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.