is
depends upon whether opportunities have respect to the consistent production of the thawed for preparation of a new WCB (and
existed for cross-contamination by other cell intended product of interest should be the the new WCB is properly qualified), as
lines. In some cases, it may be necessary to primary subject of concern. The type of appropriate. In the case when production
maintain growing cultures of different cell testing and test article(s) used for such does not take place for a long period of time,
lines in the same laboratory. During assessments will depend on the nature of the viability testing on the cell bank used as a
procedures in cell banking where open cell substrate, the cultivation methods, and source of the production substrate should be
manipulations are performed, care should be the product. For cell lines containing performed at an interval described in the
taken to ensure that simultaneous open recombinant DNA expression constructs, marketing application. If the viability of the
manipulations of other cell lines are avoided consistency of the coding sequence of the cell substrate is not significantly decreased,
to prevent cross-contamination. Whenever expression construct should be verified in generally no further testing of the MCB or
another cell line was present in the cell cells cultivated to the limit of in vitro cell age WCB is considered necessary.
banking room at the same time that open cell for production use or beyond by either 2.3.4 Tests for Karyology and Tumorigenicity
banking procedures were being performed nucleic acid testing or product analysis, as
(such as cell expansion, pooling, or described in the relevant ICH guidance. For Utilization of karyology and tumorigenicity
aliquoting of the chosen cell line), the cell nonrecombinant cell lines in which the testing for evaluating the safety of a diploid
banks should be tested for the presence of coding sequence for the desired product has cell line or characterizing a new cell line may
cells from (or products derived from) the already been analyzed at the MCB or WCB be useful depending on the cells, the nature
second cell line. In general, the methods level, invariability of the protein coding of the product, and the manufacturing
described in section 2.3.1.0 to assess cell sequence during production should be process. Extensive analysis to determine the
identity are also considered adequate tests to verified in the production cells cultivated to relative abundance of aneuploid cells has not
detect cross-contamination by other cell the proposed limit of in vitro cell age for been found to be useful. Karyology need not
lines. Additional assurance of lack of cross- production use or beyond by either nucleic be determined for rodent cell lines or new
contamination can be provided by successful acid testing or analysis of the purified protein cell lines known to be nondiploid. However,
preparation of the intended product from the product. cytogenetic analysis may be an adequate
cell substrate. Where the product cannot be analyzed as method to assess cell substrate identity or
described above, other specific traits, which purity as described in sections 2.3.1.0 and
2.3.2.2 Microbial Cells may include, for example, morphological 2.3.2.0. Repetition of tumorigenicity testing
The design and performance of specific characteristics, growth characteristics, for cells with already documented evidence
tests for adventitious microbial agents and biochemical markers, immunological of tumorigenicity is not considered
adventitious cellular contaminants in markers, productivity of the desired product, necessary.
microbial cell banks should take into account or other relevant genotypic or phenotypic For products that are highly purified and
the properties of the banked cell, the likely markers, may be useful for the assessment of that contain no cells, karyology and
contaminants based upon scientific cell substrate stability. In some cases, where tumorigenicity testing are generally not
literature, source, methods and materials direct comparison of the characteristics of the considered necessary, provided that
used for cultivation, and other organisms MCB with those of the production cells at or appropriate limits for residual host cell DNA
present in the banking laboratory. For beyond the limit of in vitro cell age is are shown to be consistently met by either Federal Register/Vol. 63, No. 182/Monday, September 21, 1998/Notices 50249
process validation studies or by lot release as the host cells. For hybridomas, it is typical III. Preparation of Primary Cell Substrates
testing. to also describe the parental cells as the cells
Methods used for isolation of cells from
In general, products for which the presence to be fused.
tissue, establishment of primary cell cultures,
of live cells cannot be excluded or which WCB (Working Cell Bank)The Working
and maintenance of cultures should be
have little downstream purification (for Cell Bank is prepared from aliquots of a
described.
example, some conventional live virus homogeneous suspension of cells obtained
vaccines) will need such characterization of from culturing the MCB under defined IV. Testing of Primary Cell Substrates
the cell substrate. The utility of culture conditions. Tests performed on primary cell substrates
tumorigenicity testing and chromosomal Appendix 1 to qualify them for use in production should
analysis for new cell substrates for be described. As noted, the nature of primary
unpurified products should be evaluated on Primary Cell Substrates cell substrates precludes extensive testing
a case-by-case basis. Use of cell lines known I. Introduction and characterization prior to use. Testing to
to be tumorigenic or to possess abnormal demonstrate the absence of adventitious
karyology should be evaluated in terms of The principles contained in this document agents in these substrates is therefore
risk-benefit for each product application apply in general to biotechnological/ conducted concurrently and may include:
when the product contains cells or when not biological products prepared from Observation of production or uninfected
characterized banked cells. However, a
highly purified. control cultures before, during, and beyond
number of biological products, in particular
Products that are manufactured in the period of production; inoculation of
certain viral vaccines, are prepared using
genetically unmodified MRC5 or WI38 culture fluids from production and
primary cells.
