Automated determination of early eluting oligonucleotide impurities using ion-pair reversed-phase liquid chromatography high resolution-mass spectrometry

Abstract A new method is presented for the automated determination of early eluting (E.E.) oligonucleotide impurities analyzed by IP-RP HPLC HRMS. E.E. are impurities shorter than the main drug component and are primarily formed by the mechanisms of coupling failure, and depurination. The method is based on the detection of the theoretically derived most abundant mass of an impurity in the experimental data. An exhaustive list of candidate impurities and their formulas is automatically generated using the parent sequence and the known mechanisms of impurity formation. The approach accounts for possible modifications in the individual oligonucleotide sequence moieties (e.g., linkage, sugar, and base, 3′, and 5′ ends). The detected ion signal is summed to provide four nested levels of impurity breakdown information. The approach allows for the rapid determination of relationships and trends of impurities in samples generated by different manufacturing processes and conditions. Representative examples are given to illustrate the capabilities and utility of the approach in synthesis and purification process optimization applications.