Characterization of oligonucleotides and related impurities to support the development of drug substances

Find, relatively quantify, and confirm the structure of oligonucleotides and their impurities using the SCIEX ZenoTOF 7600 system and Molecule Profiler software

Remco van Soest, Kerstin Pohl, Yunyun Zou and Elliott Jones


This technical note describes the identification, relative quantification and structural confirmation of oligonucleotides and related impurities. Relative quantification and full sequence coverage was achieved at levels as low as 0.3% (w/w).

Oligonucleotide therapeutics and gene therapies are rapidly gaining attention as their potency improves and delivery challenges are addressed. Modalities such as antisense oligonucleotides (ASOs) are becoming more important due to their high specificity and ability to reach formerly undruggable targets. To ensure safe drugs, methods for the identification and characterization of the full length product (FLP) and impurities are critical. High resolution mass spectrometry (HRMS) can be used for the identification of potential impurities, by comparing the measured accurate masses and isotope patterns with those calculated. However, there is a lack of powerful yet intuitive processing software, and manual interpretation is cumbersome and time consuming. Furthermore, structural confirmation leveraging MS/MS adds an additional level of complexity.

Using the Molecule Profiler software to overcome these challenges, this technical note shows the identification and relative quantification of the 5’(n-1), 5’(n-2) and 5’(n-3) impurities of a fully phosphorothioated FLP spiked into an FLP sample at levels between 0.1 and 10% (w/w). The software can perform relative quantification based on TOF-MS, and assign fragment ions of the potential impurities to confirm their structures, facilitating the characterization of drugs in development.

Key features of Molecule Profiler software for oligonucleotide impurity analysis

  • Excellent quality and high mass accuracies for TOF-MS and TOF-MS/MS data allow for confident assignment of oligonucleotide FLP and impurities in Molecule Profiler software 

  • Straightforward quantification based on TOF-MS peak areas can be achieved by grouping of charge states or alternatively UV data can be leveraged for quantification 

  • Significant improvement of identification of low abundant impurities by boost in S/N and increased fragment assignment using the Zeno trap