The DOI matches its Crossref record; the references it cites are not checked yet.
Quantification of adeno-associated virus particles and empty capsids by optical density measurement
Open accessAbstract
We show here that UV absorbance of denatured adeno-associated virus (AAV) vector provides a simple, rapid, and direct method for quantifying vector genomes and capsid proteins in solution. We determined the molar extinction coefficients of capsid protein to be 3.72 x 10(6) M(-1) cm(-1) at 260 nm and 6.61 x 10(6) M(-1) cm(-1) at 280 nm. For recombinant AAV vectors, extinction coefficients can be calculated by including the predicted absorbance of the vector DNA. Since the amount of empty capsids in purified vector preparations lowers the A(260)/A(280) ratio in a predictable manner, the vector genome (vg) and capsid particle (cp) titers in purified AAV vector preparations can be calculated from the absorbance at 260 nm and the A(260)/A(280) ratio. To validate this method, the vg and cp titers calculated by UV absorbance were compared with titers determined by quantitative (Q)-PCR and capsid ELISA. The vg titers determined by absorbance agreed well with titers determined by Q-PCR. The cp/vg ratio determined by the A(260)/A(280) method also correlated well with those determined by AAV capsid ELISA in conjunction with Q-PCR. This new method provides a simple and rapid means to determine AAV vg titers and the ratio of empty to full particles in purified virus preparations.
Overview
Integrity signals
Registry
Cites
Statistics
Verification
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
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 [22]
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 [22]
OD is limited to pure, concentrated samples above 5 × 10^11 vg/mL, requiring purification and likely concentration for in-process samples.
“The method is limited to pure, concentrated samples above 5 × 1011 vg/mL, requiring purification and likely, concentration for in-process samples, but is rapid (about 15 min) and suitable for purified rAAV.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [22]
OD is rapid (about 15 min) and suitable for purified rAAV.
“The method is limited to pure, concentrated samples above 5 × 1011 vg/mL, requiring purification and likely, concentration for in-process samples, but is rapid (about 15 min) and suitable for purified rAAV.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [22]
Although repeatability of 4% to 24% CV has been reported for qPCR plus ELISA content ratio estimation, careful consideration of error propagation and data accuracy is required for routine estimation.
“Although repeatability of 4%–24% CV has been reported,22 careful consideration of error propagation (qPCR: 5%–30% CV, ELISA: 10%–15% CV, combined: 11%–36% CV) and data accuracy is required for routine estimation of content ratio by this approach.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [22]
The accuracy and precision of indirect content ratio estimation by qPCR combined with ELISA are generally low, as they depend on the cumulative variability of the two methods.
“Accuracy and precision of this indirect method are generally low, as they depend on the cumulative variability of the two methods.22”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [22]
OD is not specific to rAAV, so removal of all other absorbing impurities is strictly required for accuracy.
“The assay is not specific to rAAV; thus, removal of all other absorbing impurities from the sample, such as residual nucleic acids and proteins, is strictly required for accuracy.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [22]
OD repeatability is as poor as 15% CV for content ratio and 22% CV for capsid titer.
“The precision of OD is comparable to the combined qPCR/ELISA workflow and repeatability reported as poor as 15% CV for content ratio and 22% CV for capsid titer.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [22]
OD precision is comparable to the combined qPCR/ELISA workflow.
“The precision of OD is comparable to the combined qPCR/ELISA workflow and repeatability reported as poor as 15% CV for content ratio and 22% CV for capsid titer.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [22]
Citations
The citation lists haven’t been fetched yet
References 22 · Cited by 230
Metadata
- Authors
- Sommer, J. Ü. M., Smith, P. H., Parthasarathy, S., Isaacs, J., Vijay, S., Kieran, J., Powell, S. K., McClelland, A., & Wright, J. F.
- Corresponding author
- Sommer, J.ürg M
- Venue
- Molecular Therapy
- Publisher
- Elsevier BV
- Published
- 2003-01
- Volume
- 7
- Issue
- 1
- Pages
- 122-128
- ISSN
- 1525-0016 (ISSN-L)
- OpenAlex
- W2058942457
- Cited by
- 230
- References
- 22
- Citations by year
- 2026: 6 · 2025: 22 · 2024: 27 · 2023: 20 · 2022: 12 · 2021: 25 · 2020: 6 · 2019: 9 · 2018: 9 · 2017: 11 · 2015: 4 · 2014: 5 · 2013: 10 · 2012: 10
- FWCI
- 3.35
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular Therapy (publishedVersion, cc-by-nc-nd)
- 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 › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Keywords
- adeno-associated virus, empty capsids, UV absorption, extinction coefficient, A260/A280 ratio, capsid protein, recombinant AAV vectors
- MeSH terms
- Dependovirus, Capsid, Cell Line, Genetic Vectors, Humans, Sensitivity and Specificity, Virion, Polymerase Chain Reaction, Optics and Photonics
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
