{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,19]],"date-time":"2026-09-19T22:03:42Z","timestamp":1789855422745,"version":"4.0.1"},"posted":{"date-parts":[[2026,9,17]]},"group-title":"Bioengineering","reference-count":49,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T00:00:00Z","timestamp":1789603200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"This study was supported by ChromaCon AG and YMC CO., LTD"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,17]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Recombinant adeno-associated virus (rAAV) manufacturing remains constrained by low yields, costly purification, and process complexity, limiting cost-effective access to gene therapy. Here, we demonstrate an integrated continuous biomanufacturing platform that transfers proven antibody-manufacturing technology to rAAV production by coupling perfusion to semi-continuous twin-column affinity capture (CaptureSMB). This architecture enables continuous harvest of extracellular rAAV without cell lysis, depth filtration, or endonuclease treatment, sustaining stable operation over five days. Relative to conventional batch processing, perfusion improved capsid or vector genome yield depending on the production process, while affinity capture alone reduced total DNA to batch-comparable levels, with no measurable loss in impurity clearance or potency. By consolidating multiple manual unit operations into one continuous workflow, the platform may reduce manual intervention and process footprint. Its modular design may generalize to other rAAV serotypes, inducible producer cell lines, and viral vector classes, offering a potential route toward more efficient gene therapy manufacturing.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Graphical abstract<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"751635v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Highlights<\/jats:title>\n                  <jats:list list-type=\"bullet\">\n                    <jats:list-item>\n                      <jats:p>An integrated continuous biomanufacturing platform for rAAV by coupling perfusion to a twin-column CaptureSMB affinity system has been established.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Bypassed traditional, costly, and yield-limiting batch operations including chemical cell lysis, depth filtration, and endonuclease treatment by capturing exclusively extracellular rAAVs.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Transfer of various scalable continuous process technologies from antibody processes to rAAV manufacturing.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Alternative platform with the potential to increase overall yields, lower cost of goods, increase automation and sustain or even improve critical quality attributes.<\/jats:p>\n                    <\/jats:list-item>\n                  <\/jats:list>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Technology readiness<\/jats:title>\n                  <jats:p>Whilst individual continuous processing units such as perfusion bioreactors and multi-column chromatography systems are mature and already employed in industry for various biologics, continuous production and purification systems for recombinant adeno-associated virus (rAAV) are still in their infancy. Recent studies suggest intensifying rAAV workflows via upstream perfusion and multi-column chromatography, however the integration of these steps into a cohesive continuous manufacturing line has remained a conceptual-stage challenge Technology Readiness Level (TRL 2). In this manuscript, we have established a proof-of-concept at laboratory scale conditions where state-of-the-art technologies from integrated continuous biomanufacturing for antibodies have been leveraged and applied to rAAV biomanufacturing. Thus, all employed technological elements used are available with scalable options for industrial implementation. Consequently, integrated continuous biomanufacturing of rAAV has advanced through TRL 3 (experimental proof of concept) to TRL 4 (technology validated in the laboratory) of the integrated continuous biomanufacturing workflow. In order to progress to TRL 5 (technology validated in relevant environment), it is necessary to demonstrate higher and consistent process yield, robust product quality, scalability and operational practicality under manufacturing conditions.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.09.14.751635","type":"posted-content","created":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T12:15:12Z","timestamp":1789647312000},"source":"Crossref","is-referenced-by-count":0,"title":["Integrated Continuous Biomanufacturing of Recombinant Adeno-Associated Virus"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-6937-6494","authenticated-orcid":false,"given":"Richard","family":"Plieninger","sequence":"first","affiliation":[{"name":"Institute of Pharma Technology and Biotechnology, FHNW University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerland"},{"name":"Institute of Applied Microbiology, RWTH Aachen University, Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-9671-4580","authenticated-orcid":false,"given":"Julia M.","family":"M\u00fcller","sequence":"additional","affiliation":[{"name":"Institute of Pharma Technology and Biotechnology, FHNW University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerland"},{"name":"Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-6359-3835","authenticated-orcid":false,"given":"Daniela","family":"Tobler","sequence":"additional","affiliation":[{"name":"Institute of Pharma Technology and Biotechnology, FHNW University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-8977-5461","authenticated-orcid":false,"given":"Efe","family":"Saygili","sequence":"additional","affiliation":[{"name":"Institute of Pharma Technology and Biotechnology, FHNW University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerland"},{"name":"Department of Chemistry, Materials and Chemical Engineering \u201cGiulio Natta\u201d, Politecnico di Milano, Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-1829-0343","authenticated-orcid":false,"given":"Patrick","family":"Werder","sequence":"additional","affiliation":[{"name":"Institute of Pharma Technology and Biotechnology, FHNW University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8349-1831","authenticated-orcid":false,"given":"Yuki","family":"Higuchi","sequence":"additional","affiliation":[{"name":"YMC CO., LTD., Kyoto, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8403-8249","authenticated-orcid":false,"given":"Ryosuke","family":"Takahashi","sequence":"additional","affiliation":[{"name":"YMC CO., LTD., Kyoto, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6890-6744","authenticated-orcid":false,"given":"Sebastian","family":"Vogg","sequence":"additional","affiliation":[{"name":"ChromaCon AG, Z\u00fcrich, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"M\u00fcller-Sp\u00e4th","sequence":"additional","affiliation":[{"name":"ChromaCon AG, Z\u00fcrich, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7082-1574","authenticated-orcid":false,"given":"Sven","family":"G\u00f6bel","sequence":"additional","affiliation":[{"name":"Institute of Pharma Technology and Biotechnology, FHNW University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0036-2522","authenticated-orcid":false,"given":"Thomas K.","family":"Villiger","sequence":"additional","affiliation":[{"name":"Institute of Pharma Technology and Biotechnology, FHNW University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026091914350456000_2026.09.14.751635v1.1","doi-asserted-by":"crossref","first-page":"821","DOI":"10.18609\/CGTI.2024.095","article-title":"Advancing AAV production with high-throughput screening and transcriptomics","volume":"10","year":"2024","journal-title":"Cell Gene Ther. 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