Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development
Abstract
Recombinant adeno-associated virus (rAAV)-mediated gene therapy is a fast-evolving field in the biotechnology industry. One of the major challenges in developing a purification process for AAV gene therapy is establishing an effective yet scalable method to remove empty capsids, or viral vectors lacking the therapeutic gene, from full capsids-viral product containing the therapeutic sequence. Several analytical methods that can quantify the empty-to-full capsid ratio have been reported in the literature. However, as samples can vary widely in viral titer, buffer matrix, and the relative level of empty capsids, understanding the specifications and limitations of different analytical methods is critical to providing appropriate support to facilitate process development. In this study, we developed a novel anion-exchange high-performance liquid chromatography assay to determine the empty-to-full capsid ratio of rAAV samples. The newly developed method demonstrated good comparability with both the transmission electron microscopy and analytical ultracentrifugation methods used in empty-to-full capsid ratio quantification, while providing much higher assay throughput and reducing the minimum sample concentration requirement to 2.7E11 viral genomes/mL.
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Limited sample quantities and matrix effects, such as buffer identity, residual DNA or protein, or ionic strength, pose challenges to rapid, high-throughput analytical methods in process development.
“However, limited sample quantities and matrix effects (e.g., buffer identity, residual DNA or protein, or ionic strength) pose challenges to such methods in process development.21, 22, 23”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [23]
AUC is well suited as an orthogonal method to validate more rapid methods with poorer resolution.
“Instead, it is well suited as an orthogonal method to validate more rapid methods with poorer resolution.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [23]
AUC throughput is limited to 7 samples in around 6 h, making routine use in process development challenging.
“With throughput limited to 7 samples in around 6 h, routine use of AUC in process development is challenging.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [23]
Multiple thousands of capsids must be imaged and analyzed for statistical significance in TEM.
“Multiple thousands of capsids must be imaged and analyzed for statistical significance, with the capsid content of individual images varying by around 2%.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [23]
TEM low accuracy is often caused by dimly stained, partially filled particles and uneven staining, leading to low contrast and ambiguity in capsid identification.
“Low accuracy is often caused by dimly stained, partially filled particles and uneven staining, leading to low contrast and ambiguity in identification of capsids.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [23]
AUC requires large sample volumes (400-500 μL) at high concentrations around 2 × 10^12-5 × 10^12 vg/mL, representing significant loss of unrecoverable material during early production stages.
“In general, AUC requires large sample volumes, 400–500 μL, at high concentrations around 2 × 1012–5 × 1012 vg/mL, representing a significant loss of unrecoverable material during early production stages.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [23]
In a single experiment, AUC assesses presence of aggregates and quantifies relative content of empty, partially filled, and full capsids.
“In a single experiment, AUC assesses the presence of aggregates and quantifies the relative content of empty, partially filled, and full capsids.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [23]
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 [23]
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 [23]
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 [23]
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 [23]
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 [23]
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 [23]
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Green, E. A., Fu, Q., Ndahiro, N., Leibiger, T. M., Wang, Y., Lee, Y.-S., Lee, K. H., Betenbaugh, M. J., Yoon, S., & McNally, D. J. (2025) · Biotechnology and Bioengineering · doi:10.1002/bit.28980 · Cited by 9 · see citing works
“Inter ‐ process comparisons are also complicated by differences in methods used to measure rAAV genome, capsid, and functional titers (François et al. 2018; Fu et al. 2019; Gimpel et al. 2021; Kontogiannis et al. 2024; Wang et al. 2020).”
Janc, M., Zevnik, K., Dolinar, A., Jakomin, T., Štalekar, M., Bačnik, K., Kutnjak, D., Žnidarič, M. T., Zentilin, L., Fedorov, D., et al. (2024) · Viruses · doi:10.3390/v16081235 · Cited by 8 · see citing works
“The content ratio (empty to full capsids) is traditionally assessed by analytical ultracentrifugation (AUC), transmission electron microscopy (TEM), qPCR/dPCR-ELISA combination and/or other novel methods that are also emerging [4–9].”
