Over the last years, the demand of MS based structural characterization approaches in quality control of oligonucleotide based pharmaceuticals increased notably. Sense strand RNAs with >30 nucleotide building blocks and labile modifications such as N-acetylgalactosamine residues represent thereby a special challenge. We will present direct, robust and easy to use techniques for full nucleotide sequence characterization of highly modified single-stranded RNAs by top down tandem mass spectrometry. This will include a case study for an N-acetylgalactosamine modified RNA sense strand.

The approach can be applied on single strand level, as well as on siRNA duplex level in a single ESI-MS experiment. Based on this technique, we also developed an efficient and highly sophisticated LC-MS(/MS) workflow for the characterization of product-related impurities in oligonucleotides such as methylation as well as PS-PO and rC/rU substitution. This approach allows us to confidently predict the modification sites in oligonucleotide impurities to support process development with important structural information.

Subject area:
Biologics

Objectives (If applicable):
NA

Target Audience (If applicable):
QA Manager, QA Staff, QC Chemist, QC Manager, R&D, Regulatory, Student