Utilization of Oxford Nanopore Technologies Direct RNA Sequencing and RNase 4 based LC-MS workflows for mRNA characterization

Wednesday, August 12th
1pm EDT

The advancement of in vitro transcribed (IVT) mRNA therapeutics has driven demand for analysis methods that support consistent product quality and safety throughout development and manufacturing. As an inherently complex molecule, the bioactivity,stability, and ultimate therapeutic potential of mRNA as a medicine requires robust examination of multiple critical quality attributes (CQAs) — including sequence identity, transcript integrity, poly(A) tail profiles, capping efficiency, and impurity detection. As mRNA therapeutics evolve toward longer constructs, incorporate modified nucleotides, and introduce sophisticated in vivo regulatory elements, there is a growing need for high-resolution methods that assess multiple CQAs using streamlined workflows.

In this webinar we highlight two complementary approaches for mRNA characterization using the National Institute of Standards and Technology Research-Grade Test Material 10202 (NIST RGTM 10202), developed to support CQA assessment of mRNA-based therapeutics. We will demonstrate how Oxford Nanopore Technologies^® (ONT) Direct RNA Sequencing enables simultaneous assessment of multiple CQAs, including sequence identity, transcript length, poly(A) tail distribution, and identify antisense impurities that may complement dsRNA detection methods. We will also present an RNase 4-based liquid chromatography-tandem mass spectrometry workflow which generates shorter oligonucleotides from long mRNA molecules for direct characterization of 5´ cap structures and poly(A) tails. Importantly, the results demonstrate the feasibility of ONT Direct RNA Sequencing and tandem LC-MS as complementary multi-attribute methods for mRNA analysis.