[Federal Register Volume 60, Number 161 (Monday, August 21, 1995)]
[Notices]
[Pages 43496-43498]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 95-20611]
[[Page 43495]]
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Part II
Department of Health and Human Services
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Food and Drug Administration
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Harmonisation International Conference; Guidelines Availability;
Notices
Federal Register / Vol. 60, No. 161 / Monday, August 21, 1995 /
Notices
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DEPARTMENT OF HEALTH AND HUMAN SERVICES
Food and Drug Administration
[Docket No. 95D-0216]
International Conference on Harmonisation; Draft Guideline on
Analysis of the Expression Construct in Cells Used for the Production
of r-DNA Derived Protein Products; Availability
AGENCY: Food and Drug Administration, HHS.
ACTION: Notice.
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SUMMARY: The Food and Drug Administration (FDA) is publishing a draft
guideline entitled ``Analysis of the Expression Construct in Cells Used
for Production of r-DNA Derived Protein Products.'' This 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 is intended to
describe the types of information that are considered valuable in
assessing the structure of the expression construct used to produce
recombinant deoxyribonucleic acid (r-DNA) derived proteins.
DATES: Written comments by October 5, 1995.
ADDRESSES: Submit written comments on the draft guideline to the
Dockets Management Branch (HFA-305), Food and Drug Administration, rm.
1-23, 12420 Parklawn Dr., Rockville, MD 20857. Copies of the draft
guideline are available from the CDER Executive Secretariat Staff (HFD-
8), Center for Drug Evaluation and Research, Food and Drug
Administration, 7500 Standish Pl., Rockville, MD 20855, as well as the
CBER Congressional and Consumer Affairs Branch (HFM-12), Center for
Biologics Evaluation and Research, Food and Drug Administration, 1401
Rockville Pike, Rockville, MD 20852.
FOR FURTHER INFORMATION CONTACT:
Regarding the guideline: Kenneth Seamon, Center for Biologics
Evaluation and Research (HFM-20), Food and Drug Administration, 1401
Rockville Pike, Rockville, MD 20852, 301-827-0377.
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.
At a meeting held on March 28, 1995, the ICH Steering Committee
agreed that a draft guideline entitled ``Analysis of the Expression
Construct in Cells Used for Production of r-DNA Derived Protein
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 Expert
Working Group. Ultimately, FDA intends to adopt the ICH Steering
Committee's final guideline.
This draft guideline presents guidance regarding the
characterization of the expression construct for the production of r-
DNA protein products in eukaryotic and prokaryotic cells. The draft
guideline is intended to describe the types of information that are
considered valuable in assessing the structure of the expression
construct used to produce recombinant DNA derived proteins. The draft
guideline is not intended to cover the whole quality aspect of r-DNA-
derived medicinal products.
In the past, guidelines have generally been issued under
Sec. 10.90(b) (21 CFR 10.90(b)), which provides for the use of
guidelines to state procedures or standards of general applicability
that are not legal requirements but are acceptable to FDA. The agency
is now in the process of revising Sec. 10.90(b). Therefore, this
guideline is not being issued under the authority of Sec. 10.90(b), and
it does not create or confer any rights, privileges, or benefits for or
on any person, nor does it operate to bind FDA in any way.
Interested persons may, on or before October 5, 1995, 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.
The text of the draft guideline follows:
Analysis of the Expression Construct in Cells Used for Production of r-
DNA Derived Protein Products
I. Introduction
This document presents guidance regarding the characterization
of the expression construct for the production of recombinant DNA
protein products in eukaryotic and prokaryotic cells. This document
is intended to describe the types of information that are considered
valuable in assessing the structure of the expression construct used
to produce recombinant DNA derived proteins. This document is not
intended to cover the whole quality aspect of r-DNA derived
medicinal products.
