Cell identity testing beyond isoenzyme analysis
by Weihong Wang, Ph.D., Technology Development Manager
Recombinant cell lines are commonly utilized in the production of biopharmaceuticals. To ensure product safety, sufficient characterization of these production cell lines is a regulatory requirement. Cell line characterization is a comprehensive program that includes an array of testing, among which is the identity test. The purpose of the identity test is primarily to confirm the cell line’s species of origin, but may also assist in detecting contamination from other cell lines.
While ribosomal DNA sequencing has been well established for accurate species identification of microbial cell lines at the molecular level, biochemical analysis of isoenzyme polymorphism is considered the standard test for mammalian and insect cell line identification for quality control purposes. Despite the common use of Isoenzyme analysis, the methodology suffers several limitations, including limited species coverage, lack of sufficient sensitivity and difficulty in data interpretation, especially when trying to differentiate closely related species. Further, the test kit is provided by a single supplier (Innovative Chemistry). The technical limitations, as well as a recent supply shortage of assay reagents has heightened the need for alternative, more robust methodologies that can provide accurate speciation, and can be easily implemented in a quality control environment.
To that effort, Eurofins Lancaster Laboratories is currently developing the DNA barcoding method which is a molecular technique for species identification. This method utilizes one particular mitochondrial gene, cytochrome oxidase I (COI), as a target for molecular identification of cell lines commonly used in biopharmaceutical production. The COI gene contains substantial interspecies variation whereas intraspecies variation remains remarkably low in most species. Therefore the COI gene has been established as an excellent target for species identification. In the COI DNA barcoding method, DNA is isolated from the cell line being tested and amplified using an appropriate set of primer cocktails in a multiplex qPCR. Species identification can be made based on size of the PCR amplicon, followed by sequencing analysis of the amplicon to confirm expected species specific sequence, where necessary. For clients with human cell lines, cell line identification can be further substantiated by short tandem repeat analysis (STR). STRs are regions of DNA where short sequences, usually between 2-6 base pairs (bp), are repeated side-by-side. A small subset of known STRs have been selected and standardized for use in human identity testing. By comparing the STR profile of a given cell line to the corresponding reference cell line (such as those available through ATCC), the identity of the cell line (based on the expected donor STR profile) can be confirmed. In addition to providing better accuracy and robustness, both COI barcoding and STR analysis are expected to be more sensitive in detecting cell line cross-contamination.
The COI barcoding and STR analysis will be available later this year. For more information on cell identity testing, including sample submission instructions, pricing and turnaround time, please contact Biopharmaceutical Business Development at 717-656-2300.