Sarah Mutchek, Vladimir V. Papov, Scott Pennino, Daniele Fabris
-acetylgalactosamine (GalNAc) modifiers. Typical collision-induced dissociation (CID) spectra obtained from the intact constructs were invariably dominated by fragments corresponding to the loss of GalNAc moieties and neutral nucleobases, which were not sequence-informative. These outcomes were attributed to the effects of interstrand pairing interactions that masked backbone cleavage events in the paired regions. To mitigate these effects, we implemented in-source activation to induce strand dissociation and enable separate MS/MS analyses of the released strands. Although this strategy afforded full sequence coverage for simple duplex constructs, more complex stemloop structures resisted dissociation and subsequent analysis. This observation was consistent with the inability of our in-source conditions to induce sufficient destabilization of the intrastrand pairs that define these higher-order structures. Sufficient destabilization was instead accomplished by adding a denaturing agent directly to the initial sample solutions, which led to the observation of new and abundant backbone fragments. Used in concert, the different strategies allowed us to corroborate not only the sequences of all oligonucleotide components, but also the sites of GalNAc conjugation and their respective structures. Therefore, the study demonstrated that top-down analysis can support the comprehensive characterization of leading pharmaceutical forms of siRNAs, provided that the selected strategy is adjusted according to their morphologies. The absence of hydrolysis and front-end separations also resulted in modest sample consumption and reduced analysis time, which should promote the application of this minimalistic approach to all phases of discovery, development, compliance, and manufacturing of this emerging class of biotherapeutics.