Thomas Franz, Study Director Biopharmaceutical MS at A&M Stabtest examined Stabtest’s QC methods for identity and purity analysis of oligonucleotides by LC-ToF-MS. The main purpose is to offer clients all relevant analytical parameters for small molecules, large molecules, and ATMPs under one roof.
In the case of oligonucleotides, the BioAccord system is used with the latest Waters Connect platform. Franz gave a brief overview of how the mass spectrometer works and explained that since MS is equipped with a negative ion mode, it is possible to analyse proteins and oligos with the same instrument. However, the team exercised caution since they did not want to destroy the performance of intact proteins by using incompatible elements and additives for oligo analysis.
Franz explained that as a result of this predicament, the team searched for alternatives. They aimed to explore the influence of different kinds of alkylamines in oligonucleotide analysis. The study used LC-MS to analyse multiple factors including oligonucleotide identity, purity, and stability. The study showed that DBA (dibutylamine) displayed higher sensitivity levels than TEA (triethylamine) and optimising cone voltage eliminated unwanted adducts. For stability testing, a thiophosphoryl oligonucleotide stored for 30 days showed degradation due to desulphuration, with the degradation products characterised successfully. Furthermore, the study found that adjusting LC-MS conditions like the UPLC gradient and flow rate significantly improved the separation and detection of oligonucleotides.
Analysing fragmentation patterns was explored for sequence confirmation and the integrated Waters Connect oligo sequencing application assisted with sequence coverage. Franz commented: “The higher the modification of the oligos, the fewer fragmentation pathways and cleaner spectra are obtained.”
In summary, the LC-MS technique was effective for the detailed characterisation of oligonucleotides, including impurity profiling, sequence confirmation, and stability assessment. Franz successfully showcased the flexibility and precision of LC-MS for oligo-therapeutic development.
