Special approach of droplet digital polymerase chain reaction (ddPCR) for transgene stability of a Chinese hamster ovary (CHO) cell line.

Cell line stability of mammalian cell lines is an important concern for manufacturing of biopharmaceuticals. Characterization of the master cell bank (MCB) for monoclonal antibody A (Mab A) via multiplex fluorescence in situ hybridization (M-FISH) analysis identified two distinct karyotypic cell populations. As a result, a regulatory agency requested additional analysis to support a single clonal lineage of the MCB and demonstrate control of the two cell populations in subsequently derived cell banks. Next Generation Sequencing (NGS) revealed 11 transgene integration site sequences. A multiplex (measure two amplicons) Droplet Digital PCR (ddPCR) method was developed and characterized those 11 integration site copy numbers in the MCB. Analysis of 12 subclones from the MCB displayed that among 7 subclones there was loss of at least one of five specific integration sites. The characterization of these five unstable sites in the end production cells (EPCs) of four manufacturing batches are consistent with the MCB. The conservation of five other integration sites among all clones supported that these cells were derived from the same population and that loss of the unstable integration sites contributed to the two distinct karyotypic populations.