Genetic stability testing ensures product integrity

Weihong Wang, PhD, Technology Development Manager, Eurofins Lancaster Laboratories, WeihongWang@eurofinsus.com

Genetic stability testing is a key component of GMP production cell bank characterisation and a regulatory requirement. Typical mammalian production cell lines are created by stable transfection of the expression vector into the host cell line. During subsequent cell culture, genomic events such as deletions, rearrangements and point mutations may occur and result in an altered cell phenotype and/or gene expression profile. The instability of the cell line is of great concern as it may negatively impact product integrity, posing a risk to patients. Even when product integrity is not immediately impacted, the possible reduction of productivity and the elevated risk of future events still raise concerns from an operational perspective.

Genetic stability testing includes an array of assays that are typically performed on a manufacturer’s master cell banks (MCB) and representative lot(s) of end of production cells (EOPC). Genetic stability testing of the working cell banks (WCB) may also be performed at a manufacturer’s discretion. Typical assays include, but are not limited to, those intended to confirm the integrity of the product transcript (mRNA/cDNA sequencing and Northern analysis), the genomic structure at the integration site (restriction digestion map via Southern analysis), and the ratio of insert gene copy number relative to host genome (via qPCR). Testing results from the EOPC and WCB are compared to those of the MCB to allow the detection of any changes that may be indicative of cell line instability.

The Molecular Biology Testing Team at Eurofins Lancaster Laboratories has established generic methods of cDNA sequencing, restriction digestion mapping via Southern analysis, and gene copy number determination via qPCR. A generic method for transcript size determination via Northern analysis is currently under development and is expected to be available soon. These generic methods for genetic stability testing can be adapted quickly to each client’s unique cell line/cell bank. Product specific method validation can also be performed per client request to support CMC filings for later phase clinical trials and/or commercialisation.