Characterization of siRNA product-related impurities by hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry

Quality control of therapeutic oligonucleotides is mainly achieved by Ion-Pair Reversed Phase Liquid Chromatography (IP-RPLC) coupled to Mass Spectrometry and Anion-Exchange Liquid Chromatography. As manufacturing requirements increase during clinical development, so too do the requirements for the separation of impurities from full-length product. Hence, there is a need for a wider range of analytical methods for therapeutic oligonucleotides. Hydrophilic Interaction Liquid Chromatography (HILIC) is emerging as an orthogonal method for oligonucleotide analysis. We present an Ultra-Performance Liquid Chromatography Mass Spectrometry HILIC method with good separation of truncation impurities in modified RNA. We share an example where the method is applied to partly phosphorothioated single strands that are used to form GalNAc-conjugated double stranded siRNA. Truncation impurities arising from incomplete coupling were well separated from their parent FLP, both for incomplete couplings in the sequences as well as in the GalNAc-cluster, which is built up by successive couplings of a brancher and GalNAc phosphoramidite. The results showed both comparability of HILIC with IP-RPLC and orthogonality of HILIC towards IP-RPLC for the separation of GalNAc-cluster-related truncations.