A TaqMan® Reverse Transcription Polymerase Chain Reaction (RT-PCR) In Vitro Potency Assay for Plasmid-based Vaccine Products

Abstract

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.

Vaccine Analysis: Strategies, Principles, and Control

“…Recently, Mahajan et al (2008) reported a Taqman RT-PCR-based in vitro potency assay for DNA plasmid products and Badger et al (2011) reported the development of a flow cytometry based potency assay for a Venezuelan equine encephalitis virus DNA plasmid vaccine that assesses the in vivo expression of the transgene product in host cells. The FDA guidance document provides developers considerable flexibility in the choice of assays early in development.…”

Product Specifications and Quality Control

“…104 This parameter is critical since it is the only one that actually provides important information about the effi cacy of individual lots of a given plasmid biopharmaceutical. 104 This parameter is critical since it is the only one that actually provides important information about the effi cacy of individual lots of a given plasmid biopharmaceutical.…”

“…104 This parameter is critical since it is the only one that actually provides important information about the effi cacy of individual lots of a given plasmid biopharmaceutical. 104 In general, potency assays will evaluate the effi ciency of gene transfer, the level and stability of expression of the encoded transgene, and its effects. 15 The existence of such assays is crucial to decide whether a specifi c production lot can be released, to check for manufacturing consistency, and to control product shelf stability.…”

“…These assays are basically designed to monitor transcription and/or translation of the gene(s) encoded in the plasmid 105 (Figure 12.3 ). 104 As described in the previous section, when the issue of the quantifi cation of residual E. coli RNA was discussed, the most sensitive method available today to detect RNA is RT -PCR. Following incubation of the plates for a predetermined amount of time, the transfection effi ciency is examined.…”

“…Following incubation of the plates for a predetermined amount of time, the transfection effi ciency is examined. 104 With this methodology, the relative potency of a specifi c plasmid lot can be assessed by comparing results with the Ct values obtained when transfecting cells with a reference plasmid standard. 104 As described in the previous section, when the issue of the quantifi cation of residual E. coli RNA was discussed, the most sensitive method available today to detect RNA is RT -PCR.…”