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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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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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. |
format | Online Article Text |
id | pubmed-10218170 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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