MethodHenrickson, A., Ding, X., Seal, A. G., Qu, Z., Tomlinson, L., Forsey, J., Gradinaru, V., Oka, K., & Demeler, B. (2023) · Nanomedicine · doi:10.2217/nnm-2023-0156 · Cited by 7 · see citing works
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“28,29 30–32 Traditionally, verification of the rAAV genome size has been conducted through de-naturing agarose gel electrophoresis.”
Deng, J. Z., Rustandi, R. R., Barbacci, D. C., Swartz, A. R., Gulasarian, A., & Loughney, J. W. (2022) · Human Gene Therapy · doi:10.1089/hum.2022.013 · Cited by 7 · see citing works
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Adeno-associated virus genome quantification with amplification-free CRISPR-Cas12a
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“In addition, the separation conditions are optimized for one specific serotype of AAV and will likely require further optimization for other serotypes (23).”
BackgroundSelf-citationEnd-to-end characterization of AAV manufacturing process using charge detection mass spectrometry
Young, L. M., Draper, B. E., Jarrold, M. F., Froelich, C. A., & Motevalian, S. P. (2025) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2025.101607 · Cited by 3 · see citing works
Accelerating AAV to the Clinic with Purification Improvements
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“23 Since AAVX resin required near-saturated loading to maximize recovery, 24 a large volume (>100 mL) was required for purification; thus, only scales of 250 mL and above were evaluated.”
Exploring Development Options of a Polishing Chromatography Step for AAV7 and AAV8
Fu, X., Haws, C., Begley, D. W., & Atta, R. G. (2025) · Human Gene Therapy · doi:10.1177/10430342251408498 · Cited by 2 · see citing works
Self-citationOptimization and scale up strategies for reproducible AAV enrichment step on CIMmultus® QA HR line
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“The preparative chromatogram reveals that the resolution between empty and full peaks has improved with the latter.”
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Particle Analytics: Comparative Approaches for Analysis of Viral Vectors
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Defining a Simplified Process in Yeast for Production of Enveloped VLP Dengue Vaccine
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“Low resolution between full and empty AAV capsids in AEX chromatography is an obstacle to optimal quality control and purification of an AAV-based viral vector manufacturing process [11].”
InfluentialLow voltage electron microscopy: An emerging tool for AAV characterization
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“Empty capsids are well-defined product-related impurities that can act as decoys, potentially eliciting undesired immune responses and reducing the effective dose of functional vectors [8].”
- Authors
- Fu, X., Chen, W.-C., Argento, C., Clarner, P., Bhatt, V., Dickerson, R., Bou-Assaf, G., Bakhshayeshi, M., Lu, X., Bergelson, S., & Pieracci, J.
- Affiliations
- Biogen (United States) (US)
- Corresponding author
- Fu, Xiaotong
- Venue
- Human Gene Therapy Methods
- Publisher
- SAGE Publications
- Published
- 2019-08
- Volume
- 30
- Issue
- 4
- Pages
- 144-152
- ISSN
- 1946-6536 (ISSN-L)
- 1946-6544
- OpenAlex
- W2965347002
- Cited by
- 77
- References
- 24
- Citations by year
- 2026: 2 · 2025: 15 · 2024: 14 · 2023: 18 · 2022: 7 · 2021: 19 · 2020: 1 · 2019: 1
- FWCI
- 4.22
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Human Gene Therapy Methods (publishedVersion)
- PubMed (publishedVersion)
- 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
- recombinant adeno-associated virus, empty capsids, viral vector purification, gene therapy vectors, method comparability, transmission electron microscopy, analytical ultracentrifugation, process development
- MeSH terms
- Dependovirus, Capsid, Chromatography, High Pressure Liquid, Genetic Therapy, Microscopy, Electron, Transmission
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