{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,21]],"date-time":"2026-02-21T05:00:24Z","timestamp":1771650024402,"version":"3.50.1"},"reference-count":20,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2024,2,26]],"date-time":"2024-02-26T00:00:00Z","timestamp":1708905600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia\/Minist\u00e9rio da Ci\u00eancia, Tecnologia e Ensino Superior (FCT\/MCTES, Portugal)","award":["UIDB\/04462\/2020"],"award-info":[{"award-number":["UIDB\/04462\/2020"]}]},{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia\/Minist\u00e9rio da Ci\u00eancia, Tecnologia e Ensino Superior (FCT\/MCTES, Portugal)","award":["UIDB\/04462\/2020"],"award-info":[{"award-number":["UIDB\/04462\/2020"]}]},{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia\/Minist\u00e9rio da Ci\u00eancia, Tecnologia e Ensino Superior (FCT\/MCTES, Portugal)","award":["UIDP\/04462\/2020"],"award-info":[{"award-number":["UIDP\/04462\/2020"]}]},{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia\/Minist\u00e9rio da Ci\u00eancia, Tecnologia e Ensino Superior (FCT\/MCTES, Portugal)","award":["LA\/P\/0087\/2020"],"award-info":[{"award-number":["LA\/P\/0087\/2020"]}]},{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia\/Minist\u00e9rio da Ci\u00eancia, Tecnologia e Ensino Superior (FCT\/MCTES, Portugal)","award":["UIDB\/04462\/2020"],"award-info":[{"award-number":["UIDB\/04462\/2020"]}]},{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia\/Minist\u00e9rio da Ci\u00eancia, Tecnologia e Ensino Superior (FCT\/MCTES, Portugal)","award":["UIDB\/04462\/2020"],"award-info":[{"award-number":["UIDB\/04462\/2020"]}]},{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia\/Minist\u00e9rio da Ci\u00eancia, Tecnologia e Ensino Superior (FCT\/MCTES, Portugal)","award":["UIDP\/04462\/2020"],"award-info":[{"award-number":["UIDP\/04462\/2020"]}]},{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia\/Minist\u00e9rio da Ci\u00eancia, Tecnologia e Ensino Superior (FCT\/MCTES, Portugal)","award":["LA\/P\/0087\/2020"],"award-info":[{"award-number":["LA\/P\/0087\/2020"]}]},{"name":"Associate Laboratory LS4FUTURE","award":["UIDB\/04462\/2020"],"award-info":[{"award-number":["UIDB\/04462\/2020"]}]},{"name":"Associate Laboratory LS4FUTURE","award":["UIDB\/04462\/2020"],"award-info":[{"award-number":["UIDB\/04462\/2020"]}]},{"name":"Associate Laboratory LS4FUTURE","award":["UIDP\/04462\/2020"],"award-info":[{"award-number":["UIDP\/04462\/2020"]}]},{"name":"Associate Laboratory LS4FUTURE","award":["LA\/P\/0087\/2020"],"award-info":[{"award-number":["LA\/P\/0087\/2020"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Separations"],"abstract":"<jats:p>Adeno-associated viruses (AAV) are currently predominant viral transfer tools for gene therapy, and efforts are being made to design faster and more efficient methods and technologies for their manufacturing. The early selection of high-performing filters is essential for developing an ultrafiltration and diafiltration (UF\/DF) process, especially when feed material is scarce, and timelines are short. However, few methods and technologies exist to enable process optimization with multiple variations in a single run. In this study, we explored the potential of Ambr\u00ae Crossflow for high-throughput, automated screening of different membrane materials, pore sizes and different process conditions for the UF\/DF step of AAV8. The best overall performance was obtained with a 100 kDa PES flat sheet cassette. The UF\/DF process was further transferred to a larger scale to the Sartoflow\u00ae Smart Tangential Flow Filtration (TFF) system using a 100 kDa PES Sartocon\u00ae Slice 200 cassette and compared to a 100 kDa PES hollow fiber. Virus recovery, permeate flux and total protein removal values of the flat sheet cassette were similar to those achieved in small-scale devices, and higher than those of the hollow fiber, thus demonstrating similar performance at a larger process scale. The high-throughput, automated method described herein allowed to screen multiple materials and process parameters of a UF\/DF process in a time- and resource-efficient way, making it a useful tool to accelerate early-stage downstream process development of AAV.