Accurate Quantification and Characterization of Adeno-Associated Viral Vectors
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One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. This requires optimization for upstream and downstream processes as well as methods that enable the step-by step analytical characterization of the virus, the results of which inform the iterative refinement of production for yield, purity and potency. The biggest problem here is a plethora of viral vector formulations, many of which interfere with analytical techniques. We took adeno-associated virus (AAV) as an example and showed benefits of combined use of molecular methods and transmission electron microscopy (TEM) for viral vectors' characterization and quantification. Results of the analyses showed that droplet digital PCR (ddPCR) performs better than quantitative real-time PCR (qPCR), in terms of robustness and assay variance, and this was especially relevant for partially purified (in-process) samples. Moreover, we demonstrate the importance of sample preparation prior to PCR analysis. We evaluated viral structure, presence of aggregates and impurities with TEM analysis and found that these impacted the differences in viral titers observed by qPCR and ddPCR and could be altered by sample preparation. These results serve as a guide for the establishment of the analytical methods required to provide measures of identity and purity for AAV viral vectors.
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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 [21]
Excess residual byproducts limits the suitability of analytical methods with insufficient specificity unless extensive sample purification is performed.
“This excess of residual byproducts limits the suitability of methods with insufficient specificity, unless extensive sample purification is performed.21,22”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [21]
qPCR and ddPCR are currently the most widely used methods for quantification of genome titer because of their simplicity, specificity, and robustness.
“They are currently the most widely used methods for the quantification of the genome titer because of their simplicity, specificity, and robustness.21,35,71,79”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [21]
Genome titers determined by qPCR and ddPCR may differ, complicating the switch from qPCR to ddPCR during development or manufacturing.
“Moreover, genome titers determined by qPCR and ddPCR may differ,21 complicating the switch from qPCR to ddPCR during development or manufacturing.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [21]
ddPCR is significantly more precise than qPCR, with repeatability of 2% to 10% CV and intermediate precision of 3% to 8% CV, and is more tolerant to matrix effects and inhibitors.
“Thus, ddPCR is significantly more precise than qPCR—at repeatability of 2%–10% CV and intermediate precision of 3%–8% CV—and more tolerant to matrix effects and inhibitors.21,35,57,58”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [21]
TEM is the de facto reference method for determination of the content ratio in a laboratory setting but has also been used for identification of aggregates, deformed capsids, and non-capsid particles such as host-cell debris.
“It is the de facto reference method for determination of the content ratio in a laboratory setting but has also been used for the identification of aggregates, deformed capsids, and non-capsid particles, such as host-cell debris.21,76,77”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [21]
Current use of different PCR assay protocols renders titer results incomparable, jeopardizing accuracy and reproducibility of rAAV dosing.
“As a result, the current use of different assay protocols renders titer results incomparable, jeopardizing accuracy and reproducibility of rAAV dosing.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [21]
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AFM‐TEM correlation microscopy and its application to lipid nanoparticles
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Assessment of Key Factors Impacting Variability in AAV Vector Genome Titration by Digital PCR
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rAAV Production and Titration at the Microscale for High-Throughput Screening
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- Authors
- Dobnik, D., Kogovšek, P., Jakomin, T., Košir, N., Tušek Žnidarič, M., Leskovec, M., Kaminsky, S. M., Mostrom, J., Lee, H., & Ravnikar, M.
- Affiliations
- National Institute of Biology (SI)
- BIA Separations (Slovenia) (SI)
- Cornell University (US)
- Corresponding author
- Dobnik, David
- Venue
- Frontiers in Microbiology
- Publisher
- Frontiers Media SA
- Published
- 2019-07-17
- Volume
- 10
- Pages
- 1570-1570
- ISSN
- 1664-302X (ISSN-L)
- PMC
- PMC6650692
- OpenAlex
- W2962334628
- Cited by
- 123
- References
- 29
- Citations by year
- 2026: 15 · 2025: 16 · 2024: 23 · 2023: 28 · 2022: 13 · 2021: 22 · 2020: 6
- FWCI
- 6.18
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Frontiers in Microbiology (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DiRROS repository (University of Maribor) (publishedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Javna Agencija za Raziskovalno Dejavnost RS
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- adeno-associated virus vectors, gene therapy, droplet digital PCR, quantitative real-time PCR, transmission electron microscopy, sample preparation, viral titer
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