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Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9

Removal of empty capsids from adeno-associated virus (AAV) manufacturing lots remains a critical step in the downstream processing of AAV clinical-grade batches. Because of similar physico-chemical characteristics, the AAV capsid populations totally lacking or containing partial viral DNA are diffic...

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Autores principales: Joshi, Pranav R.H., Bernier, Alice, Moço, Pablo D., Schrag, Joseph, Chahal, Parminder S., Kamen, Amine
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Gene & Cell Therapy 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056178/
https://www.ncbi.nlm.nih.gov/pubmed/33898632
http://dx.doi.org/10.1016/j.omtm.2021.03.016
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author Joshi, Pranav R.H.
Bernier, Alice
Moço, Pablo D.
Schrag, Joseph
Chahal, Parminder S.
Kamen, Amine
author_facet Joshi, Pranav R.H.
Bernier, Alice
Moço, Pablo D.
Schrag, Joseph
Chahal, Parminder S.
Kamen, Amine
author_sort Joshi, Pranav R.H.
collection PubMed
description Removal of empty capsids from adeno-associated virus (AAV) manufacturing lots remains a critical step in the downstream processing of AAV clinical-grade batches. Because of similar physico-chemical characteristics, the AAV capsid populations totally lacking or containing partial viral DNA are difficult to separate from the desired vector capsid populations. Based on minute differences in density, ultracentrifugation remains the most effective separation method and has been extensively used at small scale but has limitations associated with availabilities and operational complexities in large-scale processing. In this paper, we report a scalable, robust, and versatile anion-exchange chromatography (AEX) method for removing empty capsids and subsequent enrichment of vectors of AAV serotypes 5, 6, 8, and 9. On average, AEX resulted in about 9-fold enrichment of AAV5 in a single step containing 80% ± 5% genome-containing vector capsids, as verified and quantified by analytical ultracentrifugation. The optimized process was further validated using AAV6, AAV8, and AAV9, resulting in over 90% vector enrichment. The AEX process showed comparable results not only for vectors with different transgenes of different sizes but also for AEX runs under different geometries of chromatographic media. The herein-reported sulfate-salt-based AEX process can be adapted to different AAV serotypes by appropriately adjusting elution conditions to achieve enriched vector preparations.
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spelling pubmed-80561782021-04-23 Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9 Joshi, Pranav R.H. Bernier, Alice Moço, Pablo D. Schrag, Joseph Chahal, Parminder S. Kamen, Amine Mol Ther Methods Clin Dev Original Article Removal of empty capsids from adeno-associated virus (AAV) manufacturing lots remains a critical step in the downstream processing of AAV clinical-grade batches. Because of similar physico-chemical characteristics, the AAV capsid populations totally lacking or containing partial viral DNA are difficult to separate from the desired vector capsid populations. Based on minute differences in density, ultracentrifugation remains the most effective separation method and has been extensively used at small scale but has limitations associated with availabilities and operational complexities in large-scale processing. In this paper, we report a scalable, robust, and versatile anion-exchange chromatography (AEX) method for removing empty capsids and subsequent enrichment of vectors of AAV serotypes 5, 6, 8, and 9. On average, AEX resulted in about 9-fold enrichment of AAV5 in a single step containing 80% ± 5% genome-containing vector capsids, as verified and quantified by analytical ultracentrifugation. The optimized process was further validated using AAV6, AAV8, and AAV9, resulting in over 90% vector enrichment. The AEX process showed comparable results not only for vectors with different transgenes of different sizes but also for AEX runs under different geometries of chromatographic media. The herein-reported sulfate-salt-based AEX process can be adapted to different AAV serotypes by appropriately adjusting elution conditions to achieve enriched vector preparations. American Society of Gene & Cell Therapy 2021-03-23 /pmc/articles/PMC8056178/ /pubmed/33898632 http://dx.doi.org/10.1016/j.omtm.2021.03.016 Text en © 2021 The Authors https://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 Original Article
Joshi, Pranav R.H.
Bernier, Alice
Moço, Pablo D.
Schrag, Joseph
Chahal, Parminder S.
Kamen, Amine
Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9
title Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9
title_full Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9
title_fullStr Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9
title_full_unstemmed Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9
title_short Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9
title_sort development of a scalable and robust aex method for enriched raav preparations in genome-containing vcs of serotypes 5, 6, 8, and 9
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056178/
https://www.ncbi.nlm.nih.gov/pubmed/33898632
http://dx.doi.org/10.1016/j.omtm.2021.03.016
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