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. 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. 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. Our purification data indicate the effectiveness of dual chromatography method to obtain rAAV9 vectors with DNA-containing capsid content over 90%.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11852678 | PMC |
| http://dx.doi.org/10.3390/biomedicines13020361 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Consecutive Affinity and Ion-Exchange Chromatography for AAV9 Vectors Purification.
Biomedicines
February 2025
The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA.
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. 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.
View Article and Find Full Text PDFRetention mechanism in slalom chromatography: Perspectives on the characterization of large DNA and RNA biopolymers in cell and gene therapy.
J Chromatogr A
February 2025
Waters Corporation, Instrument/Core Research/Fundamental, Milford, MA, 01757, USA. Electronic address:
Significant progress has been made in the last two decades in producing small (<2μm), high-purity, and low-adsorption particles, columns and system hardware, for ultra-high pressure liquid chromatography (UHPLC). Simultaneously, the recent rapid expansion of cell and gene therapies for treating diseases necessitates novel analytical technologies for analyzing large (>2 kbp) plasmid double-stranded (ds) DNA (which encodes for the in vitro transcription (IVT) of single-stranded (ss) mRNA therapeutics) and dsRNAs (related to IVT production impurities) biopolymers. In this context, slalom chromatography (SC), a retention mode co-discovered in 1988, is being revitalized using the most advanced column technologies for improved determination of the critical quality attributes (CQAs) of such new therapeutics.
View Article and Find Full Text PDFMixed-mode separation of antisense oligonucleotides using a single column with complementary anion-exchange and hydrophobic interaction chromatography approaches.
J Chromatogr A
January 2025
Genetics Guided Dementia Discovery (G2D2), Eisai, Inc. 35 Cambridge Park Drive, Suite 200, Cambridge, MA, 02140, USA.
The current study investigates the use of mixed-mode chromatography as a combination of anion-exchange (AEX) and hydrophobic interaction chromatography (HIC) for the analysis and purification of single-stranded antisense oligonucleotides with stereo-controlled phosphorothioate inter- nucleotide linkages. Initially a Scherzo-SS-C18 trimodal stationary phase with reversed-phase/AEX/ cation-exchange (CEX) functionalities is systematically evaluated to reveal the presence of U-shaped retention composed of two retention modes namely AEX and HIC, where the latter was also observed on related trimodal Scherzo SM and SW analogues. For the first time, retention and separation of deprotected oligonucleotides was described on a single mixed-mode column using a combination of AEX and HIC.
View Article and Find Full Text PDFIntegrated multidimensional chromatography on preparative scale for oligonucleotides purification.
J Chromatogr A
November 2024
Department of Environmental and Prevention Sciences, University of Ferrara, via L. Borsari 46, Ferrara 44121, Italy.
Therapeutic oligonucleotides represent a recent breakthrough in the pharmaceutical industry due to their ability to regulate gene expression with great specificity. This aspect allows treatment of a wide range of diseases. However, since oligonucleotides are used for therapeutic purposes, the Active Pharmaceutical Ingredient (API) must fulfill strict purity levels which require intensive purification steps.
View Article and Find Full Text PDFCoupling cation and anion exchange chromatography for fast separation of monoclonal antibody charge variants.
J Chromatogr A
September 2024
Department of Chemical and Process Engineering, Rzeszów University of Technology, Rzeszów/PL, Poland. Electronic address:
A design procedure for the separation of charge variants of a monoclonal antibody (mAb) was developed, which was based on the coupling of cation-exchange chromatography (CEX) and anion-exchange chromatography (AEX) under high loading conditions. The design of the coupled process was supported by a dynamic model. The model was calibrated on the basis of band profiles of variants determined experimentally for the mAb materials of different variant compositions.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!