cells do not need characterization of these uninfected control cultures into various
Because primary cell cultures are used
cell substrates by karyology or tumorigenicity susceptible indicator cell cultures capable of
within the first passage after establishment
since extensive characterization has already detecting a wide range of relevant viruses,
from the tissue of origin, it is not possible to
been performed and published for these cell followed by examination for cytopathic
carry out extensive characterization of the
lines. However, for each MRC5 and WI38 changes and testing for the presence of
cells prior to their use as is done for banked
WCB generated, manufacturers should hemadsorbing viruses; and other tests for
cell substrates. In addition, biological
confirm, once, that the cells grown in the specific agents (such as relevant retroviruses)
products produced using primary cell
manner to be used in production are diploid as necessary. Additional information
substrates often do not undergo extensive
and have the expected lifespan. processing (e.g., purification). Despite these concerning specific viral tests may be found
For new or previously uncharacterized differences, the approach taken to ensure the in the relevant national/regional/
diploid cell substrates, confirmation of suitability and safety of primary cell international guidances.
diploid karyology should be presented and substrates for production of biologicals is Appropriate testing regimens and test
tumorigenic potential should be established, analogous, in many respects, to that outlined methods for cells used in the production of
using cells from the MCB. in this document and in other guidances. specific products will vary depending on the
3.0 Glossary This annex outlines cell substrate-related donor species used as a source of tissue,
information that should be included in adventitious agents potentially present, the
Cell bankA cell bank is a collection of marketing applications for biological nature of the product, its intended clinical
appropriate containers, whose contents are of products prepared using primary cells. This use, aspects of the manufacturing process,
uniform composition, stored under defined information falls into three general and the extent of testing performed on the
conditions. Each container represents an categories: (1) Information concerning the final product. Applicants should explain and
aliquot of a single pool of cells. source tissue (or organ) and other animal- justify the approach taken with respect to
Cell lineType of cell population that derived raw materials used for the their specific product.
originates by serial subculture of a primary
cell population, which can be banked. establishment of primary cell substrates, (2) Dated: August 28, 1998.
information concerning the preparation of
Continuous cell lineA cell line having an William B. Schultz,
primary cell substrates, and (3) testing
infinite capacity for growth. Often referred to performed on primary cell substrates to Deputy Commissioner for Policy.
as immortal and previously referred to as
ensure the safety of the product. [FR Doc. 9825108 Filed 91898; 8:45 am]
established.
Diploid cell lineA cell line having a II. Source Tissue and Other Raw Materials BILLING CODE 416001F
finite in vitro lifespan in which the Information should be provided about the DEPARTMENT OF HEALTH AND
chromosomes are paired (euploid) and are animals used as a source of tissue for the HUMAN SERVICES
structurally identical to those of the species preparation of primary cell substrates. Tissue
from which they were derived. should be derived from healthy animals
Food and Drug Administration
Host cellsSee parental cells. subjected to veterinary and laboratory
In vitro cell ageMeasure of time between monitoring to certify the absence of
Medical Gas Workshop
thaw of the MCB vial(s) and harvest of the pathogenic agents. Whenever possible, donor
production vessel measured by elapsed animals should be obtained from closed, AGENCY: Food and Drug Administration,
chronological time, by population doubling specific pathogen-free (when available) HHS.
level of the cells, or by passage level of the colonies or flocks. Animals used as tissue
cells when subcultivated by a defined donors should not have been used previously ACTION: Notice of workshop.
procedure for dilution of the culture. for experimental studies. Animals should be
The Food and Drug Administration
MetazoanOrganism of multicellular adequately quarantined for an appropriate
(FDA) is announcing the following
animal nature. period of time prior to use for the preparation
MCB (Master Cell Bank)An aliquot of a of cells. In some countries, animals may need workshop: Medical Gas Workshop. The
single pool of cells which generally has been to be quarantined in the country where the topics to be discussed are good
prepared from the selected cell clone under primary cells are prepared. Manufacturers manufacturing practices (GMPs) issues
defined conditions, dispensed into multiple should consult with national/regional for the medical gas industry, including
containers, and stored under defined authorities for specific requirements. air liquefaction, both process and
conditions. The MCB is used to derive all Information on materials and components computer validation, transfilling of both
working cell banks. The testing performed on used for the preparation of primary cell
liquid and high pressure cylinders, and
a new MCB (from a previous initial cell substrates should be provided, including the
hospital installations.
clone, MCB, or WCB) should be the same as identity and source of all reagents of human
Date and Time: The workshop will be
for the original MCB unless justified. or animal origin. A description of testing
Parental cellsCells to be manipulated to performed on components of animal origin to held on Tuesday, November 10, 1998,
give rise to a cell substrate or an intermediate certify the absence of detectable 8:30 a.m. to 4:30 p.m.
cell line. For microbial expression systems, it contaminants and adventitious agents should Location: The workshop will be held
is typical to also describe the parental cells be included. at the Century Center, Convention Hall