CONFIRM Sequence: A waters_connect™ Application For Sequencing Of Synthetic Oligonucleotides And Their Impurities

By Catalin E. Doneanu, Chris Knowles, Matt Gorton, Henry Shion, Joseph Fredette, and Ying Qing Yu, Waters Corporation

Synthetic oligonucleotides are a class of therapeutics that emerged in the past decade as an alternative to small molecule and protein therapeutics. Manufacturing and quality control of oligonucleotide therapeutics requires highly selective and sensitive LC-MS methods.

In addition to intact mass confirmation, another cornerstone of complete oligonucleotide characterization is the sequence verification/validation process. Sequence accuracy is crucial for the activity of therapeutic oligonucleotides and other nucleic acid therapeutics (e.g. mRNA), as their nucleotide sequence is directly linked to their biological function within cells. Achieving a complete sequence coverage (100%) for unambiguous sequence assignment is desired. Following gas phase fragmentation of oligonucleotide precursors by tandem mass spectrometry, all the detected fragment ions are analyzed in order to confirm the expected oligonucleotide sequence. This can be a tedious, time-consuming process, when performed manually, because of the many possibilities of oligonucleotide fragmentation and the large variety of a, b, c, d, w, x, y, and z ions10 typically observed after collision-induced dissociation (CID) of oligonucleotide precursors. Due to this variety of fragment ions, combined with the presence of sequence non-informative fragments (such as the loss of nucleobases), the unambiguous sequencing of oligonucleotides is clearly more challenging than peptide sequencing.

This application note demonstrates the recently introduced waters_connect application CONFIRM Sequence. This is an automated, compliance-ready liquid chromatography-mass spectrometry (LC-MS) workflow for sequence confirmation of oligonucleotides and their impurities.