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

Lead Research Organisation: Imperial College London
Department Name: Chemical Engineering


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.


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Description The aim of this project is to optimise an existing assay to measure RNA length and 5' cap modifications to support RNA vaccine manufacturing. There are three objectives. The first is to optimise the existing assay using design of experiments approaches. This has been completed and led to a 3-fold decrease in the limit of detection of the assay, which is a significant achievement if this were to be applied as a quality control assay in manufacturing. We have taken steps to automate this version of the assay using the widely available, open source robotics platform OpenTrons. The work is currently being prepared for publication as both a research paper (detailing the DoE approaches as well as the optimised assay) and a protocol book chapter (outlining the step-by-step procedure for others to follow). Objective 2 aimed to reverse the capture and detection procedures to opposite ends of the RNA molecule compared to the first design. We have prepared the necessary reagents to develop this approach further-- a protein to capture the RNA and a fusion protein to detect the other end. This is currently being optimised via a combination of biolayer interferometry measurements and design of experiments approaches. The third objective was to develop a format that omits the capture step in the assay, which we believe is the rate limiting step. This work is ongoing and due to a change in staffing on the project, we have asked for a no-cost extension (result pending).
Exploitation Route We are preparing a manuscript and a book chapter with the new assay protocol and will publish this in an open access journal to allow others to use it. Any lab or manufacturer who is producing RNA can use the assay to measure the amount of capped and full length RNA in their sample.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology