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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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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American Society of Gene & Cell Therapy
2021
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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. |
format | Online Article Text |
id | pubmed-8056178 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Society of Gene & Cell Therapy |
record_format | MEDLINE/PubMed |
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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