Identification and Relative Quantitation of Oligonucleotides and Impurities by a Data Dependent LC-MS/MS Method Using an Orbitrap Exploris 240 Mass Spectrometer
Purpose: Oligonucleotide therapeutics have been successfully developed for treatment of rare diseases, such as neurological and hepatic diseases, as well as common muscle and cardiovascular diseases. The potential of oligonucleotide therapies for alleviating the coronavirus disease-19 (COVID-19) pandemic was also discussed very recently. The advancement of oligonucleotide therapeutics drives the need to develop robust analytical approaches for confident sequence mapping and impurity assessment of these novel drugs. The LC-MS has proven to be a viable analytical tool for characterization and quantitation of therapeutic oligonucleotides and their impurities. However, a robust LC-MS/MS method is currently lacking due to challenge with automatic interpretation and annotation of MS/MS data. Methods: Synthetic oligonucleotides and their impurities were separated by ion pairing liquid chromatography using a Vanquish™ Horizon UHPLC system coupled with a DNAPac™ RP column. The DDA LC-MS/MS data were acquired using an Orbitrap Exploris™ 240 mass spectrometer. The MS/MS data were analyzed by the Oligonucleotide Analysis workflow in BioPharma Finder™ 4.0 software. Results: The DDA LC-MS/MS method coupled with the new Oligonucleotide Analysis tool of BioPharma Finder 4.0 software provides confident identification and complete or near-complete mapping of oligonucleotides as large as 45mer. Comparative analysis of the DDA LC-MS/MS data facilitated fast method development and optimization for oligonucleotide analysis. The HRAM MS and MS/MS data acquired also allowed identification and relative quantitation of impurities including those at very low abundance that were hardly discernible chromatographically (Figure 1). In a proof of concept study, a synthetic oligonucleotide purified with two different methods was analyzed. The comparative data analysis revealed a drastic decrease of all impurities in the sample purified with a more refined method (Figure 2). In another study, heat treatment of oligonucleotides led to almost complete depletion of their intact and large n-x forms while generating abundant small n-x forms and products associated with depurination and base loss. Conclusion: The HRAM-based DDA LC-MS/MS method allows confident identification, mapping, and relative quantitation of oligonucleotides and their impurities in a single experiment. The new oligonucleotide features available in BioPharma Finder 4.0 software not only facilitate method optimization, but they also allow quick assessment of different purification methods and monitoring of degradation products.