{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T07:52:32Z","timestamp":1791273152884,"version":"4.1.0"},"reference-count":51,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"},{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"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;amp; Bioengineering"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    The glutamine synthetase expression system is widely used for therapeutic protein biomanufacturing in Chinese hamster ovary (CHO) cells. Here, we present the design of an enhanced glutamine synthetase vector for generating CHOK1SV GS\u2010KO stable pools that produce recombinant proteins at multigram\u2010per\u2010liter levels. We first developed a synthetic promoter, LHP\u20101, for high\u2010level constitutive recombinant gene transcription, achieving up to 123% increase in titer across monoclonal and bispecific antibody formats compared to the viral CMV promoter. We then demonstrate, for the first time, that the bacterial origin of replication (Ori) can function as a core promoter to drive glutamine synthetase selection in CHO cells. In this novel glutamine synthetase vector, Ori and LHP\u20101 each play dual roles: Ori acts as both the plasmid replication origin\n                    <jats:italic>and<\/jats:italic>\n                    a minimal glutamine synthetase promoter, while LHP\u20101 drives product expression\n                    <jats:italic>and<\/jats:italic>\n                    optimal enhancement of glutamine synthetase activity without the use of methionine sulfoximine (MSX) supplementation. Together, these two elements further increased product titers by up to 73% compared to the vector harboring the SV40 promoter to drive glutamine synthetase gene expression with 50\u2009\u00b5M MSX. Notably, the LHP\u20101 Ori\u2010GS system effectively maintained high bulk pool titers of up to 5.7\u2009g\/L with no decline after 30 days\u2014supporting consistent product yield and quality during culture scale\u2010up for toxicology material supply. Further cell line development showed that clones derived from the LHP\u20101 Ori\u2010GS pool achieved a 23% higher median titer while maintaining long\u2010term expression stability compared to those generated with the SV40\u2010GS vector, with the top\u2010performing Ori\u2010GS clone reaching 8.3\u2009g\/L. Overall, the MSX\u2010free LHP\u20101 Ori\u2010GS system offers a robust platform for both rapid, high\u2010yield material generation from bulk pools and efficient progression to high\u2010producing clonal cell lines.\n                  <\/jats:p>","DOI":"10.1002\/bit.70405","type":"journal-article","created":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T07:22:07Z","timestamp":1791271327000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Next\u2010Generation Glutamine Synthetase Expression Vector for Therapeutic Protein Manufacturing Using CHO Cells"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9933-5764","authenticated-orcid":false,"given":"Yusuf B.","family":"Johari","sequence":"first","affiliation":[{"name":"Lonza Biologics Cambridge UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sophie 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