New DNA sizing and barcoding methods are robust molecular alternatives to Isoenzyme analysis
Jeri Ann Boose, PhD, Senior Director, Eurofins Lancaster Laboratories BioPharmaceutical Services, jeriannboose@eurofinsus.com
Cell line identity testing is a critical regulatory requirement for recombinant cell lines in order to confirm the cell line’s species of origin, as well as to assist in detecting contamination from other cell lines.
Biochemical analysis of isoenzyme polymorphism has been considered the standard test for mammalian and insect cell line identification. However, the methodology suffers several limitations. With limited species coverage, insufficient sensitivity, and reagent supply shortages of the historically standard isoenzyme ID test, Eurofins Lancaster Laboratories has developed DNA Sizing and Barcoding methods to combat these challenges. For species identification, these robust methods utilise the mitochondrial genes Cytochrome Oxidase 1 (CO1) and Cytochrome B (Cyt B) as targets for the molecular identification of cell lines commonly used in biopharmaceutical production.
Why are the DNA Sizing and Barcoding methods superior? For CO1/Cyt B Sizing, genomic DNA is isolated from the cells being tested and amplified using a set of species specific primers. Species identification and detection of cross contamination can be determined based upon the presence/absence of PCR amplification by given sets of primers and the size of the PCR amplicon. Cell lines of cow, mouse, dog, rat, monkey, hamster, cat, human and pig origin can be tested using CO1/Cyt B Sizing. Identification of cell lines derived from a wider variety of animal and insect species can be made by CO1 DNA Barcoding. The comparison between the determined CO1 sequence of tested cell lines and the confirmed species specific sequences deposited in the Consortium for the Barcode of Life database allows for unambiguous genetic identification.
The Sizing and Barcoding methods are based on a more robust technology than that of the isoenzyme assay and can provide more accurate speciation. They can also be easily implemented in a quality control environment. Further, the PCR amplification and DNA sequencing techniques used for the Sizing and Barcoding methods decrease dependency on a single supplier for test reagents. And the Sizing and Barcoding methods are more sensitive with regard to the detection of cell line cross contamination than isoenzyme analysis.
To see how Eurofins Lancaster Laboratories’ comprehensive cell line characterisation services, along with its cell banking capabilities, provide clients with a single-source solution for all cell line needs, visit www.EurofinsLancasterLabs.com
