Biophysical characterization of the structure of a SARS-CoV-2 self-amplifying RNA (saRNA) vaccine

Abstract

The current SARS-Covid-2 pandemic has led to an acceleration of messenger—ribonucleic acid (mRNA) vaccine technology. The development of production processes for these large mRNA molecules, especially self-amplifying mRNA (saRNA) has required concomitant development of analytical characterisation techniques. Characterising the purity, shape and structure of these biomolecules is key to their successful performance as drug products. This paper describes the biophysical characterisation of the Imperial College London Self-amplifying viral RNA vaccine (IMP-1) developed for SARS-CoV-2. A variety of analytical techniques have been used to characterise the IMP-1 RNA molecule. In this paper we use UV spectroscopy, dynamic light scattering (DLS), size-exclusion chromatography small angle scattering (SEC-SAXS) and circular dichroism (CD) to determine key biophysical attributes of IMP-1. Each technique provides important information about the concentration, size, shape, structure and purity of the molecule.

Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides

“…Moreover, separation-based techniques lose resolution as mRNA length increases, exacerbating this challenge in e.g. very long saRNA [ 14 ]. Although more advanced CGE systems [ 15 ] or long-read Nanopore sequencing [ 16 ] show great promise in more sensitively identifying changes in mRNA integrity, these methods require a considerable amount of time, expertise, and financial resources to run.…”

“…This was beyond the scope of the current investigation, but we do show that the PNPase assay is amenable to high throughput setups ( Figure 3J ) and that the assay can be employed to assess very long mRNA constructs such as 10 kb self-amplifying mRNA ( Figure 3I ). Although saRNA vaccines were recently approved for clinical use in Japan [ 19 ], its large size and complex structure makes saRNA notoriously challenging to analyse using conventional bioanalytical methods [ 14 ]. Compared to shorter mRNA, our experiments also exposed a higher susceptibility of 10 kb saRNA for strand breaks.…”

Medical and molecular biophysical techniques as substantial tools in the era of mRNA-based vaccine technology

“…These biophysical techniques have played a critical role in the development of mRNA vaccines for COVID-19 by allowing a better understanding of the structure and interactions of the SARS-CoV-2 virus and the spike protein, as well as the interactions between the mRNA vaccine and the immune system. 166,167 The development of mRNA vaccines has been a breakthrough in immunization against infectious diseases, including COVID-19. Several medical and biophysical techniques have optimized mRNA vaccine production, purification, and formulation.…”

“…These biophysical techniques have played a critical role in the development of mRNA vaccines for COVID-19 by allowing a better understanding of the structure and interactions of the SARS-CoV-2 virus and the spike protein, as well as the interactions between the mRNA vaccine and the immune system. 166,167…”