A TaqMan reverse transcription polymerase chain reaction (RT-PCR) in vitro potency assay for plasmid-based vaccine products.

A TaqMan-based reverse transcription polymerase chain reaction (RT-PCR) assay has been developed as an in vitro potency assay to measure the most immediate biological activity of plasmid DNA (pDNA)-based products. The assay measures transgene-specific messenger RNA (mRNA) from cultured cells transfected with VCL-CB01, a bivalent pDNA-based human cytomegalovirus (CMV) vaccine. The forward and reverse primers have been designed to make the RT-PCR reaction selective for plasmid-derived mRNA and to allow discrimination of expression levels of individual plasmids in a multivalent pDNA vaccine. The relative potency of a vaccine lot is assessed by transfecting reference and test samples into cultured cells in parallel and analyzing total RNA from the cells by RT-PCR. Statistical analysis of dose response data from reference material supports a parallel-line model for calculating relative potency. Preliminary data demonstrate the ability of this assay to distinguish product potencies at 50, 75, 150, and 200% of the reference material. In addition, forced degradation of pDNA demonstrates that a decrease in relative potency as measured by the RT-PCR assay in vitro correlates well with a decrease in CMV DNA vaccine-mediated humoral immune responses in mice injected with the same material.