| Abstract: | Phosphorothioate oligonucleotide drugs typically contain product‐related impurities that are difficult to resolve chromatographically from the parent oligonucleotide due to the size of these compounds and the large number of stereoisomers that comprise the parent. The presence of co‐eluting impurities hinders the process of determining assay based on chromatographic separation alone. A mass spectrometry‐based purity assessment of the main chromatography peak can be used to quantify co‐eluting impurities and enable the accurate determination of assay, but a more direct measure of assay was desired due to the complexity of measuring all co‐eluting impurities by mass spectrometry. Therefore, we developed an assay method that utilizes the specificity of mass spectrometry to measure the amount of active pharmaceutical ingredient in a sample, which eliminates the need for chromatographic separation of impurities from the product. This procedure uses a single quadrupole mass spectrometer and incorporates an internal standard that is co‐sprayed with the analyte to compensate for the drift commonly associated with mass spectrometry‐based quantitation. Using the mass spectrometry response ratio for sample to internal standard enables the method to achieve excellent linearity (R2 = 0.998), repeatability (relative standard deviation = 0.5%), intermediate precision (0.6%), and accuracy, with measured assay values consistently within 2.0% of expected. The results indicate the method possesses the accuracy and precision required for measuring assay in clinical and commercial stage pharmaceutical products. Since the method is based on the specificity of the mass spectrometer, and does not rely on chromatographic separation of impurities, the procedure should be applicable to a wide variety of oligonucleotide therapeutics regardless of sequence or chemical modifications. |
