Detailed characterization of nucleic acid products is an essential part of the development program for recombinant products. A comprehensive array of analytical services to support this characterization is available at Charles River Biopharmaceutical Services (BPS). These methods are applied to determine the genetic stability of recombinant products and for the characterization of plasmid products. Additionally, multiple methods are available for the detection of residual host cell DNA.
Genetic Stability Testing
The genetic stability of recombinant cell lines needs to be demonstrated according to regulatory guidelines. The identity and integrity of recombinant genes has to be verified and shown to be stable throughout the production process. Stability is supported by characterization studies of cells at the end of production and comparing those attributes to those of the master cell bank. Identity may be addressed by DNA sequencing and comparing the results to a reference sequence. Genetic integrity is often confirmed by restriction enzyme analysis followed by Southern blotting and comparing the resulting fragment sizes to the expected sizes.
Determination of Copy Number
Genetically modified cell banks are characterized to quantify the number of copies of a recombinant gene or plasmid. Charles River has experience performing these types of studies on animal, bacterial and yeast cell banks. Both quantitative Southern blot analysis and quantitative PCR can be used to determine the copy number of one or more target sequences.
DNA Sequencing
BPS offers validated, current Good Manufacturing Practice (cGMP)-compliant DNA sequencing of recombinant gene constructs, plasmids and viruses. Sequencing can be accomplished using either the LiCORTM or Applied Biosystems DNA sequencing systems. Both systems provide at least two-fold, and in some cases three-fold, coverage on each DNA strand to ensure accuracy.
Detection of Residual DNA
The detection and quantification of minute amounts of residual host cell DNA can be accomplished using any one of three highly sensitive, validated methods. These methods encompass the entire range of residual DNA from nonspecific detection of total DNA to highly specific detection of single target sequences. The methods used are:
- The ThresholdTM System from Molecular Devices® uses DNA binding proteins with high affinity for single-stranded DNA for nonspecific quantification of total DNA.
- A hybridization-based method for the detection of the specific DNA of defined origin with dot blots and hybridization of radioisotope-labeled DNA probes uses random hexamers to generate representative probes covering the whole genome of the host cells.
- A quantitative PCR-based method for the detection of specific DNA of defined origin targets a specific gene sequence for amplification and is calibrated using purified, species-matched, genomic DNA.
The extensive experience of Charles River with sample purification and preparation enables us to analyze samples in many different matrices. Samples are generally tested with and without the addition of known, small amounts of genomic DNA (“spikes”) to detect and control for potential interference.
For more information, please contact us at askcharlesriver@crl.com.