Purification of N-acetylgalactosamine-modified-oligonucleotides using orthogonal anion-exchange and mixed-mode chromatography approaches.

N-acetylgalactosamine (GalNAc)-modified small interfering ribonucleic acids (siRNA) have shown promising outcomes for targeted siRNA delivery resulting in gene silencing in vivo; however, their structural complexity requires development of new purification methods to address high purity and recovery requirements. The current study evaluates complementary purification approaches using a mixed-mode Scherzo SS-C18 and anion-exchange (AEX) TSK-gel SuperQ-5PW for a range of single-stranded triantennary GalNAc-oligonucleotides. Initially, the semi-preparative mixed-mode support (10 × 250 mm, 3 µm) was compared against the preparative AEX analogue (21.5 × 300 mm, 13 µm), with the former affording double the recovery and higher purity of 95% over its AEX counterpart displaying 91% for a selected siRNA conjugate. An assortment of GalNAc-modified oligonucleotides was later purified using the mixed-mode resin revealing good recoveries (∼30-60%) and high purities of 90-94% ranging from straightforward to more challenging purifications. High sample loading in the 20 mg range was achieved, which was comparable with the larger preparative TSKgel SuperQ-5PW support. The Scherzo-SS-C18 resin also afforded some degree of resolution between diastereomers containing phosphorothioate functionalities. The TSKgel SuperQ-5PW support was later investigated to provide orthogonal separation selectivity to the Scherzo-SS-C18 column enabling purification of a selected, GalNAc-siRNA conjugate. The developed pH (8.5-11) and salt (0.3-0.7 M) gradients method provided enhanced separation selectivity between the free and conjugated siRNA, while minimizing formation of secondary structures and highlighting a complementary approach to deal with challenging purifications of oligonucleotide-GalNAc conjugates. Together, the use of AEX and mixed-mode columns provide much needed orthogonality to deal with complex GalNAc-modified oligonucleotides and potentially other upcoming modalities.

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