Full comprehensive ion-pair-mediated anion exchange chromatography × ion-pair reversed-phase liquid chromatography fingerprinting of digested messenger ribonucleic acid drug substances

• LC × LC of RNase T1 digested mRNA. • Ion-pair mediated anion exchange chromatography coupled to ion-pair reversed phase. • Convex hull plots to evaluate orthogonality of LC × LC contour plots. • Profiling of different mRNA digests by LC × LC. The growing interest in messenger RNA (mRNA) based therapeutics has increased the need for analytical method development to meet the desired standards for quality control of these novel drugs. The identity of the mRNA drug substance is usually confirmed via indirect approaches such as Sanger sequencing or next generation sequencing (NGS), or more direct approaches such as LC-MS/MS after digestion with specific RNases. These conventional bottom-up MS methodologies, however, usually require several partial digests with various RNases to get sufficient sequence coverage. In the present study, a comprehensive two-dimensional liquid chromatography (LC × LC) platform is presented, utilizing ion-pair-mediated anion exchange chromatography in the first dimension and ion-pair reversed-phase liquid chromatography in the second dimension and evaluated using the convex hull model for orthogonality determination. This platform can be employed for the fingerprinting of RNase T1 digested mRNA, enabling the distinction between different mRNAs based on the fingerprint of a full digestion with a single RNase. It could also be used to confirm its identity or help with sequencing efforts, when combined with high-resolution MS.