Decoding Complexity in Synthetic Oligonucleotides: Unraveling Coeluting Isobaric Impurity Ions by High Resolution...

Area of Science:

• Analytical Chemistry
• Mass Spectrometry
• Oligonucleotide Therapeutics

Background:

• Liquid chromatography-mass spectrometry (LC-MS) struggles with separating coeluting impurities in synthetic oligonucleotides due to similar masses.
• Impurities like uracil or cytosine nucleotide additions/deletions present significant analytical challenges.
• Existing methods lack the capability to quantify diverse sequence variants within isobaric impurity ions.

Purpose of the Study:

• To develop a direct method for identifying and quantifying isobaric impurity ions in synthetic oligonucleotides.
• To overcome the limitations of traditional LC-MS for analyzing complex impurity mixtures.
• To enable accurate impurity profiling in oligonucleotide therapeutics.

Main Methods:

• Utilized high-resolution mass spectrometry (HRMS) to resolve isotopic envelopes of impurity ions.
• Employed full or targeted MS analysis to measure chemical formula-dependent isotopic distributions.
• Quantified isobaric impurity ions using measured or theoretically adjusted isotopic distributions, bypassing standard curves.

Main Results:

• Successfully identified and quantified isobaric impurity ions resulting from nucleotide additions or deletions.
• Demonstrated the ability to quantify multiple sequence variants within a single impurity ion type.
• Validated the use of theoretical isotopic distributions with correction factors when standards are unavailable.

Conclusions:

• The HRMS-based method effectively resolves coeluting isobaric impurities in synthetic oligonucleotides.
• This approach provides a robust solution for in-depth impurity profiling, crucial for oligonucleotide therapeutics development.
• The technique offers enhanced quantification capabilities for complex impurity mixtures compared to conventional MS/MS methods.