Synthetic oligonucleotides are a new and emerging class of drugs that have gained lots of interest. As a consequence of the complex solid-phase synthesis of therapeutic oligonucleotides, the full length product (FLP) typically contains several related substances, including deletion (shortmers) and addition (longmers) sequences.
Additionally, large mRNA constructs have evolved rapidly into an important modality for treatment of oncogenic and inflammatory diseases. The rapid development of mRNA based vaccines and therapeutics is supported by advances in analytical methodologies.
HRMS is a powerful tool to assess the critical quality attributes such as: (I) poly A length determination at 3’ end,
(II) capping efficiency at 5’ end,
(III) confirmation of identity, purity & modification through mRNA sequence mapping.
In this case study, we have setup a single LC-UV-HRMS analytical platform that allows synthetic oligonucleotide impurity profiling, poly A length analysis and determination of capping efficiency (data not shown), as well as sequence confirmation of both small oligonucleotides and large mRNA constructs. Despite the wide mass range of analytes, a single column and mobile phase composition is used for all experiments. In addition, the added value of MS2 analysis, allowing confirmation of the sequence, is demonstrated. For all applications, commercially available oligonucleotides were used.
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