RNA-based therapeutics are emerging as powerful therapeutics for the treatment and prevention of various diseases. Synthetic oligonucleotides, including antisense oligonucleotides and small interfering RNA (siRNA), are a subset of RNA-based medicines ranging in molecular weights from ~6000 to 30,000 Da. Owing to their synthetic nature and relatively large size compared to small molecules, the impurity profile of synthetic oligonucleotides can be quite complex and can contain very structurally similar impurities. Thus, high resolution analytical capabilities are needed to obtain in-depth characterization of oligonucleotides. We have investigated various one- and two-dimensional liquid chromatography-mass spectrometry strategies for the characterization of single and double stranded siRNA. The utility and orthogonality between hydrophilic interaction chromatography, anion exchange chromatography, and ion pair reversed phase chromatography was evaluated for a model phosphorothioate oligonucleotide. Use of the second dimension for desalting prior to MS allowed for online mass spectrometry analysis with high inorganic salt concentrations in the first dimension. Finally, highly targeted second dimension gradients coupled with high resolution mass spectrometry analysis allowed for improved resolution and identification of co-eluting impurities from the first dimension.

Development of single and multidimensional liquid chromatography-mass spectrometry techniques for the characterization of oligonucleotides

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