Creation of a High-Yield AAV Vector Production Platform in Suspension Cells Using a Design-of-Experiment Approach
Open accessAbstract
Recombinant adeno-associated virus (rAAV) vectors are a leading gene delivery platform, but vector manufacturing remains a challenge. New methods are needed to increase rAAV yields and reduce costs. Past efforts to improve rAAV production have focused on optimizing a single variable at a time, but this approach does not account for the interactions of multiple factors that contribute to vector generation. Here, we utilized a design-of-experiment (DOE) methodology to optimize rAAV production in a HEK293T suspension cell system. We simultaneously varied the transgene, packaging, and helper plasmid ratios, the total DNA concentration, and the cell density to systematically evaluate the impact of each variable across 52 conditions. The results revealed a unique set of parameters with a lower concentration of transgene plasmid, a higher concentration of packaging plasmid, and a higher cell density than previously described protocols. Using this DOE-optimized protocol, we achieved unpurified yields approaching 3 × 10 14 viral genomes (VGs)/L of cell culture. Additionally, we incorporated polyethylene glycol (PEG)-based virus precipitation, pH-mediated protein removal, and affinity chromatography to our downstream processing, enabling average purified yields of >1 × 10 14 VGs/L for rAAV-EGFPs across 13 serotypes and capsid variants.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
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?
Laboratory-scale production yields only a few milliliters of purified rAAV, equivalent to 10^12 to 10^14 vector genomes, such that the required sample volumes for analysis can cause significant loss of product.
“Commonly, laboratory-scale production yields only few milliliters (mL) of purified rAAV—equivalent to 1012–1014 vector genomes (vgs)—such that the required sample volumes for analysis can cause significant loss of product.6,7,20,23,24”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [20]
A fractional factorial design used for early screening of a rAAV production platform with only five process parameters at three levels required rapid, highly parallelized analysis of 32 experiments.
“For example, a fractional factorial design used for early screening of a rAAV production platform—with only five process parameters at three levels—required rapid, highly parallelized analysis of 32 experiments.20”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [20]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
The scanning model for translation: an update.
Kozak, M. (1989) · The Journal of Cell Biology · doi:10.1083/jcb.108.2.229 · Cited by 3,485 · see citing works
Adeno-associated virus vector as a platform for gene therapy delivery
Wang, D., Tai, P. W. L., & Gao, G. (2019) · Nature Reviews Drug Discovery · doi:10.1038/s41573-019-0012-9 · Cited by 2,444 · see citing works
Adenovirus-Associated Virus Vector–Mediated Gene Transfer in Hemophilia B
Nathwani, A. C., Tuddenham, E. G. D., Rangarajan, S., Rosales, C., McIntosh, J., Linch, D. C., Chowdary, P., Riddell, A., Pie, A. J., Harrington, C., et al. (2011) · New England Journal of Medicine · doi:10.1056/nejmoa1108046 · Cited by 1,864 · see citing works
Xiao, X., Li, J., & Samulski, R. J. (1998) · Journal of Virology · doi:10.1128/jvi.72.3.2224-2232.1998 · Cited by 1,381 · see citing works
Emerging issues in AAV-mediated in vivo gene therapy
Colella, P., Ronzitti, G., & Mingozzi, F. (2018) · Molecular Therapy - Methods & Clinical Development · doi:10.1016/j.omtm.2017.11.007 · Cited by 881 · see citing works
