Structural Fingerprinting of Antisense Oligonucleotide Therapeutics by Solution NMR Spectroscopy.

Purpose: Antisense oligonucleotide (ASO) therapeutics are an emerging class of biopharmaceuticals to treat and prevent diseases, particularly those involving "undruggable" protein targets. Impurities generated throughout the ASO drug manufacturing and formulation pipeline can be detrimental to drug safety and efficacy. Therefore, analytical techniques are needed to rigorously characterize these molecules for quality assurance purposes.

Methods: We demonstrate 1D and 2D nuclear magnetic resonance (NMR) spectroscopy methods that can generate high-resolution structural "fingerprints" of ASOs.

Results And Conclusions: 1D H and P measurements are shown to provide rapid initial assessment of the ASO integrity. In particular, a well-resolved pair of P signals arising from the 5´-end of the phosphorodiamidate morpholino oligomer (PMO) are sensitive to complex formation and oligomerization state. 2D H-H, H-C, and H- N experiments, although less sensitive, are further shown to enable resonance assignment, which will allow the tracking of structural changes at high-resolution during the drug development and manufacturing processes. We further anticipate that the described NMR approaches will be broadly applicable to fully formulated ASO therapeutics, including modalities other than PMOs.