Buffer Valency Engineering Enables High-concentration and Shelf-stable DNA Transfection Particles for Viral Vector Production

Buffer Valency Engineering Enables High-concentration and Shelf-stable DNA Transfection Particles for Viral Vector Production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Buffer Valency Engineering Enables High-concentration and Shelf-stable DNA Transfection Particles for Viral Vector Production Hai-Quan Mao, Jinghan Lin, Yizong Hu, Turash Pial, Kailei Ding, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7042059/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Cost-effective and scalable production is critical for advancing the clinical translation of viral vector-mediated gene therapy. The widely used transient transfection method for the production of adeno-associated virus (AAVs) and lentivirus utilizes polyethyleneimine (PEI)/DNA particles loaded with multiple plasmids; however, the current pDNA/PEI particles must be prepared under dilute concentrations (< 20 µg mL⁻¹) and used immediately, hampering scale-up and reproducibility. Here, we introduce a kinetic-gating strategy in which transient binding of trivalent citrate ions slows complexation, enabling a streamlined and scalable strategy to generate shelf-stable, highly concentrated pDNA/poly(ethylenimine) (PEI) transfection particles. By incorporating trivalent citrate ions in the assembly buffers, we kinetically modulate electrostatic complexation to achieve uniform particle assembly and prevent aggregation at high concentrations. This enables a tenfold increase in pDNA concentration in stabilized transfection particles from a typical range of 10–20 µg/mL to 200 µg/mL, while reducing the required dosing volume from 5–10% to 0.5% of the cell culture medium. The particle assembly approach is mixer-agnostic, GMP-compatible, and compatible with standard workflows. We demonstrate equivalent AAV production efficiencies to standard methods and consistent performance in various production scales, which confirms the practical utility of this assembly method in developing robust, scalable, and cost-effective AAV manufacturing processes. Biological sciences/Biotechnology/Gene delivery/Transfection Physical sciences/Engineering/Biomedical engineering Biological sciences/Biological techniques/Nanobiotechnology/Nanoparticles Viral vector production Adeno-associated virus Plasmid DNA Poly(ethylenimine) Kinetic controlled assembly Shelf-stable transfection particles Scalable manufacturing Full Text Additional Declarations Yes there is potential Competing Interest. J.L., Y.H., and H.-Q.M. are co-inventors on a patent application covering the pDNA/PEI particle assembly technique described in this study, filed through and managed by the Johns Hopkins Office of Technology Ventures. P.B. and M.C. are employees of Biogen; M.G. is an employee of Polyplus Sartorius. The other authors declare no competing interests. Supplementary Files 2ManuscriptSI250620.pdf Buffer Valency Engineering Enables High-concentration and Shelf-stable DNA Transfection Particles for Viral Vector Production Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7042059","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":483567728,"identity":"016b9c48-aaf6-4b17-a6ab-b863182dd8ee","order_by":0,"name":"Hai-Quan 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The widely used transient transfection method for the production of adeno-associated virus (AAVs) and lentivirus utilizes polyethyleneimine (PEI)/DNA particles loaded with multiple plasmids; however, the current pDNA/PEI particles must be prepared under dilute concentrations (\u0026lt;\u0026thinsp;20 \u0026micro;g mL⁻\u0026sup1;) and used immediately, hampering scale-up and reproducibility. Here, we introduce a kinetic-gating strategy in which transient binding of trivalent citrate ions slows complexation, enabling a streamlined and scalable strategy to generate shelf-stable, highly concentrated pDNA/poly(ethylenimine) (PEI) transfection particles. By incorporating trivalent citrate ions in the assembly buffers, we kinetically modulate electrostatic complexation to achieve uniform particle assembly and prevent aggregation at high concentrations. This enables a tenfold increase in pDNA concentration in stabilized transfection particles from a typical range of 10\u0026ndash;20 \u0026micro;g/mL to 200 \u0026micro;g/mL, while reducing the required dosing volume from 5\u0026ndash;10% to 0.5% of the cell culture medium. The particle assembly approach is mixer-agnostic, GMP-compatible, and compatible with standard workflows. We demonstrate equivalent AAV production efficiencies to standard methods and consistent performance in various production scales, which confirms the practical utility of this assembly method in developing robust, scalable, and cost-effective AAV manufacturing processes.\u003c/p\u003e","manuscriptTitle":"Buffer Valency Engineering Enables High-concentration and Shelf-stable DNA Transfection Particles for Viral Vector Production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-23 04:01:06","doi":"10.21203/rs.3.rs-7042059/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1e557275-7742-41e3-99b3-1a1d2fdc6327","owner":[],"postedDate":"October 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":51346145,"name":"Biological sciences/Biotechnology/Gene delivery/Transfection"},{"id":51346146,"name":"Physical sciences/Engineering/Biomedical engineering"},{"id":51346147,"name":"Biological sciences/Biological techniques/Nanobiotechnology/Nanoparticles"}],"tags":[],"updatedAt":"2025-10-23T04:01:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-23 04:01:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7042059","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7042059","identity":"rs-7042059","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}