posted on 2023-12-30, 03:29authored byA. M. Abdullah, Cynthia Sommers, Jason D. Rodriguez, Deyi Zhang, Darby Kozak, Jessica Hawes, Mohan Sapru, Kui Yang
Analyzing
coeluting impurities with similar masses in synthetic
oligonucleotides by liquid chromatography–mass spectrometry
(LC-MS) poses challenges due to inadequate separation in either dimension.
Herein, we present a direct method employing fully resolved isotopic
envelopes, enabled by high resolution mass spectrometry (HRMS), to
identify and quantify isobaric impurity ions resulting from the deletion
or addition of a uracil (U) or cytosine (C) nucleotide from or to
the full-length sequence. These impurities may each encompass multiple
sequence variants arising from various deletion or addition sites.
The method utilizes a full or targeted MS analysis to measure accurate
isotopic distributions that are chemical formula dependent but nucleotide
sequence independent. This characteristic enables the quantification
of isobaric impurity ions involving sequence variants, a capability
typically unavailable in sequence-dependent MS/MS methods. Notably,
this approach does not rely on standard curves to determine isobaric
impurity compositions in test samples; instead, it utilizes the individual
isotopic distributions measured for each impurity standard. Moreover,
in cases where specific impurity standards are unavailable, the measured
isotopic distributions can be adequately replaced with the theoretical
distributions (calculated based on chemical formulas of standards)
adjusted using experiment-specific correction factors. In summary,
this streamlined approach overcomes the limitations of LC-MS analysis
for coeluting isobaric impurity ions, offering a promising solution
for the in-depth profiling of complex impurity mixtures in synthetic
oligonucleotide therapeutics.