The expression construct is defined as the expression vector
containing the coding sequence of the recombinant protein. Segments
of the expression construct should be analyzed using nucleic acid
techniques in conjunction with other tests performed on the purified
recombinant protein for assuring the quality and consistency of the
final product. Analysis of the expression construct at the nucleic
acid level should be considered as part of the overall evaluation of
quality, taking into account that this testing only evaluates the
coding sequence of a recombinant gene and not the translational
fidelity nor other characteristics of the recombinant protein, such
as secondary structure, tertiary structure, and post-translational
modifications.
II. Rationale for Analysis of the Expression Construct
The purpose of analyzing the expression construct is to
establish that the correct
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coding sequence of the product has been incorporated into the host cell
and is maintained during culture to the end of production. The
genetic sequence of recombinant proteins produced in living cells
can undergo mutations that could alter the properties of the protein
with potential adverse consequences to patients. No single
experimental approach can be expected to detect all possible
modifications to a protein. Protein analytical techniques can be
used to assess the amino acid sequence of the protein and structural
features of the expressed protein due to post-translational
modifications such as proteolytic processing, glycosylation,
phosphorylation, and acetylation. Data from nucleic acid analysis
may be useful since protein analytical methods may not detect all
changes in protein structure resulting from mutations in the
sequence coding for the recombinant protein. The relative importance
of nucleic acid analysis and protein analysis will vary from product
to product.
Nucleic acid analysis can be used to verify the coding sequence
and the physical state of the expression construct. The nucleic acid
analysis is performed to ensure that the expressed protein will have
the correct amino acid sequence but is not intended to detect low
levels of variant sequences. Where the production cells have
multiple integrated copies of the expression construct, not all of
which may be transcriptionally active, examination of the
transcription product itself by analysis of mRNA or cDNA may be more
appropriate than analysis of genomic DNA. Analytical approaches that
examine a bulk population of nucleic acids, such as those performed
on pooled clones or material amplified by the polymerase chain
reaction, may be considered as an alternative to approaches that
depend on selection of individual DNA clones. Other techniques could
be considered that allow for rapid and sensitive confirmation of the
sequence coding for the recombinant protein in the expression
construct.
The following sections describe information that should be
supplied regarding the characterization of the expression construct
during the development and validation of the production system.
Analytical methodologies should be validated for the intended
purpose of confirmation of sequence. The validation documentation
should at a minimum include estimates of the limits of detection for
variant sequences. This should be performed for either nucleic acid
or protein sequencing methods. The philosophy and recommendations
for analysis expressed in this document should be periodically
reviewed to take advantage of new advances in technology and
scientific information.
III. Characterization of the Expression System
A. Expression Construct and Cell Clone Used to Develop the Master Cell
Bank (MCB)
The manufacturer should describe the origin of the nucleotide
sequence coding for the protein. This should include identification
and source of the cell from which the nucleotide sequence was
originally obtained. Methods used to prepare the DNA coding for the
protein should be described.
The steps in the assembly of the expression construct should be
described in detail. This description should include the source and
function of the component parts of the expression construct, e.g.,
origins of replication, antibiotic resistance genes, promoters,
enhancers, whether or not the protein is being synthesized as a
fusion protein. A detailed component map and a complete annotated
sequence of the plasmid should be given, indicating those regions
that have been sequenced during the construction and those taken
from the literature. Other expressed proteins encoded by the plasmid
should be indicated. The nucleotide sequence of the coding region of
the gene of interest and associated flanking regions that are
inserted into the vector, up to and including the junctions of
insertion, should be determined by DNA sequencing of the construct.
A description of the method of transfer of the expression
construct into the host cell should be provided. In addition,
methods used to amplify the expression construct and criteria used
to select the cell clone for production should be described in
detail.
