SEC x IP two-dimensional LCMS for the analysis of non-denatured and denatured cyclic-peptide siRNAs in a single step

The increased effectiveness of small interfering RNAs (siRNAs) to induce gene silencing has brought a great therapeutic promise to many diseases. siRNAs are under highly active current research and development. Ligand conjugation and chemical modifications of the sense (SS) and antisense (AS) strands of the siRNA duplex improve stability and facilitate delivery, but significantly increase the complexity of the analytical requirements. Two chromatographic methods are needed to guide synthesis and formulation: (1) a non-denaturing method to analyze the duplex, residual sense and antisense strands, their impurities, and those of the duplex, and (2) a denaturing method for each strand and its impurities. In this work, ion-pair reversed phase (IP-RP) and strong anion exchange (SAX) methods were not successful in the analysis of a cyclic-peptide (CP) siRNA, in the non-denaturing mode. Selection of the most appropriate chromatographic method is greatly challenged by the chemical properties of the conjugated ligands. However, separation was possible by size exclusion chromatography (SEC). The non-denaturing SEC method was implemented, using a 2D-LC system, in the ¹D dimension of the analysis, coupled with a denaturing IP-RP method in the ²D dimension. The 2D-LC system greatly simplified the siRNA analysis by combining, for the first time, the non-denaturing and denaturing methods into a single-instrument, one sample injection method. An additional benefit of the 2D-LC system is the interfacing of MS-incompatible methods (e.g., SAX, SEC) to a mass spectrometer, broadening thus the analytical options, by coupling with MS-compatible methods (IP-RP, HILIC) in the ²D dimension. Application of the approach was exemplified in a CP-siRNA duplex formulation study to determine the optimal mixing ratio of the individual strands. A duplex maximum was reached at a sample solution AS:SS ratio of 0.9. The method was found to be independent of the amount and concentration of sample injected. A duplex annealing study found no significant temperature or salt effects in the formulation of the CP-siRNA duplex.