Automated online direct mRNA sequence mapping using partial RNase T1 digests

Mass spectrometry-based approaches have emerged as powerful tools for the analysis of a wide range of critical quality attributes of mRNA medicines, including sequence identity, 5' capping efficiency, and 3' poly(A) tail length and heterogeneity. These critical quality attributes can impact the quality, safety, and efficacy of mRNA medicines. In this study, we have utilized online partial RNase T1 digests in conjunction with two-dimensional liquid chromatography mass spectrometry (2D LC-MS) for the direct sequence mapping of mRNA. Automated online partial RNase T1 digests are performed in conjunction with ion-pair reversed-phase liquid chromatography. No sample or solvent manipulation is required following online RNase digestions, demonstrating the simplicity of the method. High-resolution tandem mass spectrometry was used to identify the corresponding oligoribonucleotides and generate mRNA sequence maps. High sequence coverage (93-99%) for eGFP mRNA was obtained in <60 min based only on unique oligoribonucleotide identifications. Moreover, the online partial RNase T1 digests result in controlled, fully automated and reproducible mRNA digests, enabling high-throughput, direct mRNA sequence mapping studies. The online partial RNase digests offer significant advantages over existing methods for rapid, automated mRNA identity testing. Furthermore, precise control of the digest conditions via flow rate and temperature of the online RNase T1 digest, enables multiattribute monitoring of 5' capping efficiency, mRNA sequence mapping, and 3' poly(A) tail length and heterogeneity in a fully automated 2D LC-MS workflow.