RNA platform technologies have emerged as powerful new classes of crop protection active ingredients and therapeutic drug substances. To realise the potential of these emerging technologies, large-scale manufacturing and rapid, high-throughput analytical methods are required. This thesis presents advancements in the development of novel analytical methods and the utilisation of mass spectrometry-based approaches to optimise the production and characterisation of dsRNA biocontrols and mRNA medicines.
The production of dsRNA biocontrols is routinely performed in E. coli; however, large-scale economic manufacturing of high-quality dsRNA remains a significant challenge. For the first time, quantitative proteomics was used to study differential expression during dsRNA production. Nucleotide biosynthetic pathways were found to be significantly upregulated, and candidate enzymes were identified for future studies to alleviate metabolic bottlenecks and increase dsRNA yield.
Mass spectrometry (MS) is emerging as a powerful tool for the analysis of RNA. This is, however, an emerging area with distinct and unique challenges compared to the analysis of proteins and oligonucleotides. Novel MS methods were developed to characterise dsRNA produced in vitro and in vivo. Intact mass analysis provided important new mechanistic insight into transcriptional termination in dsRNA produced from multi-terminator DNA templates and identified differences in dsRNA manufactured in vivo compared to in vitro.
Novel MS methods were also developed for the analysis of mRNA CQAs, including identity, capping efficiency, and poly(A) tail length and heterogeneity. A middle-up approach using MazF digestion enabled the development of the first rapid multi-attribute method, resulting in 100% sequence coverage, quantification of 5’-capping efficiency and nucleotide resolution of the poly(A) tail. Finally, novel LC-MS methods were used for the first direct analysis of both DNA template poly(A/T) sequences and corresponding mRNA poly(A) tails, providing important insight into how tail length and heterogeneity are affected by template design, quality, and RNA manufacturing conditions.
