Developing a rapid quality control and long-term stability assay for RNA vaccine candidates

RNA vaccines against SARS-Cov2 have shown great promise with early results from clinical trials indicating >90% protection conferred. The RNA component of these vaccines is very long, up to 10,000 nucleotides, which introduces analytical challenges as standard methods such as gel electrophoresis are not sufficient to detect small differences in chain length. In addition, assays to detect the presence of the 5' cap that is necessary for efficient translation rely on slow and laborious methods. Thus, there is a need to develop new analytical technologies that can be applied to the QC/QA of RNA vaccines to support both manufacturing and the assessment of the long-term stability of vaccines during storage. We have previously demonstrated a proof-of-concept for a new RNA assay that is able to capture the molecule by one end and then probe specifically for the other end, leading to a measurable signal only when the RNA molecule is intact and contains a 5' cap. However, when applied to long RNA molecules such as vaccines, the assay loses sensitivity due to steric hindrance in the initial capture step. Therefore, high concentrations of RNA are required for analysis, which might limit the overall application of the assay. In this project, we aim to explore a series of interventions designed to maximise RNA capture. We will compare three strategies in the project. First, we will use statistical design of experiments to optimise our existing assay by varying the concentrations of the different molecules involved to maximise the signal-to-noise ratio. Second, we will try reversing the assay so that we capture the RNA molecule by the 5' cap and probe for the opposite end. Finally, we will examine whether it is possible to omit the capture step entirely and form a complex that bridges both ends of the RNA molecule leading to a fluorescence signal. As a proof-of-concept we will apply the assay to the Imperial College saRNA vaccine candidate that is currently undergoing clinical trials. To build on this proof-of-concept study, we aim to engage with a broad variety of stakeholders to enable uptake by vaccine manufacturers and will seek regulatory approval to enable the assay to be used for batch release testing.