Genetic Stability Testing
Genetic stability testing is required to demonstrate that the genetic sequence of an expression construct is stable throughout the production life cycle to ensure the quality and consistency of the product and production process.
Next Generation Sequencing (NGS) is now recognized as one of the most powerful and reliable tools for full genome sequencing. Using NGS for the cell line characterization purpose will allow you to run one study rather than performing multiple assays to achieve the same results.
Webinar: Using NGS to Characterize Cell Lines
Learn about the use of next generation sequencing technologies for the characterization of your cell lines and for verifying clonality and identity.
Regulations for Genetic Stability Testing
According to ICH Q5B guidelines, regulating agencies request that cell substrates must be analyzed at two time points (i.e., on the level of the Master Cell Bank and on the level of the End of Production cells) to show their genetic stability. Methods must be applied to confirm the coding sequence of the expression construct or the corresponding RNA transcripts. The copy number of the transgene has to be determined, and its integrity and integration pattern must be demonstrated by restriction endonuclease mapping.
The strategy for genetic characterization and stability testing varies and depends on the production system and the project phase. Our regulatory experts can provide guidance to your program to meet regulatory requirements.
Genetic Stability Methods
Charles River applies the following methods to determine the genetic stability of recombinant production cell lines and to characterize plasmid products:
- Restriction endonuclease mapping
- Southern blotting
- Northern blotting
- Copy number determination by qPCR
- DNA and RNA sequencing
- Plasmid sequencing
- Retention of recombinant construct
Charles River has partnered with PathoQuest, a genomics expert, to provide Next Generation Sequencing (NGS) services that can also be used for genetic stability testing. Our partnership can accelerate your drug development by offering quick and comprehensive testing.
Frequently Asked Questions (FAQs) About Genetic Stability
Why is genetic stability important?
Transgenes, expressed in recombinant production cells, are subjected to mutations which may impact the product quality and consistency of the process; therefore, it is a regulatory requirement to demonstrate the stability of the expression construct over the life span of the production cells.
Where can I find regulatory guidelines on genetic stability?
Supplement to the Points to Consider in the Production and Testing of New Drugs and Biologics Produced by Recombinant DNA Technology: Nucleic Acid Characterization and Genetic Stability, Food and Drug Administration, Center for Biologics Evaluation and Research Food and Drug Administration, 1992
What is restriction enzyme analysis of DNA?
The purpose of the restriction enzyme analysis method is to confirm that no changes have occurred in the restriction digestion pattern with respect to the integrated expression construct. The genomic DNA is extracted from the sample cells and then fragmented with a set of selected suitable restriction endonucleases.
The resulting DNA fragments are separated using agarose gel electrophoresis. The restricted genomic DNA is then subjected to the Southern blotting procedure using labelled, sequence-specific probes. The length of the resulting fragments is determined and compared to the theoretical values.