[Federal Register Volume 61, Number 37 (Friday, February 23, 1996)]
[Notices]
[Pages 7006-7008]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 96-4064]
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DEPARTMENT OF HEALTH AND HUMAN SERVICES
[Docket No. 95D-0216]
International Conference on Harmonisation; Final Guideline on
Quality of Biotechnological Products: Analysis of the Expression
Construct in Cells Used for 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 is publishing a final
guideline on the quality of biotechnological products entitled
``Analysis of the Expression Construct in Cells Used for Production of
r-DNA Derived Protein Products.'' The guideline was prepared under the
auspices of the International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for Human Use (ICH).
The 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: Effective February 23, 1996. Submit written comments at any
time.
ADDRESSES: Submit written comments on the guideline to the Dockets
Management Branch (HFA-305), Food and Drug Administration, 12420
Parklawn Dr., rm. 1-23, Rockville, MD 20857. Copies of the guideline
are available from the Division of Communications Management (HFD-210),
Center for Drug Evaluation and Research, Food and Drug Administration,
7500 Standish Pl., Rockville, MD 20855, 301-594-1012. An electronic
version of this guideline is also available via Internet by connecting
to the CDER file transfer protocol (FTP) server (CDVS2.CDER.FDA.GOV).
FOR FURTHER INFORMATION CONTACT:
Regarding the guideline: Elaine C. Esber, Center for Biologics
Evaluation and Research (HFM-30), Food and Drug Administration, 1401
Rockville Pike, Rockville, MD 20852, 301-827-0641.
Regarding ICH: Janet J. Showalter, Office of Health Affairs (HFY-
1), Food and Drug Administration, 5600 Fishers Lane, Rockville, MD
20857, 301-443-1382.
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
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from both regulatory and industry representatives. FDA also seeks input
from consumer representatives and others. ICH is concerned with
harmonization of technical requirements for the registration of
pharmaceutical products among three regions: The European Union, Japan,
and the United States. The six ICH sponsors are the European
Commission, the European Federation of Pharmaceutical Industries
Associations, the Japanese Ministry of Health and Welfare, the Japanese
Pharmaceutical Manufacturers Association, the Centers for Drug
Evaluation and Research and Biologics Evaluation and Research, FDA, and
the Pharmaceutical Research and Manufacturers of America. The ICH
Secretariat, which coordinates the preparation of documentation, is
provided by the International Federation of Pharmaceutical
Manufacturers Associations (IFPMA).
The ICH Steering Committee includes representatives from each of
the ICH sponsors and the IFPMA, as well as observers from the World
Health Organization, the Canadian Health Protection Branch, and the
European Free Trade Area.
In the Federal Register of August 21, 1995 (60 FR 43496), FDA
published a draft tripartite guideline entitled ``Analysis of the
Expression Construct in Cells Used for Production of r-DNA Derived
Protein Products.'' The notice gave interested persons an opportunity
to submit comments by October 5, 1995.
After consideration of the comments received and revisions to the
guideline, a final draft of the guideline was submitted to the ICH
Steering Committee and endorsed by the three participating regulatory
agencies at the ICH meeting held on November 29, 1995.
The guideline presents guidance regarding the characterization of
the expression construct for the production of r-DNA protein products
in eukaryotic and prokaryotic cells. The guideline is intended to
describe the types of information that are considered valuable in
assessing the structure of the expression construct used to produce r-
DNA derived proteins. The guideline is not intended to cover the entire
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). Although this
guideline does not create or confer any rights for or on any person and
does not operate to bind FDA, it does represent the agency's current
thinking on the production of r-DNA derived protein products.
As with all of FDA's guidelines, the public is encouraged to submit
written comments with new data or other new information pertinent to
this guideline. The comments in the docket will be periodically
reviewed, and, where appropriate, the guideline will be amended. The
public will be notified of any such amendments through a notice in the
Federal Register.
Interested persons may, at any time, submit written comments on the
final guideline to the Dockets Management Branch (address above). Two
copies of any comments are to be submitted, except that individuals may
submit one copy. Comments are to be identified with the docket number
found in brackets in the heading of this document. The 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 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
(r-DNA) protein products in eukaryotic and prokaryotic cells. The
document is intended to describe the types of information that are
considered valuable in assessing the structure of the expression
construct used to produce r-DNA derived proteins. The document is
not intended to cover the entire 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 posttranslational
modifications.
II. Rationale for Analysis of the Expression Construct
The purpose of analyzing the expression construct is to
establish that the correct 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
posttranslational modifications such as proteolytic processing,
glycosylation, phosphorylation, and acetylation. Data from nucleic
acid analysis may be useful because 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 m-RNA or c-DNA 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
reviewed periodically 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.
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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, and 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 MCB and Working Cell Banks (WCB). 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 MCB is generally derived from the selected cell clone containing
the expression construct. The 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 c-DNA 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 plant-
scale or full-scale conditions to the proposed in vitro cell age or
beyond. Generally, the production cells are obtained by expansion of
the WCB; the MCB 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 that have been expanded to an in vitro cell age that
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 r-DNA derived product. As described above,
analytical data derived from both nucleic acid analysis and
evaluation of the final purified protein should be evaluated 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 that 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 that 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.
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 of the
cell line that should include the expression of the recombinant
protein or presence of the expression construct.
Working Cell Bank (WCB)
The WCB is prepared from aliquots of a homogeneous suspension of
cells obtained from culturing the MCB under defined culture
conditions.
Dated: February 16, 1996.
William K. Hubbard,
Associate Commissioner for Policy Coordination.
[FR Doc. 96-4064 Filed 2-22-96; 8:45 am]
BILLING CODE 4160-01-F