Characterisation of a modified oligonucleotide together with its synthetic impurities using accurate mass measurements

Oligonucleotide-based drugs are beginning to establish themselves within the pharmaceutical industry as important agents in the treatment of various disease states with the potential of exhibiting high specificity with gene targeted therapies. Recent studies regarding RNA interference has stimulated interest in this field. There are now an increasing number of oligonucleotide-based pharmaceutical products in various stages of clinical development for the treatment of life-threatening diseases. As a result, the production of synthetic oligonucleotides has become increasingly important, with both antisense and RNAi-related oligonucleotides under development as therapeutic agents. One potential drug candidate currently under development at GlaxoSmithKline, is a 2'-O-methyl phosphorothioate in which the non-bridging oxygens of the phosphate diester are replaced with sulphur. Oligonucleotides are polymeric sequences made from an array of nucleotides (RNA, DNA and their respective analogs) usually ranging from 20-100 nucleotides. The polar nature, low thermal stability, complexity and large molecular weights of oligonucleotides have posed a challenge for the analysis of oligonucleotides by mass spectrometry. This paper demonstrates the use of negative ion electrospray with a combination of high resolution and high mass accuracy for the characterisation of oligonucleotides with the intention of supporting an evidence of structure document for a regulatory submission. This is a new area within the mass spectrometry field and as such there is limited software amongst the instrument companies for the data processing for the analysis of these compounds. Therefore, many of the examples in the literature only use mass spectrometry to generate average molecular weights by deconvoluting the multiple charged states observed to give an average molecular weight; under-utilizing the capability of high-resolution instruments. Copyright (C) 2011 John Wiley & Sons, Ltd.