New Sanger DNA sequencing for genetic stability testing
by Klaudia Shick, M.Sc., Ph.D., Principal Scientist/Group Leader
Eurofins Lancaster Laboratories, Inc. (ELLI) has added a new service: the Sanger DNA sequencing analysis, which is performed in compliance with current Good Manufacturing Practice (GMP). Sanger sequencing is a critical component of genetic stability testing, a regulatory requirement for cell lines used to produce recombinant DNA derived biopharmaceutical products.
The purpose of the genetic stability testing is to ensure that correct coding sequence of the product has been incorporated into the host cell and is maintained during culture to the end of the production. The nucleic sequence of both the coding region of the gene of interest and associated flanking regions should be confirmed by sequencing before the expression construct is used to establish the production cell line. Subsequently, the integrity and stability of the expression construct is also confirmed for Master Cell Bank (MCB), Working Cell Bank (WCB), and End of Production Cells (EOPC).
For extrachromosomal expression systems, the expression construct should be isolated and directly sequenced without further cloning. For production systems in which expression construct is integrated into the host cell genome, sequencing may be performed on PCR product amplified from cDNA that are reverse transcribed from cellular RNA. To ensure sufficient sequencing coverage, the PCR product is typically analyzed bidirectionally (sense and antisense strand) with overlapping sequencing generated from different reactions at each position.
At ELLI, the cGMP sequencing is delivered on Beckman Coulter GenomeLab GeXP Genetic Analysis System using the Dye Termination Cycle Sequencing method. The Dye Termination Cycle Sequencing method offers multiple sequencing chemistries that allow efficient DNA sequencing on various templates such as PCR products, plasmids, single and double stranded complementary DNA (cDNA) with different G/C content. The GenomeLab GeXP Genetic Analysis System is an automated instrument able to analyze fluorescently labeled DNA fragments by capillary electrophoresis. The sequencing analysis software interprets the result, calling the base based on fluorescence signal collected at each data point. The DNA sequencing method was developed and validated for system suitability, specificity, and robustness at ELLI. In validation, the resulting sample sequences were unambiguous with all regions fully resolved. The average percent accuracy base calling achieved for the system suitability samples at 700 base pairs was 99%, and the average length of determined DNA sequence with precision base calling of >97% was ~800 base pairs, respectively. Depending on the exact sequence of the gene of interest, sequencing efficiency may vary.
For difficult samples, our clients can rely on our scientists’ more than 15 years of experience in delivering DNA sequencing services to maximize the efficiency and accuracy of the output. For more information on GMP DNA sequencing capabilities, including sample submission instructions, pricing and turnaround time, please contact Biopharmaceutical Business Development at 717-656-2300.