<\/jats:p>","DOI":"10.3390\/separations11030073","type":"journal-article","created":{"date-parts":[[2024,2,26]],"date-time":"2024-02-26T10:40:17Z","timestamp":1708944017000},"page":"73","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Accelerated Development of AAV Purification Process Using a High-Throughput and Automated Crossflow System"],"prefix":"10.3390","volume":"11","author":[{"given":"S\u00f3nia","family":"Mendes","sequence":"first","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"ITQB-NOVA, Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal"}]},{"given":"Tiago Q.","family":"Faria","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"ITQB-NOVA, Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal"}]},{"given":"Andr\u00e9","family":"Nascimento","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"ITQB-NOVA, Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal"}]},{"given":"Marc","family":"Noverraz","sequence":"additional","affiliation":[{"name":"Sartorius Stedim Switzerland AG, 8317 Tagelswangen, Switzerland"}]},{"given":"Franziska","family":"Bollmann","sequence":"additional","affiliation":[{"name":"Sartorius Stedim Biotech GmbH, 37079 G\u00f6ttingen, Germany"}]},{"given":"Piergiuseppe","family":"Nestola","sequence":"additional","affiliation":[{"name":"Sartorius Stedim Switzerland AG, 8317 Tagelswangen, Switzerland"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0414-4899","authenticated-orcid":false,"given":"Ant\u00f3nio","family":"Rold\u00e3o","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"ITQB-NOVA, Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal"}]},{"given":"Cristina","family":"Peixoto","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"ITQB-NOVA, Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2959-102X","authenticated-orcid":false,"given":"Ricardo J. S.","family":"Silva","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"ITQB-NOVA, Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2024,2,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"El Andari, J., and Grimm, D. (2021). Production, Processing, and Characterization of Synthetic AAV Gene Therapy Vectors. Biotechnol. J., 16.","DOI":"10.1002\/biot.202000025"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1080\/14712598.2020.1693541","article-title":"Current challenges in biotherapeutic particles manufacturing","volume":"20","author":"Moleirinho","year":"2020","journal-title":"Expert. Opin. Biol. Ther."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"809118","DOI":"10.3389\/fmed.2021.809118","article-title":"Gene Therapy Advances: A Meta-Analysis of AAV Usage in Clinical Settings","volume":"8","author":"Au","year":"2021","journal-title":"Front. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"R2","DOI":"10.1093\/hmg\/ddr141","article-title":"Large-scale recombinant adeno-associated virus production","volume":"20","author":"Kotin","year":"2011","journal-title":"Hum. Mol. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"869","DOI":"10.1016\/j.biotechadv.2011.07.004","article-title":"Rational design and optimization of downstream processes of virus particles for biopharmaceutical applications: Current advances","volume":"29","author":"Vicente","year":"2011","journal-title":"Biotechnol. Adv."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Loewe, D., Dieken, H., Grein, T.A., Salzig, D., and Czermak, P. (2022). A Combined Ultrafiltration\/Diafiltration Process for the Purification of Oncolytic Measles Virus. Membranes, 12.","DOI":"10.3390\/membranes12020105"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/j.coche.2011.08.008","article-title":"Downstream bioprocessing: Recent advances and future promise","volume":"1","author":"Cramer","year":"2011","journal-title":"Curr. Opin. Chem. Eng."},{"key":"ref_8","first-page":"489","article-title":"High throughput screening techniques in protein purification","volume":"54","author":"Lacki","year":"2011","journal-title":"Methods Biochem. Anal."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1002\/bit.26859","article-title":"An ultra scale-down method to investigate monoclonal antibody processing during tangential flow filtration using ultrafiltration membranes","volume":"116","author":"Rayat","year":"2019","journal-title":"Biotechnol. Bioeng."