| Autor: |
Abdullah AM; Division of Complex Drug Analysis, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, St. Louis, Missouri 63110, United States., Sommers C; Division of Complex Drug Analysis, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, St. Louis, Missouri 63110, United States., Rodriguez JD; Division of Complex Drug Analysis, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, St. Louis, Missouri 63110, United States., Zhang D; Division of Therapeutic Performance I, Office of Research and Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland 20903, United States., Kozak D; Division of Therapeutic Performance I, Office of Research and Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland 20903, United States., Hawes J; Division of Systems Biology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas 72079, United States., Sapru M; Division of New Drug Product III, Office of New Drug Product, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland 20903, United States., Yang K; Division of Complex Drug Analysis, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, St. Louis, Missouri 63110, United States. |
| Abstrakt: |
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. |
