{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T15:27:01Z","timestamp":1787239621563,"version":"build-2736575974"},"reference-count":25,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T00:00:00Z","timestamp":1787184000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Healthcare of the Republic of Kazakhstan","award":["BR25293293"],"award-info":[{"award-number":["BR25293293"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Bioengineering"],"abstract":"<jats:p>The growing demand for high-quality plasmid DNA (pDNA) in cell and gene therapy, including chimeric antigen receptor T-cell therapy (CAR-T) manufacturing, is constrained by the limited availability of transfection-grade plasmids. This study describes a practical, scalable, and cost-effective acidic phenol extraction method based on a modified alkaline lysis protocol. This approach efficiently removes endotoxins and residual genomic DNA without requiring chromatography or ultracentrifugation. When benchmarked against CsCl density gradient ultracentrifugation and a commercial midiprep kit, the acidic phenol method delivered comparable plasmid purity and functional performance while providing superior scalability, yielding 5\u201310 mg of pDNA per 500 mL culture. Notably, residual endotoxin levels were negligible for downstream applications such as transfection and lentiviral vector packaging. Lentiviral vectors produced with these plasmids reached titers exceeding 5 \u00d7 106 TU\/mL, with transduction efficiencies statistically indistinguishable from those obtained with CsCl-purified DNA. The acidic phenol extraction method is a robust, scalable, and cost-effective alternative to industry-standard methods, matching them in yield and purity while being readily scalable, making it a practical tool for both academic research and preparative laboratory production.<\/jats:p>","DOI":"10.3390\/bioengineering13080939","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T09:48:31Z","timestamp":1787219311000},"page":"939","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Scalable Production of Transfection-Grade Plasmid DNA by Liquid\u2013Liquid Extraction Without Chromatography: Application to CAR-T Vector Packaging"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-7868-3396","authenticated-orcid":false,"given":"Gaziza","family":"Nigmatulla","sequence":"first","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin hwy 3\/5, 010000 Astana, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-2199-5756","authenticated-orcid":false,"given":"Aitolkyn","family":"Kydyrbayeva","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin hwy 3\/5, 010000 Astana, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tolganay","family":"Kulatay","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin hwy 3\/5, 010000 Astana, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1514-9302","authenticated-orcid":false,"given":"Gulzat","family":"Zauatbayeva","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin hwy 3\/5, 010000 Astana, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bakytkali","family":"Ingirbay","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin hwy 3\/5, 010000 Astana, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lyazzat","family":"Sagyndykova","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin hwy 3\/5, 010000 Astana, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8885-2387","authenticated-orcid":false,"given":"Viktoriya","family":"Keyer","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin hwy 3\/5, 010000 Astana, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dinara","family":"Zharlyganova","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin hwy 3\/5, 010000 Astana, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maral","family":"Zhumabekova","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin hwy 3\/5, 010000 Astana, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9880-9382","authenticated-orcid":false,"given":"Alexandr V.","family":"Shustov","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Korgalzhin hwy 3\/5, 010000 Astana, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"865","DOI":"10.1016\/S0141-0229(03)00205-9","article-title":"Industrial scale production of plasmid DNA for vaccine and gene therapy: Plasmid design, production, and purification","volume":"33","author":"Prather","year":"2003","journal-title":"Enzym. 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