Introduction Antisense oligonucleotides (ASOs) are a class of therapeutic molecules being evaluated for a wide range of disease areas. Most ASO impurities arise from the starting materials and the synthetic process used to make the ASO. The impurities are structurally diverse and present in low quantities, making their identification and quantification a challenge. Currently available methods can’t profile isobaric impurities, such as ASOs missing the same nucleotide at different positions, which are prevalent in ASO products. Methods A MS-based method was developed to identify and quantify individual isobaric species in ASOs. In this approach, the main product (mixture of full-length product, P=O, n-1, n+1, etc.) is first separated from shortmers and branchmers via ion-pair reversed phase liquid chromatography. Subsequently, isobaric impurities within the main product are selected from the mixture using a quadrupole mass filter and fragmented in a trap collision cell. The characteristic fragment ions are then separated by ion-mobility based on their size/charge and detected by a time-of-flight mass spectrometer (Waters Synapt). The correlation between concentration and peak area was established using authentic standards to quantify the isobars. Representative Results Impurities which are one nucleotide shorter than the desired 20-mer ASO product, termed n-1 impurities, were evaluated. The impurity mixture contained two isobaric 19-mer species, both missing a methoxyethyl modified MeC (n-moe-MeC) but from two different positions in the sequence, and another impurity missing a methoxyethyl modified T, which isn’t resolved from n-moe-MeC (1 Da difference) via single scan MS. Parent ions were fragmented and separated by ion-mobility. DriftScope visualization was used to differentiate fragment ions of the same m/z but with different charge states and characteristic fragment ions were selected to quantify the individual n-1 impurities. The validity of the method was demonstrated by performing impurity spiking studies and accurately quantifying them. Novel Aspect Quantification of isobaric ASO impurities utilizing orthogonal separation methodologies and unique MS/MS fragmentation patterns

Characterization and quantification of isobaric antisense oligonucleotide impurities by tandem mass spectrometry with ion mobility

256th National Meeting (2018)

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