Consecutive Affinity and Ion-Exchange Chromatography for AAV9 Vectors Purification
<b>Background:</b> Irrespective of the rapid development of AAV-based gene therapy, the production of clinical-grade vectors has a bottleneck resulting from product-related impurities such as empty and partially filled capsids, which lack a functional recombinant genome. <b>Methods...
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MDPI AG
2025-02-01
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| author | Ozgun Firat Duzenli George Aslanidi |
| author_facet | Ozgun Firat Duzenli George Aslanidi |
| author_sort | Ozgun Firat Duzenli |
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| description | <b>Background:</b> Irrespective of the rapid development of AAV-based gene therapy, the production of clinical-grade vectors has a bottleneck resulting from product-related impurities such as empty and partially filled capsids, which lack a functional recombinant genome. <b>Methods:</b> In the current study, we applied the sequential affinity chromatography (AC)- and anion-exchange chromatography (AEX)-based method for purification of AAV9 vector harboring single-stranded genome encoding the fusion of firefly luciferase (fLuc)-yellow fluorescent protein (YFP) under chicken beta actin (CBA) promoter. We assessed the efficiency of two different pre-packed cross-linked sepharose and one monolithic AEX columns following AC purification to separate fully encapsulated with recombinant DNA AAV vectors from byproducts. <b>Results:</b> We showed the possibility to achieve approximately 20–80% recovery and over 90% calculated DNA-containing/empty capsid ratio depending on column and buffers composition. Additionally, we confirmed the infectivity of AAV by in vitro luciferase assay regardless of recovery method from different AEX columns. <b>Conclusions:</b> Our purification data indicate the effectiveness of dual chromatography method to obtain rAAV9 vectors with DNA-containing capsid content over 90%. |
| format | Article |
| id | doaj-art-926afd1535dd48f4b4ac7b2ea88e1ee8 |
| institution | DOAJ |
| issn | 2227-9059 |
| language | English |
| publishDate | 2025-02-01 |
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| series | Biomedicines |
| spelling | doaj-art-926afd1535dd48f4b4ac7b2ea88e1ee82025-08-20T02:44:49ZengMDPI AGBiomedicines2227-90592025-02-0113236110.3390/biomedicines13020361Consecutive Affinity and Ion-Exchange Chromatography for AAV9 Vectors PurificationOzgun Firat Duzenli0George Aslanidi1The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USAThe Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA<b>Background:</b> Irrespective of the rapid development of AAV-based gene therapy, the production of clinical-grade vectors has a bottleneck resulting from product-related impurities such as empty and partially filled capsids, which lack a functional recombinant genome. <b>Methods:</b> In the current study, we applied the sequential affinity chromatography (AC)- and anion-exchange chromatography (AEX)-based method for purification of AAV9 vector harboring single-stranded genome encoding the fusion of firefly luciferase (fLuc)-yellow fluorescent protein (YFP) under chicken beta actin (CBA) promoter. We assessed the efficiency of two different pre-packed cross-linked sepharose and one monolithic AEX columns following AC purification to separate fully encapsulated with recombinant DNA AAV vectors from byproducts. <b>Results:</b> We showed the possibility to achieve approximately 20–80% recovery and over 90% calculated DNA-containing/empty capsid ratio depending on column and buffers composition. Additionally, we confirmed the infectivity of AAV by in vitro luciferase assay regardless of recovery method from different AEX columns. <b>Conclusions:</b> Our purification data indicate the effectiveness of dual chromatography method to obtain rAAV9 vectors with DNA-containing capsid content over 90%.https://www.mdpi.com/2227-9059/13/2/361adeno-associated vectorschromatographydownstream purificationAEX |
| spellingShingle | Ozgun Firat Duzenli George Aslanidi Consecutive Affinity and Ion-Exchange Chromatography for AAV9 Vectors Purification Biomedicines adeno-associated vectors chromatography downstream purification AEX |
| title | Consecutive Affinity and Ion-Exchange Chromatography for AAV9 Vectors Purification |
| title_full | Consecutive Affinity and Ion-Exchange Chromatography for AAV9 Vectors Purification |
| title_fullStr | Consecutive Affinity and Ion-Exchange Chromatography for AAV9 Vectors Purification |
| title_full_unstemmed | Consecutive Affinity and Ion-Exchange Chromatography for AAV9 Vectors Purification |
| title_short | Consecutive Affinity and Ion-Exchange Chromatography for AAV9 Vectors Purification |
| title_sort | consecutive affinity and ion exchange chromatography for aav9 vectors purification |
| topic | adeno-associated vectors chromatography downstream purification AEX |
| url | https://www.mdpi.com/2227-9059/13/2/361 |
| work_keys_str_mv | AT ozgunfiratduzenli consecutiveaffinityandionexchangechromatographyforaav9vectorspurification AT georgeaslanidi consecutiveaffinityandionexchangechromatographyforaav9vectorspurification |