B. Cell Bank System
Production of the recombinant protein should be based on well-
defined Master and Working Cell Banks. A cell bank is a collection
of ampoules of uniform composition stored under defined conditions
each containing an aliquot of a single pool of cells. The Master
Cell Bank (MCB) is generally derived from the selected cell clone
containing the expression construct. The Working Cell Bank (WCB) is
derived by expansion of one or more ampoules of the MCB. The cell
line history and production of the cell banks should be described in
detail including methods and reagents used during culture, in-vitro
cell age, and storage conditions. All cell banks should be
characterized for relevant phenotypic and genotypic markers which
could include the expression of the recombinant protein or presence
of the expression construct.
The expression construct in the MCB should be analyzed as
described below. If the testing cannot be carried out on the MCB, it
should be carried out on each WCB.
Restriction endonuclease mapping or other suitable techniques
should be used to analyze the expression construct for copy number,
for insertions or deletions, and for the number of integration
sites. For extrachromosomal expression systems, the percent of host
cells retaining the expression construct should be determined.
The protein coding sequence for the recombinant protein product
of the expression construct should be verified. For extrachromosomal
expression systems, the expression construct should be isolated and
the nucleotide sequence encoding the product should be verified
without further cloning. For cells with chromosomal copies of the
expression construct, the nucleotide sequence encoding the product
could be verified by recloning and sequencing of chromosomal copies.
Alternatively, the nucleic acid sequence encoding the product could
be verified by techniques such as sequencing of pooled cDNA clones
or material amplified by the polymerase chain reaction. The nucleic
acid sequence should be identical, within the limits of detection of
the methodology, to that determined for the expression construct as
described in Section III.A. and should correspond to that expected
for the protein sequence.
C. Limit for In-Vitro Cell Age for Production
The limit for in-vitro cell age for production should be based
on data derived from production cells expanded under pilot or full
scale conditions to the proposed in-vitro cell age or beyond.
Generally, the production cells are obtained by expansion of the
Working Cell Bank; the Master Cell Bank could be used to prepare the
production cells with appropriate justification.
The expression construct of the production cells should be
analyzed once for the MCB as described in Section III.B., except
that the protein coding sequence of the expression construct in the
production cells could be verified by either nucleic acid testing or
analysis of the final protein product. Increases in the defined
limit for in-vitro cell age for production should be supported by
data from cells which have been expanded to an in-vitro cell age
which is equal to or greater than the new limit for in-vitro cell
age.
IV. Conclusion
The characterization of the expression construct and the final
purified protein are both important to ensure the consistent
production of a recombinant DNA derived product. As described above,
it is considered that analytical data derived from both nucleic acid
analysis and evaluation of the final purified protein are necessary
to ensure the quality of a recombinant protein product.
Glossary of Terms
Expression Construct
The expression vector that contains the coding sequence of the
recombinant protein and the elements necessary for its expression.
Flanking Control Regions
Noncoding nucleotide sequences that are adjacent to the 5' and
3' end of the coding sequence of the product which contain important
elements that affect the transcription, translation, or stability of
the coding sequence. These regions include, e.g., promoter,
enhancer, and splicing sequences and do not include origins of
replication and antibiotic resistance genes.
Integration Site
The site where one or more copies of the expression construct is
integrated into the host cell genome.
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 in
culture, 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.
Master Cell Bank (MCB)
An aliquot of a single pool of cells which generally has been
prepared from the selected cell clone under defined conditions,
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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.
Pilot Plant Scale
The production of a recombinant protein by a procedure fully
representative of and simulating that to be applied on a full
commercial manufacturing scale. The methods of cell expansion,
harvest, and product purification should be identical except for the
scale of production.
Relevant Genotypic and Phenotypic Markers
Those markers permitting the identification of the strain or the
cell line which should include the expression of the recombinant
protein or presence of the expression construct.
Working Cell Bank (WCB)
The Working Cell Bank is prepared from aliquots of a homogeneous
suspension of cells obtained from culturing the MCB under defined
culture conditions.
Dated: August 14, 1995.
William K. Hubbard,
Acting Deputy Commissioner for Policy.
[FR Doc. 95-20611 Filed 8-18-95; 8:45 am]
BILLING CODE 4160-01-F