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.

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.

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.

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.