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Analysis of AAV-Extracted DNA by Charge Detection Mass Spectrometry Reveals Genome Truncations
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    Analysis of AAV-Extracted DNA by Charge Detection Mass Spectrometry Reveals Genome Truncations
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    • Lauren F. Barnes
      Lauren F. Barnes
      Chemistry Department, Indiana University, 800 E Kirkwood Avenue, Bloomington, Indiana 47405, United States
    • Benjamin E. Draper
      Benjamin E. Draper
      Chemistry Department, Indiana University, 800 E Kirkwood Avenue, Bloomington, Indiana 47405, United States
    • Justin Kurian
      Justin Kurian
      Analytical Research and Development, Pfizer Inc., 875 Chesterfield Pkwy. West, Chesterfield, Missouri 63017, United States
    • Yu-Ting Chen
      Yu-Ting Chen
      Analytical Research and Development, Pfizer Inc., 875 Chesterfield Pkwy. West, Chesterfield, Missouri 63017, United States
      More by Yu-Ting Chen
    • Tatiana Shapkina
      Tatiana Shapkina
      Analytical Research and Development, Pfizer Inc., 875 Chesterfield Pkwy. West, Chesterfield, Missouri 63017, United States
    • Thomas W. Powers
      Thomas W. Powers
      Analytical Research and Development, Pfizer Inc., 875 Chesterfield Pkwy. West, Chesterfield, Missouri 63017, United States
    • Martin F. Jarrold*
      Martin F. Jarrold
      Chemistry Department, Indiana University, 800 E Kirkwood Avenue, Bloomington, Indiana 47405, United States
      *Email: [email protected]
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    Analytical Chemistry

    Cite this: Anal. Chem. 2023, 95, 9, 4310–4316
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    https://doi.org/10.1021/acs.analchem.2c04234
    Published February 22, 2023
    Copyright © 2023 American Chemical Society

    Abstract

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    Adeno-associated virus (AAV) is a widely used gene therapy vector. The intact packaged genome is a critical quality attribute and necessary for an effective therapeutic. In this work, charge detection mass spectrometry (CDMS) was used to measure the molecular weight (MW) distribution for the genome of interest (GOI) extracted from recombinant AAV (rAAV) vectors. The measured MWs were compared to sequence masses for a range of rAAV vectors with different GOIs, serotypes, and production methods (Sf9 and HEK293 cell lines). In most cases, the measured MWs were slightly larger than the sequence masses, a result attributed to counterions. However, in a few cases, the measured MWs were significantly smaller than the sequence masses. In these cases, genome truncation is the only reasonable explanation for the discrepancy. These results suggest that direct analysis of the extracted GOI by CDMS provides a rapid and powerful tool to evaluate genome integrity in gene therapy products.

    Copyright © 2023 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.2c04234.

    • Mass distributions and charge versus mass scatter plots for AAV8 empty and for vectors not shown in the manuscript (PDF)

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    This article is cited by 28 publications.

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    Analytical Chemistry

    Cite this: Anal. Chem. 2023, 95, 9, 4310–4316
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acs.analchem.2c04234
    Published February 22, 2023
    Copyright © 2023 American Chemical Society

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