Concept for high-throughput mRNA sequence confirmation by atmospheric pressure MALDI technique applied to RNA digestion products

Development and manufacturing of mRNA vaccines require frequent quality control measures to ensure product safety and efficacy. Mass Spectrometry in combination with Liquid Chromatography (LC-MS) is known as a powerful tool for sequence confirmation and assessment of post-transcriptional modifications. LC-MS methods, while accurate, suffer from long acquisition times, making them typically impractical for high-throughput applications.

This study introduces a novel approach for RNA oligonucleotide sequence confirmation using UV-MALDI at atmospheric pressure (AP-MALDI) coupled with high-resolution accurate mass (HRAM) Orbitrap™ mass analyzer technology. By using an Orbitrap detector, we achieve a mass accuracy below 2 ppm for the digestion products of a RNA digest, suitable for verifying oligonucleotide sequences from complex mixtures without prior separation. We employ RNase T1 owing its G-specific cleavage, an ammonium-activated cation exchange resin for purification, followed by AP-UV-MALDI MS analysis. The method is validated with a synthetic 119mer RNA oligonucleotide, confirming sequence identity through MS/MS in both positive and negative ion modes. The approach is further applied to in silico analysis of mRNA sequences from vaccine variants, showcasing its potential for high-throughput, rapid sequence confirmation, and variant differentiation in RNA, particularly relevant for mRNA vaccine development and quality control in biopharmaceutical manufacturing.