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Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode

Gene therapy is opening unprecedented opportunities for novel therapeutic approaches. Based on the concept of rescuing function mutations by co-expressing the correct gene to allow biological functions to be restored, it requires the use of viral vectors to ensure the proper delivery of therapeutic...

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Autores principales: Aebischer, Megane K., Gizardin-Fredon, Hugo, Lardeux, Honorine, Kochardt, Dominik, Elger, Carsten, Haindl, Markus, Ruppert, Raphael, Guillarme, Davy, D’Atri, Valentina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9604245/
https://www.ncbi.nlm.nih.gov/pubmed/36293189
http://dx.doi.org/10.3390/ijms232012332
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author Aebischer, Megane K.
Gizardin-Fredon, Hugo
Lardeux, Honorine
Kochardt, Dominik
Elger, Carsten
Haindl, Markus
Ruppert, Raphael
Guillarme, Davy
D’Atri, Valentina
author_facet Aebischer, Megane K.
Gizardin-Fredon, Hugo
Lardeux, Honorine
Kochardt, Dominik
Elger, Carsten
Haindl, Markus
Ruppert, Raphael
Guillarme, Davy
D’Atri, Valentina
author_sort Aebischer, Megane K.
collection PubMed
description Gene therapy is opening unprecedented opportunities for novel therapeutic approaches. Based on the concept of rescuing function mutations by co-expressing the correct gene to allow biological functions to be restored, it requires the use of viral vectors to ensure the proper delivery of therapeutic genes. In this context, recombinant adeno-associated viruses (rAAV) are the most widely used vectors. Their biomanufacturing process requires the insertion of the therapeutic gene into the rAAV (full capsids). However, a percentage of rAAV that do not contain the desired gene (empty capsids), as well as partly filled capsids, might also be produced, potentially impacting the efficiency of the therapy. Therefore, the determination of the rAAV capsids’ full/empty ratio needs to be monitored to ensure consistent product quality and efficacy. Anion-exchange chromatography (AEX) can serve this need. In this contribution, thorough AEX method development, including a mobile phase, a stationary phase and gradient conditions, has highlighted its potential in supporting gene therapy. Taking advantage of the fact that viral capsids follow an “on/off” retention behavior, the application of a step gradient approach to the rAAV serotype 8 (rAAV8) allowed the unprecedented separation of rAAV8 full/empty capsids, with a resolution gain of 3.7 as compared to the resolution obtained with a fully optimized linear gradient. Finally, the developed analytical approach allowed a precise and accurate baseline separation and quantification of full and empty rAAV8 capsids, with the potential to be applied as a high-throughput quality control (QC) method.
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spelling pubmed-96042452022-10-27 Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode Aebischer, Megane K. Gizardin-Fredon, Hugo Lardeux, Honorine Kochardt, Dominik Elger, Carsten Haindl, Markus Ruppert, Raphael Guillarme, Davy D’Atri, Valentina Int J Mol Sci Article Gene therapy is opening unprecedented opportunities for novel therapeutic approaches. Based on the concept of rescuing function mutations by co-expressing the correct gene to allow biological functions to be restored, it requires the use of viral vectors to ensure the proper delivery of therapeutic genes. In this context, recombinant adeno-associated viruses (rAAV) are the most widely used vectors. Their biomanufacturing process requires the insertion of the therapeutic gene into the rAAV (full capsids). However, a percentage of rAAV that do not contain the desired gene (empty capsids), as well as partly filled capsids, might also be produced, potentially impacting the efficiency of the therapy. Therefore, the determination of the rAAV capsids’ full/empty ratio needs to be monitored to ensure consistent product quality and efficacy. Anion-exchange chromatography (AEX) can serve this need. In this contribution, thorough AEX method development, including a mobile phase, a stationary phase and gradient conditions, has highlighted its potential in supporting gene therapy. Taking advantage of the fact that viral capsids follow an “on/off” retention behavior, the application of a step gradient approach to the rAAV serotype 8 (rAAV8) allowed the unprecedented separation of rAAV8 full/empty capsids, with a resolution gain of 3.7 as compared to the resolution obtained with a fully optimized linear gradient. Finally, the developed analytical approach allowed a precise and accurate baseline separation and quantification of full and empty rAAV8 capsids, with the potential to be applied as a high-throughput quality control (QC) method. MDPI 2022-10-15 /pmc/articles/PMC9604245/ /pubmed/36293189 http://dx.doi.org/10.3390/ijms232012332 Text en © 2022 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
Aebischer, Megane K.
Gizardin-Fredon, Hugo
Lardeux, Honorine
Kochardt, Dominik
Elger, Carsten
Haindl, Markus
Ruppert, Raphael
Guillarme, Davy
D’Atri, Valentina
Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode
title Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode
title_full Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode
title_fullStr Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode
title_full_unstemmed Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode
title_short Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode
title_sort anion-exchange chromatography at the service of gene therapy: baseline separation of full/empty adeno-associated virus capsids by screening of conditions and step gradient elution mode
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9604245/
https://www.ncbi.nlm.nih.gov/pubmed/36293189
http://dx.doi.org/10.3390/ijms232012332
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