{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T09:47:16Z","timestamp":1791798436899,"version":"4.3.4"},"reference-count":42,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T00:00:00Z","timestamp":1791763200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T00:00:00Z","timestamp":1791763200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Biotech &amp; Bioengineering"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>High\u2010quality, properly linearized plasmid DNA (pDNA) is a decisive upstream determinant of in vitro transcription (IVT) performance, impacting mRNA yield, integrity, and batch\u2010to\u2010batch consistency. We developed immobilized restriction enzyme reactors (IMERs) on monolithic supports and benchmarked EcoRI, NdeI, and HindIII to evaluate suitability for IVT\u2010grade template preparation. Across all three enzymes, IMERs quantitatively converted supercoiled and open\u2010circular pDNA to the linear form and delivered a clean product, as verified by baseline\u2010resolved chromatography and agarose gels. To further verify the functionality of the IMER platform, BspQI was immobilized and used to linearize a second plasmid construct. IVT initiated from all four IMER\u2010processed templates produced mRNA with kinetics and titers indistinguishable from conventional in\u2010solution digestion, underscoring that template quality was preserved. Long\u2010term stability studies showed enzyme\u2010dependent stability with HindIII\u2010based IMER retaining ~20% activity for more than 4 months. While the normalized productivity of the IMER matched that of in\u2010solution reactions, the possibility of reusing IMER across multiple linearization cycles significantly increased cumulative yield. Further, linearization by IMERs eliminated the need for post\u2010reaction enzyme removal and harsh inactivation, thereby reducing degradation risk. Collectively, these results establish restriction enzyme\u2010based IMERs as a robust, scalable, automation\u2010ready tool for producing high\u2010purity, IVT\u2010ready linear pDNA, securing the template quality required by the current mRNA manufacturing process.<\/jats:p>","DOI":"10.1002\/bit.70406","type":"journal-article","created":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T09:06:00Z","timestamp":1791795960000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of Immobilized Restriction Enzyme Reactors on a Monolith Backbone for Streamlined Plasmid DNA Linearization"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-9800-027X","authenticated-orcid":false,"given":"Ma\u0161a","family":"Nabergoj","sequence":"first","affiliation":[{"name":"Sartorius BIA Separations, Mirce 21 Ajdov\u0161\u010dina Slovenia"},{"name":"University of Nova Gorica, Vipavska 13 Nova Gorica Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"\u0160pela","family":"Per\u0161i\u010d Debenjak","sequence":"additional","affiliation":[{"name":"Sartorius BIA Separations, Mirce 21 Ajdov\u0161\u010dina Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Polona","family":"Skrt","sequence":"additional","affiliation":[{"name":"Sartorius BIA Separations, Mirce 21 Ajdov\u0161\u010dina Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tina","family":"Vodopivec Seravalli","sequence":"additional","affiliation":[{"name":"Sartorius BIA Separations, Mirce 21 Ajdov\u0161\u010dina Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Polona","family":"Megu\u0161ar","sequence":"additional","affiliation":[{"name":"Sartorius BIA Separations, Mirce 21 Ajdov\u0161\u010dina Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2890-3856","authenticated-orcid":false,"given":"Manuela","family":"Bartolini","sequence":"additional","affiliation":[{"name":"Department of Pharmacy and Biotechnology Alma Mater Studiorum University of Bologna, Via Belmeloro 6 Bologna Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rok","family":"Sekirnik","sequence":"additional","affiliation":[{"name":"Sartorius BIA Separations, Mirce 21 Ajdov\u0161\u010dina Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ale\u0161","family":"\u0160trancar","sequence":"additional","affiliation":[{"name":"Sartorius BIA Separations, Mirce 21 Ajdov\u0161\u010dina Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9307-070X","authenticated-orcid":false,"given":"Sebastijan","family":"Peljhan","sequence":"additional","affiliation":[{"name":"Sartorius BIA Separations, Mirce 21 Ajdov\u0161\u010dina Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,10,12]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1016\/j.chroma.2007.05.034","article-title":"Influence of the Methacrylate Monolith Structure on Genomic DNA Mechanical Degradation, Enzymes Activity and Clogging","volume":"1160","author":"Ben\u010dina K.","year":"2007","journal-title":"Journal of Chromatography A"},{"key":"e_1_2_8_3_1","doi-asserted-by":"crossref","first-page":"811","DOI":"10.1002\/jssc.200401800","article-title":"Enzyme Immobilization on Epoxy\u2010 and 1,1\u2032\u2010carbonyldiimidazole\u2010activated Methacrylate\u2010Based Monoliths","volume":"27","author":"Ben\u010dina K.","year":"2004","journal-title":"Journal of Separation Science"},{"key":"e_1_2_8_4_1","first-page":"130","article-title":"Study on EcoRI and HindIII Immobilization Using Sodium 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