Comparison of Three LC-MS Platforms for Oligonucleotide Therapeutic Quantification

Oligonucleotide therapeutics is an evolving drug class that is composed of short stranded RNA or DNA used to treat diseases that were once thought to be undruggable. For the most part the evolution of the drug class has focused on modifications of the backbone of the sequence strand to increase specificity to its target and increase stability while in circulation. Another unique aspect of oligonucleotides is that the bioanalysis used to support preclinical and clinical studies can be approached using a variety of techniques that include mass spectrometry, hybridization LBA, or qPCR. Historically both LBA and qPCR have been the most sensitive platforms and mass spectrometry has been the more specific platform, recent development that utilizes a hybridization approach to LC/MS analysis has increased the sensitivity of oligonucleotide analysis on a mass spectrometry platform. In this presentation we will discuss the analysis of three types of oligonucleotides: an antisense oligonucleotide (Fomivirsen), a GalNAc-conjugated siRNA (Lumasiran), and a phosphorodiamidate morpholino oligomer (Viltolarsen). All three compounds have been analyzed on three mass spectrometry platforms which include an Orbitrap, Time of flight, and the more traditional triple quadruple. The goal of the analysis is to determine across the different platforms the overall gain in sensitivity and specificity of the analysis. By using oligonucleotides that have already been approved we are also able to talk about probe design related to the sequence of the oligonucleotide to aid in future discussions. This specific project is part of an overall collaboration between participants in industry where the same set of samples of each of the three oligonucleotides will be analyzed using mass spectrometry, hybridization LBA, and qPCR.