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

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

[[Page 43497]]

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,

[[Page 43498]]

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