Anion exchange HPLC monitoring of mRNA in vitro transcription reactions to support mRNA manufacturing process development

作者：Emma N. Welbourne, Kate A. Loveday, Adithya Nair, Ehsan Nourafkan, Jixin Qu, Ken Cook, Zoltán Kis, Mark J. Dickman · 发表于：Frontiers in Molecular Biosciences · 年份：2024 · DOI：10.3389/fmolb.2024.1250833 · 被引用次数：28 · 研究领域：RNA Interference and Gene Delivery、RNA and protein synthesis mechanisms、Virus-based gene therapy research

mRNA technology has recently demonstrated the ability to significantly change the timeline for developing and delivering a new vaccine from years to months. The potential of mRNA technology for rapid vaccine development has recently been highlighted by the successful development and approval of two mRNA vaccines for COVID-19. Importantly, this RNA-based approach holds promise for treatments beyond vaccines and infectious diseases, e.g., treatments for cancer, metabolic disorders, cardiovascular conditions, and autoimmune diseases. There is currently significant demand for the development of improved manufacturing processes for the production of mRNA therapeutics in an effort to increase their yield and quality. The development of suitable analytical methods for the analysis of mRNA therapeutics is critical to underpin manufacturing development and the characterisation of the drug product and drug substance. In this study we have developed a high-throughput, high-performance liquid chromatography (HPLC) workflow for the rapid analysis of mRNA generated using in vitro transcription (IVT). We have optimised anion exchange (AEX) HPLC for the analysis of mRNA directly from IVT. Chromatography was performed in under 6 min enabling separation of all of the key components in the IVT, including nucleoside triphosphates (NTPs), Cap analogue, plasmid DNA and mRNA product. Moreover, baseline separation of the NTPs was achieved, which facilitates accurate quantification of each NTP such t...