Effects of Intercistronic Length on the Efficiency of Reinitiation by Eucaryotic Ribosomes
Kozak, M. (1987) · Molecular and Cellular Biology · doi:10.1128/mcb.7.10.3438-3445.1987 · Cited by 537 · see citing works
Adeno-associated virus vectors can be efficiently produced without helper virus
Matsushita, T., Elliger, S., Elliger, C., Podsakoff, G., Villarreal, L., Kurtzman, G., Iwaki, Y., & Colosi, P. (1998) · Gene Therapy · doi:10.1038/sj.gt.3300680 · Cited by 507 · see citing works
Ylä‐Herttuala, S. (2012) · Molecular Therapy · doi:10.1038/mt.2012.194 · Cited by 465 · see citing works
Bioprocess optimization using design-of-experiments methodology
Mandenius, C., & Brundin, A. (2008) · Biotechnology Progress · doi:10.1002/btpr.67 · Cited by 422 · see citing works
Onasemnogene Abeparvovec: First Global Approval
Hoy, S. M. (2019) · Drugs · doi:10.1007/s40265-019-01162-5 · Cited by 337 · see citing works
Grieger, J. C., Soltys, S. M., & Samulski, R. J. (2015) · Molecular Therapy · doi:10.1038/mt.2015.187 · Cited by 326 · see citing works
Cloning of infectious adeno-associated virus genomes in bacterial plasmids
Laughlin, C. A., Tratschin, J.-D., Coon, H., & Carter, B. J. (1983) · Gene · doi:10.1016/0378-1119(83)90217-2 · Cited by 266 · see citing works
Role for highly regulated rep gene expression in adeno-associated virus vector production
Li, J., Samulski, R. J., & Xiao, X. (1997) · Journal of Virology · doi:10.1128/jvi.71.7.5236-5243.1997 · Cited by 227 · see citing works
Venkatakrishnan, B., Yarbrough, J., Domsic, J. F., Bennett, A. D., Bothner, B. B., Kozyreva, O. G., Samulski, R. J., Muzyczka, N., McKENNA, R. V., & Agbandje‐McKenna, M. (2013) · Journal of Virology · doi:10.1128/jvi.02524-12 · Cited by 225 · see citing works
Schwartz, R. S., Ting, C. S., & King, J. W. (2001) · Genome Research · doi:10.1101/gr.158701 · Cited by 217 · see citing works
Grimm, D., Kern, A., Pawlita, M., Ferrari, F. K., Samulski, R. J., & Kleinschmidt, J. A. (1999) · Gene Therapy · doi:10.1038/sj.gt.3300946 · Cited by 195 · see citing works
Schwartz, R. S., Ting, C. S., & King, J. W. (2001) · Genome Research · doi:10.1101/gr.gr-1587r · Cited by 181 · see citing works
Lock, M., Alvira, M. R., Chen, S.-J., & Wilson, J. M. (2014) · Human Gene Therapy Methods · doi:10.1089/hgtb.2013.131 · Cited by 163 · cited 5× · see citing works
“Our lab routinely titers rAAVby theCyQuantmethod andddPCRusing protocols adapted from Lock et al.30 with the primers and probe for BGHpA.”
MethodInfluentialAllay, J. A., Sleep, S., Long, S., Tillman, D. M., Clark, R., Carney, G., Fagone, P., McIntosh, J. H., Nienhuis, A. W., Davidoff, A. M., et al. (2011) · Human Gene Therapy · doi:10.1089/hum.2010.202 · Cited by 152 · see citing works
Vandenberghe, L. H., Xiao, R., Lock, M., Lin, J., Korn, M., & Wilson, J. M. (2010) · Human Gene Therapy · doi:10.1089/hum.2010.107 · Cited by 151 · see citing works
Transient Gene Expression in Suspension HEK-293 Cells: Application to Large-Scale Protein Production
Baldi, L., Müller, N., Picasso, S., Jacquet, R., Girard, P., Thanh, H.-P., Derow, E., & Wurm, F. M. (2005) · Biotechnology Progress · doi:10.1021/bp049830x · Cited by 142 · see citing works
Pham, P. L., Perret, S., Cass, B., Carpentier, E., St‐Laurent, G., Bisson, L., Kamen, A., & Durocher, Y. (2005) · Biotechnology and Bioengineering · doi:10.1002/bit.20428 · Cited by 112 · see citing works
Chahal, P. S., Schulze, É. A., Tran, R., Montes, J., & Kamen, A. (2013) · Journal of Virological Methods · doi:10.1016/j.jviromet.2013.10.038 · Cited by 111 · see citing works
Chromatin remodeling by nuclear receptors
Hebbar, P. B., & Archer, T. (2003) · Chromosoma · doi:10.1007/s00412-003-0232-x · Cited by 106 · see citing works
