OligoDistiller:\nA Platform Agnostic Software Tool\nfor MS and MS<sup>2</sup> Data Analysis of Complex Oligonucleotides\nand Their Impurities

Oligonucleotides are currently one of the most rapidly\nadvancing\nclasses of therapeutic modalities. Understanding critical quality\nattributes, such as the impurity profile, stability, potential metabolites,\nand sequence conformity, is the key to their ultimate success. To\nobtain the information presented above, liquid chromatography–mass\nspectrometry (LC–MS) is often employed. However, data interpretation\ncan be challenging due to multiple charge states, unknown species,\nchemical noises, and overlapping isotope envelopes (OIEs) of distinct\nbut unresolved species. To address these challenges, we have developed\nan MS-platform agnostic, multifunctional R package and a web-tool\nfor automated and interactive MS and MS<sup>2</sup> data analysis, <i>OligoDistiller</i>. From the MS spectrum of a complex mixture\nof two synthetic strands, our tool <i>OligoDistiller</i> annotated and quantified 45 oligonucleotide-related features including\n13 unknown impurities and 6 OIEs, which all together explained 90.8%\nof the detected peaks. Also, major product ions were assigned from\nthe MS<sup>2</sup> spectrum of a 47-mer DNA strand, covering 80.3%\nof the oligonucleotide sequence. We provide not only diverse isotope\nquality metrics for each annotated feature but also an interactive\ndata review module allowing for direct inspection of the part of raw\nspectrum linked to a selected feature. This tool <i>OligoDistiller</i> is freely available at https://github.com/daniellyz/OligoDistiller.