Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry

Large RNA including
mRNA (mRNA) has emerged as an important new
class of therapeutics. Recently, this has been demonstrated by two
highly efficacious vaccines based on mRNA sequences encoding for a
modified version of the SARS-CoV-2 spike protein. There is currently
significant demand for the development of new and improved analytical
methods for the characterization of large RNA including mRNA therapeutics.
In this study, we have developed an automated, high-throughput workflow
for the rapid characterization and direct sequence mapping of large
RNA and mRNA therapeutics. Partial RNase digestions using RNase T1
immobilized on magnetic particles were performed in conjunction with
high-resolution liquid chromatography–mass spectrometry analysis.
Sequence mapping was performed using automated oligoribo­nucleotide
annotation and identifications based on MS/MS spectra. Using this
approach, a >80% sequence of coverage of a range of large RNAs
and
mRNA therapeutics including the SARS-CoV-2 spike protein was obtained
in a single analysis. The analytical workflow, including automated
sample preparation, can be completed within 90 min. The ability to
rapidly identify, characterize, and sequence map large mRNA therapeutics
with high sequence coverage provides important information for identity
testing, sequence validation, and impurity analysis.