Highly Efficient Ultracentrifugation-free Chromatographic Purification of Recombinant AAV Serotype 9
Open accessAbstract
Recombinant adeno-associated virus serotype 9 (rAAV9) can specifically transduce muscle and neuronal tissues; thus, rAAV9 can potentially be used in gene therapy. However, rAAV9 is the most challenging rAAV serotype to purify. Traditionally, rAAV9 has been purified by ultracentrifugation, which is not scalable. We recently described a chromatographic purification protocol for rAAV1; this protocol can achieve scalable purifications. In this study, we attempted to optimize this protocol for purifying rAAV9 preparations, and we developed a novel, effective method for high-yield purification of rAAV9 using quaternary ammonium anion exchangers and size-exclusion chromatography. The final purified rAAV9 contained mainly three capsid proteins, as observed by SDS-PAGE. Furthermore, negative-stain electron microscopy demonstrated that 96.1% ± 1.1% of rAAV9 particles carried the viral genome containing the EGFP transgene, indicating that impurities and empty capsids can be eliminated with our purification protocol. The final rAAV9 titer obtained by our protocol totaled 2.5 ± 0.4 × 10 15 viral genomes produced from ∼3.2 × 10 9 HEK293EB cells. We confirmed that our protocol can also be applied to purify other varied AAV genome constructs. Our protocol can scale up production of pure rAAV9, in compliance with current good manufacturing practice, for clinical applications in human gene therapy.
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TEM results are often unreliable and show poor agreement with orthogonal methods such as AUC.
“However, unreliable results are common, and poor agreement with orthogonal methods, such as analytical ultracentrifugation (AUC), has been reported.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [78]
Results between orthogonal methods are often not in agreement, and especially the accuracy of negative-staining TEM is often questioned.
“First, results between orthogonal methods are often not in agreement, and especially, the accuracy of negative-staining TEM is often questioned.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [78]
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References 42 · Cited by 55
- Authors
- Tomono, T., Hirai, Y., Okada, H., Miyagawa, Y., Adachi, K., Sakamoto, S., Kawano, Y., Chono, H., Mineno, J., Ishii, A., Shimada, T., Onodera, M., Tamaoka, A., & Okada, T.
- Affiliations
- University of Tsukuba (JP)
- Nippon Medical School (JP)
- Takara BIO Inc. (Japan) (JP)
- National Center For Child Health and Development (JP)
- Corresponding author
- Okada, Takashi
- ORCID
- Venue
- Molecular Therapy - Methods & Clinical Development
- Publisher
- Elsevier BV
- Published
- 2018-12
- Volume
- 11
- Pages
- 180-190
- ISSN
- 2329-0501 (ISSN-L)
- PMC
- PMC6276309
- OpenAlex
- W2899451645
- Cited by
- 55
- References
- 42
- Citations by year
- 2026: 2 · 2025: 10 · 2024: 10 · 2023: 11 · 2022: 10 · 2021: 6 · 2020: 3 · 2019: 3
- FWCI
- 1.84
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Molecular Therapy — Methods & Clinical Development (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Institutional Repositories DataBase (IRDB) (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Terrestrial Environment Research Center (University of Tsukuba) (submittedVersion, cc-by)
- Funders
- Japan Agency for Medical Research and Development
- A-STEP (16im0110418h0204)
- Basic Science and Platform Technology Program for Innovative Biological Medicine (16am0301021h0002, 17am0301021h0003, 18am0301021h0004)
- Japan Society for the Promotion of Science (JP16K08644)
- Takara Bio Inc. Research Fund
- 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 › Viral Infectious Diseases and Gene Expression in Insects
- Keywords
- chromatographic purification, size exclusion chromatography, scalable purification, gene therapy vectors, empty capsids, viral titer, current good manufacturing practice
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