Size Exclusion Chromatography–Mass Photometry: A New Method for Adeno-Associated Virus Product Characterization

Over the past decade, adeno-associated viruses (AAVs) have attained significant prominence in gene therapy and genome editing applications, necessitating the development of robust and precise methodologies to ensure the quality and purity of AAV products. Existing AAV characterization techniques hav...

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Main Authors: Di Wu, Xiaonan Zhao, Diego Antonio Jimenez, Grzegorz Piszczek
Format: Article
Language:English
Published: MDPI AG 2023-09-01
Series:Cells
Subjects:
Online Access:https://www.mdpi.com/2073-4409/12/18/2264
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author Di Wu
Xiaonan Zhao
Diego Antonio Jimenez
Grzegorz Piszczek
author_facet Di Wu
Xiaonan Zhao
Diego Antonio Jimenez
Grzegorz Piszczek
author_sort Di Wu
collection DOAJ
description Over the past decade, adeno-associated viruses (AAVs) have attained significant prominence in gene therapy and genome editing applications, necessitating the development of robust and precise methodologies to ensure the quality and purity of AAV products. Existing AAV characterization techniques have proven effective for the analysis of pure and homogeneous AAV samples. However, there is still a demand for a rapid and low-sample-consumption method suitable for the characterization of lower purity or heterogeneous AAV samples commonly encountered in AAV products. Addressing this challenge, we propose the SEC-MP method, which combines size exclusion chromatography (SEC) with mass photometry (MP). In this novel approach, SEC effectively separates monomeric AAV particles from impurities, while the UV detector determines the virus particle concentration. MP complements this process by estimating the fraction of fully packaged AAVs in the total population of AAV particles. This combined methodology enables accurate determination of the titer of effective, fully packaged AAVs in samples containing aggregates, incorrectly packaged AAVs with incomplete genomes, protein or DNA fragments, and other impurities. Our experimental results demonstrate that SEC-MP provides valuable guidance for sample quality control and subsequent applications in the field of AAV research.
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spelling doaj.art-d6c1e412a1f3455f8aeb13865a34f6a72023-11-19T09:59:36ZengMDPI AGCells2073-44092023-09-011218226410.3390/cells12182264Size Exclusion Chromatography–Mass Photometry: A New Method for Adeno-Associated Virus Product CharacterizationDi Wu0Xiaonan Zhao1Diego Antonio Jimenez2Grzegorz Piszczek3Biophysics Core Facility, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USALaboratory of Cell and Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USALaboratory of Cell and Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USABiophysics Core Facility, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USAOver the past decade, adeno-associated viruses (AAVs) have attained significant prominence in gene therapy and genome editing applications, necessitating the development of robust and precise methodologies to ensure the quality and purity of AAV products. Existing AAV characterization techniques have proven effective for the analysis of pure and homogeneous AAV samples. However, there is still a demand for a rapid and low-sample-consumption method suitable for the characterization of lower purity or heterogeneous AAV samples commonly encountered in AAV products. Addressing this challenge, we propose the SEC-MP method, which combines size exclusion chromatography (SEC) with mass photometry (MP). In this novel approach, SEC effectively separates monomeric AAV particles from impurities, while the UV detector determines the virus particle concentration. MP complements this process by estimating the fraction of fully packaged AAVs in the total population of AAV particles. This combined methodology enables accurate determination of the titer of effective, fully packaged AAVs in samples containing aggregates, incorrectly packaged AAVs with incomplete genomes, protein or DNA fragments, and other impurities. Our experimental results demonstrate that SEC-MP provides valuable guidance for sample quality control and subsequent applications in the field of AAV research.https://www.mdpi.com/2073-4409/12/18/2264AAV titerVg/Cp ratiosample qualitygene therapy
spellingShingle Di Wu
Xiaonan Zhao
Diego Antonio Jimenez
Grzegorz Piszczek
Size Exclusion Chromatography–Mass Photometry: A New Method for Adeno-Associated Virus Product Characterization
Cells
AAV titer
Vg/Cp ratio
sample quality
gene therapy
title Size Exclusion Chromatography–Mass Photometry: A New Method for Adeno-Associated Virus Product Characterization
title_full Size Exclusion Chromatography–Mass Photometry: A New Method for Adeno-Associated Virus Product Characterization
title_fullStr Size Exclusion Chromatography–Mass Photometry: A New Method for Adeno-Associated Virus Product Characterization
title_full_unstemmed Size Exclusion Chromatography–Mass Photometry: A New Method for Adeno-Associated Virus Product Characterization
title_short Size Exclusion Chromatography–Mass Photometry: A New Method for Adeno-Associated Virus Product Characterization
title_sort size exclusion chromatography mass photometry a new method for adeno associated virus product characterization
topic AAV titer
Vg/Cp ratio
sample quality
gene therapy
url https://www.mdpi.com/2073-4409/12/18/2264
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AT xiaonanzhao sizeexclusionchromatographymassphotometryanewmethodforadenoassociatedvirusproductcharacterization
AT diegoantoniojimenez sizeexclusionchromatographymassphotometryanewmethodforadenoassociatedvirusproductcharacterization
AT grzegorzpiszczek sizeexclusionchromatographymassphotometryanewmethodforadenoassociatedvirusproductcharacterization