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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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Autores principales: Wu, Di, Zhao, Xiaonan, Jimenez, Diego Antonio, Piszczek, Grzegorz
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10528216/
https://www.ncbi.nlm.nih.gov/pubmed/37759487
http://dx.doi.org/10.3390/cells12182264
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author Wu, Di
Zhao, Xiaonan
Jimenez, Diego Antonio
Piszczek, Grzegorz
author_facet Wu, Di
Zhao, Xiaonan
Jimenez, Diego Antonio
Piszczek, Grzegorz
author_sort Wu, Di
collection PubMed
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 pubmed-105282162023-09-28 Size Exclusion Chromatography–Mass Photometry: A New Method for Adeno-Associated Virus Product Characterization Wu, Di Zhao, Xiaonan Jimenez, Diego Antonio Piszczek, Grzegorz Cells Article 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. MDPI 2023-09-13 /pmc/articles/PMC10528216/ /pubmed/37759487 http://dx.doi.org/10.3390/cells12182264 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Wu, Di
Zhao, Xiaonan
Jimenez, Diego Antonio
Piszczek, Grzegorz
Size Exclusion Chromatography–Mass Photometry: A New Method for Adeno-Associated Virus Product Characterization
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 Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10528216/
https://www.ncbi.nlm.nih.gov/pubmed/37759487
http://dx.doi.org/10.3390/cells12182264
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