Metabolite ID And Relative Quantification Of Oligonucleotides In Plasma

By Remco van Soest, Kerstin Pohl, Todd Stawicki and Elliott Jones, SCIEX

Oligonucleotide therapeutics and gene therapies are rapidly gaining attention as their potency improves and delivery challenges are addressed. Modalities such as antisense oligonucleotides (ASOs) are becoming more important due to their high specificity and ability to reach formerly untreatable targets. To ensure safe drugs, methods for the identification and characterization of the full length product (FLP) and its metabolites are critical. High resolution mass spectrometry (HRMS) can be used for the identification of potential metabolites, by comparing the measured accurate masses and isotope patterns with those calculated. However, there is a lack of powerful yet intuitive processing software, and manual interpretation is cumbersome and time consuming. Furthermore, structural confirmation leveraging MS/MS adds an additional level of complexity.

This technical note describes the identification, relative quantification and structural confirmation of the chain-shortened metabolites of a phosphorothioated oligonucleotide. Using the Molecule Profiler software to overcome these challenges, we show the identification and relative quantification of the 5’ and 3’ (n-1, 2 and 3) metabolites of an ASO spiked into a rat plasma extract, in the presence of the FLP, at levels between 0.1% and 10% (w/w) of the FLP. The software can perform relative quantification based on TOF-MS, and assign fragment ions of the potential metabolites to confirm their structures, facilitating metabolism studies of drugs in development.