Development\nof a Flow Through-Based Limited Digestion\nApproach for High-Throughput and High-Sequence Coverage Mapping of\nTherapeutic mRNAs

Messenger RNAs (mRNAs) have rapidly emerged as a pivotal\nclass\nof biotherapeutics with great promise in the prevention and treatment\nof various diseases. As with other biotherapeutics, the sequence accuracy\nand integrity of mRNAs are critical quality attributes (CQAs), influencing\nthe translation efficiency and protein expression fidelity of mRNAs.\nDue to the generation of short and repetitive oligonucleotides, traditional\nsequence mapping methods, which utilize in-solution RNase T1 digestion\nfollowed by LC-MS/MS analysis, face challenges in achieving high sequence\ncoverage. In this study, we developed a novel flow through (FT)-based\nstrategy to achieve the limited RNase T1 digestion of therapeutic\nmRNAs, leading to improved mRNA sequence mapping by LC-MS/MS analysis.\nCompared with the traditional in-solution digestion methods, the FT-based\ndigestion method could consistently generate an increased number of\nunique oligonucleotides with miscleavages, which significantly boosted\nthe overall sequence coverage (over 93%) of therapeutic mRNAs of varying\nsizes. Moreover, the automated digestion workflow using the AssayMAP\nplatform offers significant advantages in method reproducibility and\nthroughput. The high throughput and high sequence coverage features\nof this method could facilitate its wide application in the development\nof mRNA-based therapeutics.