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Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2

Adeno-associated virus (AAV) vectors are clinically proven gene delivery vehicles that are attracting an increasing amount of attention. Non-genome-containing empty AAV capsids are by-products during AAV production that have been reported to potentially impact AAV product safety and efficacy. Theref...

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Autores principales: Wang, Chunlei, Mulagapati, Sri Hari Raju, Chen, Zhongying, Du, Jing, Zhao, Xiaohui, Xi, Guoling, Chen, Liyan, Linke, Thomas, Gao, Cuihua, Schmelzer, Albert E., Liu, Dengfeng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Gene & Cell Therapy 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838793/
https://www.ncbi.nlm.nih.gov/pubmed/31720304
http://dx.doi.org/10.1016/j.omtm.2019.09.006
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author Wang, Chunlei
Mulagapati, Sri Hari Raju
Chen, Zhongying
Du, Jing
Zhao, Xiaohui
Xi, Guoling
Chen, Liyan
Linke, Thomas
Gao, Cuihua
Schmelzer, Albert E.
Liu, Dengfeng
author_facet Wang, Chunlei
Mulagapati, Sri Hari Raju
Chen, Zhongying
Du, Jing
Zhao, Xiaohui
Xi, Guoling
Chen, Liyan
Linke, Thomas
Gao, Cuihua
Schmelzer, Albert E.
Liu, Dengfeng
author_sort Wang, Chunlei
collection PubMed
description Adeno-associated virus (AAV) vectors are clinically proven gene delivery vehicles that are attracting an increasing amount of attention. Non-genome-containing empty AAV capsids are by-products during AAV production that have been reported to potentially impact AAV product safety and efficacy. Therefore, the presence and amount of empty AAV capsids need to be characterized during process development. Multiple methods have been reported to characterize empty AAV capsid levels, including transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), charge detection mass spectrometry (CDMS), UV spectrophotometry, and measuring capsid and genome copies by ELISA and qPCR. However, these methods may lack adequate accuracy and precision or be challenging to transfer to a quality control (QC) lab due to the difficulty of implementation. In this study, we used AAV serotype 6.2 (AAV6.2) as an example to show the development of a QC-friendly anion exchange chromatography (AEX) assay for the determination of empty and full capsid percentages. The reported assay requires several microliters of material with a minimum titer of 5 × 10(11) vg/mL, and it can detect the presence of as low as 2.9% empty capsids in AAV6.2 samples. Additionally, the method is easy to deploy, can be automated, and has been successfully implemented to support testing of various in-process and release samples.
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spelling pubmed-68387932019-11-12 Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2 Wang, Chunlei Mulagapati, Sri Hari Raju Chen, Zhongying Du, Jing Zhao, Xiaohui Xi, Guoling Chen, Liyan Linke, Thomas Gao, Cuihua Schmelzer, Albert E. Liu, Dengfeng Mol Ther Methods Clin Dev Article Adeno-associated virus (AAV) vectors are clinically proven gene delivery vehicles that are attracting an increasing amount of attention. Non-genome-containing empty AAV capsids are by-products during AAV production that have been reported to potentially impact AAV product safety and efficacy. Therefore, the presence and amount of empty AAV capsids need to be characterized during process development. Multiple methods have been reported to characterize empty AAV capsid levels, including transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), charge detection mass spectrometry (CDMS), UV spectrophotometry, and measuring capsid and genome copies by ELISA and qPCR. However, these methods may lack adequate accuracy and precision or be challenging to transfer to a quality control (QC) lab due to the difficulty of implementation. In this study, we used AAV serotype 6.2 (AAV6.2) as an example to show the development of a QC-friendly anion exchange chromatography (AEX) assay for the determination of empty and full capsid percentages. The reported assay requires several microliters of material with a minimum titer of 5 × 10(11) vg/mL, and it can detect the presence of as low as 2.9% empty capsids in AAV6.2 samples. Additionally, the method is easy to deploy, can be automated, and has been successfully implemented to support testing of various in-process and release samples. American Society of Gene & Cell Therapy 2019-09-26 /pmc/articles/PMC6838793/ /pubmed/31720304 http://dx.doi.org/10.1016/j.omtm.2019.09.006 Text en © 2019 The Author(s) http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Article
Wang, Chunlei
Mulagapati, Sri Hari Raju
Chen, Zhongying
Du, Jing
Zhao, Xiaohui
Xi, Guoling
Chen, Liyan
Linke, Thomas
Gao, Cuihua
Schmelzer, Albert E.
Liu, Dengfeng
Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2
title Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2
title_full Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2
title_fullStr Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2
title_full_unstemmed Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2
title_short Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2
title_sort developing an anion exchange chromatography assay for determining empty and full capsid contents in aav6.2
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838793/
https://www.ncbi.nlm.nih.gov/pubmed/31720304
http://dx.doi.org/10.1016/j.omtm.2019.09.006
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