[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 

[[Page 7007]]
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.

[[Page 7008]]

    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