From ASO to mRNA—Oligonucleotide Therapeutics Separation and Characterization Using LC/MS

Sponsored By:

Description: Recent advancements in nucleic acid-based therapeutics have generated much excitement and have commanded the development of new technologies and analytical workflows to characterize oligonucleotides and related impurities. There is a need for analytical workflows for rapid and sensitive methods for confirming sequence and modification on shorter (20-200 mer) oligos, while analyzing of 5’ cap, sequence confirmation, characterization of heterogeneity of polyA tail is required for mRNA based therapeutics. As with any therapy, analytical characterization including identity, purity and degradation products need to be established for both the drug product (oligo/mRNA) and the delivery system (LNP/AAV). Using ion-pairing reversed phase liquid-chromatography mass spectrometry (LC-MS), we have developed a rapid and sensitive high resolution mass spectrometry method for the analysis of intact single strand oligo analysis as long as 200-mer and confirm the sequence of the oligonucleotide using top-down mass spectrometry. For longer oligonucleotides, such as mRNA a T1 RNase digestion-based approach is use to generate fragments to establish the capping efficiency at 5’ mRNA end, determination of nucleotide sequence, and the characterization of the poly A tail heterogeneity. An extension of this workflow has been used for characterization of known and unknown impurities.