Temperature-controlled transition between non-denaturing and denaturing analysis of Patisiran on a mixed-mode SAX stationary phase utilizing two-dimensional liquid chromatography

Oligonucleotides are emerging nucleic acid-based therapeutics with growing clinical interest and regulatory approvals. As their use expands, reliable analytical methods are needed for quality assessment. Amongst them siRNA are short RNA duplexes which require quality control in non-dissociated form under non-denaturing conditions. Size-exclusion chromatography, anion-exchange chromatography and ion-pair reversed-phase liquid chromatography have been mainly employed for duplex siRNA analysis. In this work, we demonstrate that the (type of) stationary phase affects the melting temperature of the siRNA Patisiran. A silica-based porous mixed-mode triphenylphosphonium-based SAX stationary phase enabled the separation of antisense and sense single strands at 10 °C under non-denaturing conditions with NaCl-gradient elution. In the presence of this stationary phase, on-column melting of Patisiran siRNA duplex was observed at around 60 °C. At around 80 °C, the injection of the duplex siRNA resulted in denaturing conditions allowing single strand analysis on the same column. Interestingly, employing the same elution conditions on a nonporous hydrophilic polymer particle-based quaternary trimethylammonium-SAX column no dissociation, i.e. no melting, of the siRNA duplex occurred in the temperature range up to 80 °C. Thermodynamic analysis exhibited a distinct retention mechanism on the triphenylphosphonium-SAX and quaternary trimethylammonium-SAX columns. Under salt-free ion-pair reversed-phase LC conditions with 25 mM dibutylammonium acetate, the duplex was stable only under 20 °C, while above denaturing conditions were obtained. A heart-cutting 2D-LC approach was finally suggested for simultaneous non-denaturing and denaturing LC analysis of Patisiran in one analytical run. Non-denaturing LC on triphenylphosphonium-SAX stationary phase at 20 °C column temperature was employed as the first dimension which allowed intact duplex analysis and single strand excess analysis. A heart cut of the duplex peak was transferred via loop interface into the second dimension where the single strands were analysed under denaturing conditions on triphenylphosphonium-SAX stationary phase at 80 °C with the same mobile phase.