Diving into the Structural Details of In Vitro Transcribed mRNA Using Liquid Chromatography–Mass Spectrometry-Based Oligonucleotide Profiling

Abstract

The production process of in vitro transcribed messenger RNA (IVT-mRNA)-based vaccines has matured in recent years, partly due to the fight against infectious diseases such as COVID-19. One key to success has been the use of modified, next to canonical, nucleotides and the efficient addition of a Cap-structure and poly A tail to the 5’ and 3’ end, respectively, of this massive biomolecule. These important features affect mRNA stability and impact translation efficiency, consequently boosting the optimization and implementation of liquid chromatography–mass spectrometry (LC–MS)-based oligonucleotide profiling methods for their characterization. This article will provide an overview of these LC–MS methods at a fundamental and application level. It will be shown how LC–MS is implemented in mRNA-based vaccine analysis to determine the capping efficiency and the poly A tail length, and how it allows, via RNA mapping, (i) to determine the mRNA sequence, (ii) to screen the fidelity of the manufactured modifications, and (iii) to identify and quantify unwanted modifications resulting from manufacturing or storage, and sequence variants resulting from mutation or transcription errors.