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

Abstract

The current SARS-Covid-2 (SARS-CoV-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 characterization techniques. Characterizing the purity, shape and structure of these biomolecules is key to their successful performance as drug products. This article describes the biophysical characterization 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 characterize the IMP-1 RNA molecule. In this article, we use ultraviolet spectroscopy, dynamic light scattering, size-exclusion chromatography small-angle X-ray scattering and circular dichroism to determine key biophysical attributes of IMP-1. Each technique provides important information about the concentration, size, shape, structure and purity of the molecule.

Keywords: biophysics; saRNA; vaccines.

Figure 1:

The UV spectrum of IMP-1 with the A260 and A280 values and A260: A280 ratio given.

Figure 2:

Capillary electrophoresis trace of IMP-1 saRNA (left) alongside a 0.5–9 kb RNA marker [right (Lonza, UK)].

Figure 3:

Capillary electrophoresis trace of IMP-1 saRNA (red trace - labelled IMP-1) overlaid with a scaled 0.5–9 kb RNA marker [Blue trace- 10 peaks (Lonza, UK)].

Figure 4:

DLS spectra of the IMP-1 RNA at 0.245 mg ml⁻¹ concentration in the SAXS (blue line) and CD buffer (red line).

Figure 5:

A plot of the R_H versus RNA nucleotide length (kb) of 19 RNA molecules (red circles) and IMP-1 (blue square) in ddH₂O. The data for the 19 RNAs are given in Borodavka et al. [28].

Figure 6:

A SEC-SAXS plot of X-ray signal intensity versus frame number of IMP-1. The frames process averaged for the buffer (blue) and sample (light green) shown with an estimated the R_g of each sample frame shown in red.

Figure 7:

The process averaged SAXS log–log curve for IMP-1 using the buffer and sample frames shown in Fig. 6, the inset shows the Guinier plot estimate of the R_g and the intensity at zero angle I(0).

Figure 8:

The distance (PDDF) of distribution plot the IMP-1 RNA sample taken using BioSAXS.

Figure 9:

The dimensionless Kratky plot of the IMP-1 RNA sample taken using BioSAXS.

Figure 10:

The CD spectrum of IMP-1 in 10 mM sodium phosphate buffer at pH 7.