Vincent, K. A., Piraino, S., & Wadsworth, S. C. (1997) · Journal of Virology · doi:10.1128/jvi.71.3.1897-1905.1997 · Cited by 106 · see citing works
Durocher, Y., Pham, P. L., St‐Laurent, G., Jacob, D., Cass, B., Chahal, P. S., Lau, C. J., Nalbantoglu, J. N., & Kamen, A. (2007) · Journal of Virological Methods · doi:10.1016/j.jviromet.2007.03.014 · Cited by 93 · see citing works
Ameri, H. (2018) · Journal of Current Ophthalmology · doi:10.1016/j.joco.2018.01.006 · Cited by 69 · see citing works
Joshi, P. R. H., Cervera, L., Ahmed, I. A., Kondratov, O., Zolotukhin, S., Schrag, J. D., Chahal, P. S., & Kamen, A. (2019) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2019.02.003 · Cited by 60 · see citing works
Strobel, B., Klauser, B., Hartig, J. S., Lamla, T., Gantner, F., & Kreuz, S. (2015) · Molecular Therapy · doi:10.1038/mt.2015.123 · Cited by 57 · see citing works
Hildinger, M., Baldi, L., Stettler, M., & Wurm, F. M. (2007) · Biotechnology Letters · doi:10.1007/s10529-007-9441-3 · Cited by 57 · see citing works
Qiao, C., Li, J., Skold, A., Zhang, X., & Xiao, X. (2002) · Journal of Virology · doi:10.1128/jvi.76.4.1904-1913.2002 · Cited by 56 · see citing works
Scalable production of adeno‐associated virus type 2 vectors via suspension transfection
Park, J. Y., Lim, B., Lee, K., Kim, Y. K., & Jo, E. (2006) · Biotechnology and Bioengineering · doi:10.1002/bit.20776 · Cited by 55 · see citing works
Self-citationHigh-Yield Production of Recombinant Antibody Fragments in HEK-293 Cells Using Sodium Butyrate
Grünberg, J., Knogler, K., Waibel, R., et al. (2003) · BioTechniques · doi:10.2144/03345st02 · Cited by 43 · see citing works
Gene therapy’s next installment
(2019) · Nature Biotechnology · doi:10.1038/s41587-019-0194-z · Cited by 26 · see citing works
International Efforts for Recombinant Adeno-associated Viral Vector Reference Standards
Moullier, P., & Snyder, R. O. (2008) · Molecular Therapy · doi:10.1038/mt.2008.125 · Cited by 24 · see citing works
Feng, L., Guo, M., Zhang, S., et al. (2008) · Biotechnology and Applied Biochemistry · doi:10.1042/ba20070081 · Cited by 10 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
Gimpel, A. L., Katsikis, G., Sha, S., Maloney, A. J., Hong, M. S., Nguyen, T. N. T., Wolfrum, J., Springs, S. L., Sinskey, A. J., Manalis, S. R., et al. (2021) · Molecular Therapy - Methods & Clinical Development · doi:10.1016/j.omtm.2021.02.010 · Cited by 185 · see citing works
Dobrowsky, T., Gianni, D., Pieracci, J., & Suh, J. (2021) · Current Opinion in Biomedical Engineering · doi:10.1016/j.cobme.2021.100353 · Cited by 90 · see citing works
- Authors
- Zhao, H., Lee, K.-J., Daris, M., Lin, Y., Wolfe, T., Sheng, J., Plewa, C., Wang, S., & Meisen, W. H.
- Affiliations
- Amgen (United States) (US)
- Corresponding author
- Meisen, W. Hans
- Venue
- Molecular Therapy - Methods & Clinical Development
- Publisher
- Elsevier BV
- Published
- 2020-09
- Volume
- 18
- Pages
- 312-320
- ISSN
- 2329-0501 (ISSN-L)
- PMC
- PMC7334306
- OpenAlex
- W3033585446
- Cited by
- 98
- References
- 36
- Citations by year
- 2026: 9 · 2025: 23 · 2024: 28 · 2023: 16 · 2022: 12 · 2021: 10
- FWCI
- 5.57
- 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)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- U.S. Department of Energy
- Amgen
- 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 › RNA Interference and Gene Delivery
- Keywords
- recombinant adeno-associated virus vectors, AAV vector production, cell suspension culture, HEK293T cells, experimental design, helper plasmid, cell density, affinity chromatography, downstream processing, AAV serotypes
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
