Analytical Quality by Design–Based Approach for the Assay Method Development of Nusinersen Using Liquid Chromatography–High‐Resolution Mass Spectrometry

ABSTRACT The quality assessment of oligonucleotide possesses challenges due to non‐distinguishable structurally similar impurities. These impurities are difficult to separate from the drug, and the assay method as per regulatory requirements must quantify only the drug to assign the potency of the oligonucleotide therapeutics. In the present study, we have proposed a hyphenated liquid chromatography (LC)–high resolution mass spectrometry (HRMS) approach for accurate quantification of oligonucleotides with an example of Nusinersen. An analytical quality by design (AQbD) approach has been proposed for “fit‐for‐purpose” LC method development through the Taguchi orthogonal array (OA) screening model and Box–Behnken optimization model. The developed LC method was capable of providing reasonable separation, where the specificity of the assay method was achieved through high resolution MS. The extracted ion chromatogram (EIC) with an accuracy range of 10 ppm from MS output has provided a reasonable symmetric peak for quantification. The method was also successfully employed for the Nusinersen formulation. The method validation was performed according to ICH Q2( R 2) regulatory guidelines. A developed method through EIC processing was found to be specific and having linear response for 1.6–3 µg/mL. The percentage relative standard deviation was found to be less than 5 for repeatability and intermediate precision. The standard addition approach was used to determine assay method accuracy, which was found to be well within 97%–103% for triplicate at three concentrations. The current method was found to be robust with respect to LC flow rate, LC injection volume, MS drying/desolvation temperature, MS gas flow, and mass accuracy. The AQbD‐based quick LC separation coupled with HRMS methodology has been identified as a promising solution to assess the quality of oligonucleotide therapeutics.