Development and Application of Capillary Gel Electrophoresis for the Analysis of RNA-based Vaccines and Therapeutics

In recent years, RNA-based technologies have become increasingly prominent, with significant applications in the medical field such as therapeutics and vaccines. Notably, such technologies were utilised heavily during the coronavirus disease pandemic, exemplifying the fast pace at which ribonucleic drug molecules can be developed and produced to tackle significant world problems. This thesis aims to develop and optimise capillary gel electrophoresis as an analytical separation technique for the analysis of RNA. High-performance analytical techniques are key to maintaining high-quality drug molecules that can perform as expected with minimal side effects. Hence, capillary gel electrophoresis, a heavily underutilised technology, was selected for investigation. By exploration of novel approaches in the capillary gel electrophoresis method design space, existing methods in literature were greatly improved to allow for much higher resolution ribonucleic separations. Most notably, resolutions of 4,000 nt and 6,000 nt RNA species was increased from 6 up to 9. These methods were then shown to have a high level of precision and accuracy for mRNA analysis, with standard deviations of sizing predictions being as low as 0.6% for CGE methods containing methanol and urea. The optimal methods were then applied to several subfields of RNA research area, broadening the application opportunities for this technology. Allowing for baseline separations of long mRNA, circRNA by-products and the four ribonucleic NTPs from mRNA. Finally, CE methodologies operating at pH values above 8 were assessed for viability in the RNA analytical space.