Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2
Open accessAbstract
Adeno-associated virus (AAV) vectors are clinically proven gene delivery vehicles that are attracting an increasing amount of attention. Non-genome-containing empty AAV capsids are by-products during AAV production that have been reported to potentially impact AAV product safety and efficacy. Therefore, the presence and amount of empty AAV capsids need to be characterized during process development. Multiple methods have been reported to characterize empty AAV capsid levels, including transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), charge detection mass spectrometry (CDMS), UV spectrophotometry, and measuring capsid and genome copies by ELISA and qPCR. However, these methods may lack adequate accuracy and precision or be challenging to transfer to a quality control (QC) lab due to the difficulty of implementation. In this study, we used AAV serotype 6.2 (AAV6.2) as an example to show the development of a QC-friendly anion exchange chromatography (AEX) assay for the determination of empty and full capsid percentages. The reported assay requires several microliters of material with a minimum titer of 5 × 10 11 vg/mL, and it can detect the presence of as low as 2.9% empty capsids in AAV6.2 samples. Additionally, the method is easy to deploy, can be automated, and has been successfully implemented to support testing of various in-process and release samples.
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AEC is one of the most widely used purification processes for separating empty and full capsids during downstream processing of rAAV and can quantify content ratio on HPLC with OD or FS detectors.
“AEC is one of the most widely used purification processes for the separation of empty and full capsids during downstream processing of rAAV and can be used on validation-friendly high-performance liquid chromatography (HPLC) systems equipped with OD or FS detectors to quantify the content ratio in the eluate.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [48]
AEC sensitivity is too low for quantification of raw harvest, with the lower end of the UV detector's linear range at around 10^11 vg/mL.
“Sensitivity is too low for quantification of raw harvest, however, with the lower end of the UV detector’s linear range at around 1011 vg/mL.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [48]
AEC is applicable to samples from downstream purification processes without additional sample purification or preparation.
“The method is applicable to samples from downstream purification processes without the need for additional sample purification or preparation.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [48]
AEC accuracy was verified by comparison to TEM and AUC, with high repeatability (<1%-4% CV) and intermediate precision (2%-5% CV) using UV detectors.
“Accuracy of AEC was verified by comparison to TEM and AUC, and high repeatability (<1%–4% CV) and intermediate precision (2%–5% CV) using UV detectors have been reported.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [48]
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 [48]
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References 31 · Cited by 105
- Authors
- Wang, C., Mulagapati, S. H. R., Chen, Z., Du, J., Zhao, X., Xi, G., Chen, L., Linke, T., Gao, C., Schmelzer, A. E., & Liu, D.
- Affiliations
- AstraZeneca (United States) (US)
- Corresponding author
- Wang, Chunlei
- Liu, Dengfeng
- Venue
- Molecular Therapy - Methods & Clinical Development
- Publisher
- Elsevier BV
- Published
- 2019-12
- Volume
- 15
- Pages
- 257-263
- ISSN
- 2329-0501 (ISSN-L)
- PMC
- PMC6838793
- OpenAlex
- W2975867742
- Cited by
- 105
- References
- 31
- Citations by year
- 2026: 6 · 2025: 19 · 2024: 21 · 2023: 26 · 2022: 8 · 2021: 16 · 2020: 9
- FWCI
- 4.97
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Molecular Therapy — Methods & Clinical Development (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Defense Advanced Research Projects Agency
- 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
- adeno-associated virus, empty capsids, anion exchange chromatography, gene therapy vectors, in-process testing, release testing
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