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Fernandez-Cerezo, L., Wismer, M.K., Han, I., and Pollard, J.M. (2020). High throughput screening of ultrafiltration and diafiltration processing of monoclonal antibodies via the ambr(R) crossflow system. Biotechnol. Prog., 36.","DOI":"10.1002\/btpr.2929"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2461","DOI":"10.1002\/app.10536","article-title":"Sulfonation of polysulfones: Suitability of the sulfonated materials for asymmetric membrane preparation","volume":"84","author":"Blanco","year":"2002","journal-title":"J. App. Polym. Sci."},{"key":"ref_12","unstructured":"Beer, H., Demmer, W., H\u00d6RL, H.-H., Melzner, D., Nussbaumer, D., Schmidt, H.-W., and W\u00fcnn, E. (2001). Hydrophilic Porous Membranes Made of Cross-Linked Cellulose Hydrate, Method of Producing Them and Their Use, European Patent Office."},{"key":"ref_13","unstructured":"Borujeni, E., Betts, J., Jenke, M., and Pollard, J. (2024, January 17). Applications of an Automated High Throughput Crossflow System for Ultrafiltration Process Development to Assess Manufacturability of Monoclonal Antibodies. Available online: https:\/\/www.sartorius.com\/download\/714524\/ambr-cf-hts-scale-down-model-mab-application-note-en-b-25463-1--data.pdf."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Fernandez-Cerezo, L., Benner, S.W., and Pollard, J.M. (2021). Streamlining process characterization efforts using the high throughput ambr\u00ae crossflow system for ultrafiltration and diafiltration processing of monoclonal antibodies. Biotechnol. Prog., 37.","DOI":"10.1002\/btpr.3118"},{"key":"ref_15","unstructured":"Gibson, D., Betts, J., Jenke, M., and Lewis, W. (2024, January 17). Optimizing Diafiltration Parameters for Maximum Recovery of a Bispecific Antibody Using Low Process Volumes and an Automated High Throughput Crossflow System. Available online: https:\/\/www.sartorius.com\/download\/714528\/appl-note-ambr-cf-hts-manufacturability-bsab-sl-4086-e-pdf-data.pdf."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"118812","DOI":"10.1016\/j.memsci.2020.118812","article-title":"Ultrafiltration behavior of recombinant adeno associated viral vectors used in gene therapy","volume":"620","author":"Arunkumar","year":"2021","journal-title":"J. Membr. Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"119602","DOI":"10.1016\/j.memsci.2021.119602","article-title":"A mini-module with built-in spacers for high-throughput ultrafiltration","volume":"637","author":"Baitalow","year":"2021","journal-title":"J. Membr. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"440","DOI":"10.1016\/j.memsci.2017.06.038","article-title":"Module design for ultrafiltration in biotechnology: Hydraulic analysis and statistical modeling","volume":"540","author":"Thiess","year":"2017","journal-title":"J. Membr. Sci."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Mendes, J.P., Bergman, M., Solbrand, A., Peixoto, C., Carrondo, M.J.T., and Silva, R.J.S. (2022). Continuous Affinity Purification of Adeno-Associated Virus Using Periodic Counter-Current Chromatography. Pharmaceutics, 14.","DOI":"10.3390\/pharmaceutics14071346"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2578","DOI":"10.1002\/bit.28394","article-title":"Towards a scalable bioprocess for rAAV production using a HeLa stable cell line","volume":"120","author":"Escandell","year":"2023","journal-title":"Biotechnol. Bioeng."}],"container-title":["Separations"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2297-8739\/11\/3\/73\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:05:12Z","timestamp":1760105112000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2297-8739\/11\/3\/73"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,2,26]]},"references-count":20,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2024,3]]}},"alternative-id":["separations11030073"],"URL":"https:\/\/doi.org\/10.3390\/separations11030073","relation":{},"ISSN":["2297-8739"],"issn-type":[{"value":"2297-8739","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,2,26]]}}}