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...
Saved in:
| Main Authors: | , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-02-01
|
| Series: | Biomedicines |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2227-9059/13/2/361 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Summary: | <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%. |
|---|---|
| ISSN: | 2227-9059 |