Determination of individual oligonucleotide impurities by small amine ion pair-RP HPLC MS and MS/MS: n - 1 impurities

Ion pair-reversed phase (IP-RP) HPLC is one of the most widely used methods for the analysis of oligonucleotide impurities. The method is compatible with mass spectrometry and has been used to guide the development of improved synthesis and purification approaches. The ability to detect and characterize impurities depends on the reagents and the IP buffer system employed, as each can directly affect the degree of chromatographic separation and the sensitivity of detection by MS. Previous work in our laboratory has shown that small alkyl amines are suitable IP reagents for the analysis of impurities in phosphate diester oligonucleotides and can be used to differentiate among individual members of composite impurity families. The addition of an alkyl acid often further enhances peak separation, but at the detriment of ion signal. An improved method with increased chromatographic performance and sensitivity of detection is presented here. Improvements were mainly realized through the use of lower concentrations of small alkyl amine (i.e., 5 mM) and acid (0.5 mM) IP reagents, and ammonium bicarbonate (20 mM) as a buffer. The improved capabilities of the new method are demonstrated by separation of the individual components of the composite n - 1 impurity in a set of four production-scale batches of a single oligonucleotide. Addition of the alkyl acid resulted in resolution of most individual n - 1 impurities. The observed enhanced sensitivity of detection allowed multiple reaction monitoring (MRM) experiments, which were used to differentiate among unresolved impurities.