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Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches

The purity of the three capsid proteins that make up recombinant adeno-associated virus (rAAV) is considered a critical quality attribute of gene therapy products. As such, there is a clear need to develop separation methods capable of rapidly characterizing these three viral proteins (VPs). In this...

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Autores principales: Aebischer, Megane K., Bouvarel, Thomas, Barrozo, Emmalyn, Kochardt, Dominik, Elger, Carsten, Haindl, Markus, Ruppert, Raphael, Guillarme, Davy, D’Atri, Valentina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10218170/
https://www.ncbi.nlm.nih.gov/pubmed/37239849
http://dx.doi.org/10.3390/ijms24108503
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author Aebischer, Megane K.
Bouvarel, Thomas
Barrozo, Emmalyn
Kochardt, Dominik
Elger, Carsten
Haindl, Markus
Ruppert, Raphael
Guillarme, Davy
D’Atri, Valentina
author_facet Aebischer, Megane K.
Bouvarel, Thomas
Barrozo, Emmalyn
Kochardt, Dominik
Elger, Carsten
Haindl, Markus
Ruppert, Raphael
Guillarme, Davy
D’Atri, Valentina
author_sort Aebischer, Megane K.
collection PubMed
description The purity of the three capsid proteins that make up recombinant adeno-associated virus (rAAV) is considered a critical quality attribute of gene therapy products. As such, there is a clear need to develop separation methods capable of rapidly characterizing these three viral proteins (VPs). In this study, the potential benefits and limitations of different electrophoretic and chromatographic methods were evaluated, including capillary electrophoresis–sodium dodecyl sulfate (CE-SDS), reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC), and hydrophobic interaction chromatography (HIC), for the analysis of VPs obtained from different serotypes (i.e., AAV2, AAV5, AAV8, and AAV9). CE-SDS is considered to be the reference method and provides a suitable separation of VP1-3 proteins using generic conditions and laser induced fluorescence detection. However, the characterization of post-translational modifications (i.e., phosphorylation, oxidation) remains difficult, and species identification is almost impossible due to the lack of compatibility between CE-SDS and mass spectrometry (MS). In contrast, RPLC and HILIC were found to be less generic than CE-SDS and require tedious optimization of the gradient conditions for each AAV serotype. However, these two chromatographic approaches are inherently compatible with MS, and were shown to be particularly sensitive in detecting capsid protein variants resulting from different post-translational modifications. Finally, despite being non-denaturing, HIC offers disappointing performance for viral capsid proteins characterization.
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spelling pubmed-102181702023-05-27 Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches Aebischer, Megane K. Bouvarel, Thomas Barrozo, Emmalyn Kochardt, Dominik Elger, Carsten Haindl, Markus Ruppert, Raphael Guillarme, Davy D’Atri, Valentina Int J Mol Sci Article The purity of the three capsid proteins that make up recombinant adeno-associated virus (rAAV) is considered a critical quality attribute of gene therapy products. As such, there is a clear need to develop separation methods capable of rapidly characterizing these three viral proteins (VPs). In this study, the potential benefits and limitations of different electrophoretic and chromatographic methods were evaluated, including capillary electrophoresis–sodium dodecyl sulfate (CE-SDS), reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC), and hydrophobic interaction chromatography (HIC), for the analysis of VPs obtained from different serotypes (i.e., AAV2, AAV5, AAV8, and AAV9). CE-SDS is considered to be the reference method and provides a suitable separation of VP1-3 proteins using generic conditions and laser induced fluorescence detection. However, the characterization of post-translational modifications (i.e., phosphorylation, oxidation) remains difficult, and species identification is almost impossible due to the lack of compatibility between CE-SDS and mass spectrometry (MS). In contrast, RPLC and HILIC were found to be less generic than CE-SDS and require tedious optimization of the gradient conditions for each AAV serotype. However, these two chromatographic approaches are inherently compatible with MS, and were shown to be particularly sensitive in detecting capsid protein variants resulting from different post-translational modifications. Finally, despite being non-denaturing, HIC offers disappointing performance for viral capsid proteins characterization. MDPI 2023-05-09 /pmc/articles/PMC10218170/ /pubmed/37239849 http://dx.doi.org/10.3390/ijms24108503 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
Aebischer, Megane K.
Bouvarel, Thomas
Barrozo, Emmalyn
Kochardt, Dominik
Elger, Carsten
Haindl, Markus
Ruppert, Raphael
Guillarme, Davy
D’Atri, Valentina
Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches
title Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches
title_full Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches
title_fullStr Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches
title_full_unstemmed Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches
title_short Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches
title_sort boosting the separation of adeno-associated virus capsid proteins by liquid chromatography and capillary electrophoresis approaches
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10218170/
https://www.ncbi.nlm.nih.gov/pubmed/37239849
http://dx.doi.org/10.3390/ijms24108503
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