Analysis of particle content of recombinant adeno-associated virus serotype 8 vectors by ion-exchange chromatography
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Advances in adeno-associated virus (AAV)-mediated gene therapy have brought the possibility of commercial manufacturing of AAV vectors one step closer. To realize this prospect, a parallel effort with the goal of ever-increasing sophistication for AAV vector production technology and supporting assays will be required. Among the important release assays for a clinical gene therapy product, those monitoring potentially hazardous contaminants are most critical for patient safety. A prominent contaminant in many AAV vector preparations is vector particles lacking a genome, which can substantially increase the dose of AAV capsid proteins and lead to possible unwanted immunological consequences. Current methods to determine empty particle content suffer from inconsistency, are adversely affected by contaminants, or are not applicable to all serotypes. Here we describe the development of an ion-exchange chromatography-based assay that permits the rapid separation and relative quantification of AAV8 empty and full vector particles through the application of shallow gradients and a strong anion-exchange monolith chromatography medium.
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AEC separates empty and full rAAV capsids of serotypes 1, 2, 5, 6, 8, 9 based on isoelectric point differences caused by the encapsulated negatively charged genome.
“AEC has been used to separate empty and full rAAV capsids of several serotypes (1, 2, 5, 6, 8, 9) on a positively charged stationary phase based on the difference in their isoelectric points caused by the encapsulated, negatively charged genome.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [47]
AEC concentration range is limited by detector sensitivity, with a lower limit around 10^11 vg/mL, but FS detectors reduce the limit of detection by an order of magnitude.
“The method’s concentration range is limited by sensitivity of the detector, with a lower limit of around 1011 vg/mL similarly to OD, but the limit of detection is reduced by an order of magnitude using FS detectors.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [47]
AEC requires development and optimization of mobile phase conditions for each serotype, and baseline separation of full and empty particles and resolution of partially filled capsids have not been achieved.
“Sufficient separation between empty and full capsids requires development and optimization of mobile phase conditions for each serotype, but baseline separation of full and empty particles, as well as resolution of partially filled capsids, has not been achieved so far.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [47]
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References 34 · Cited by 99
- Authors
- Lock, M., Alvira, M. R., & Wilson, J. M.
- Affiliations
- University of Pennsylvania (US)
- ORCID
- Wilson, James M.
- Venue
- Human Gene Therapy Methods
- Publisher
- SAGE Publications
- Published
- 2012-02
- Volume
- 23
- Issue
- 1
- Pages
- 56-64
- ISSN
- 1946-6536 (ISSN-L)
- 1946-6544
- PMC
- PMC4015067
- OpenAlex
- W2001599454
- Cited by
- 99
- References
- 34
- Citations by year
- 2026: 3 · 2025: 8 · 2024: 7 · 2023: 11 · 2022: 10 · 2021: 14 · 2020: 8 · 2019: 9 · 2018: 6 · 2017: 5 · 2016: 2 · 2015: 7 · 2014: 4 · 2013: 2 · 2012: 2
- FWCI
- 2.88
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Human Gene Therapy Methods (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Bill and Melinda Gates Foundation
- University of Pennsylvania
- U.S. Public Health Service (HHSN268200748202C)
- National Heart, Lung, and Blood Institute
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- recombinant adeno-associated virus, AAV8, ion-exchange chromatography, AAV vector production, gene therapy manufacturing
- MeSH terms
- Dependovirus, Chromatography, Ion Exchange, DNA, Recombinant, Electrophoresis, Polyacrylamide Gel, Genetic Vectors, Virion, Genetic Therapy, Polymerase Chain Reaction, Microscopy, Electron, Transmission
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