{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,29]],"date-time":"2026-08-29T00:14:46Z","timestamp":1787962486705,"version":"build-2784847793"},"reference-count":21,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,12,16]],"date-time":"2025-12-16T00:00:00Z","timestamp":1765843200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Institute of Bioengineering","award":["06-2024"],"award-info":[{"award-number":["06-2024"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background: Dulaglutide, a GLP-1-IgG4 Fc fusion, is a long-acting GLP-1 receptor agonist used for type 2 diabetes therapy and other emerging indications. It is produced commercially in Chinese hamster ovary (CHO) cells. The supply of the original drug is now limited in some regions, so creation of highly productive biosimilar manufacturing platforms is important. Methods: Two expression plasmids (p1.1-Tr2-Dul, p1.2-GS-Dul) encoding dulaglutide were sequentially transfected into apoptosis-resistant CHO 4BGD cells. Two-step transgene amplifications with methotrexate (MTX), followed by methionine sulfoximine (MSX) selection and subsequent cell cloning pipeline, were employed. Candidate clonal cell lines were selected using fed-batch culturing and long-term productivity testing. Results: Transfection with a second plasmid encoding glutamine synthetase (p1.2-GS-Dul) and selection with MSX resulted in a further ~30% increase titer in polyclonal population even after MTX-driven amplification. Top clone 4BGD\/Dul #73 reached 1.05 g\/L product titer in fed-batch culture (qP up to 22 pg\u00b7cell\u22121\u00b7day\u22121) and remained stable for 69 days in medium without MTX\/MSX. Size exclusion-high-performance liquid chromatography showed \u226595% monomer; EC50 of the purified GLP-1-Fc in a GLP-1R\/CRE-Luc assay was 52 pM for the obtained product versus 76 pM for the original reference drug. Conclusions: The sequential transfection and dual-marker selection approach enables the efficient generation of a robust, high-yield, and glutamine-independent CHO producer, representing a productive strategy suitable for industrial biosimilar development.<\/jats:p>","DOI":"10.3390\/ph18121896","type":"journal-article","created":{"date-parts":[[2025,12,16]],"date-time":"2025-12-16T17:22:58Z","timestamp":1765905778000},"page":"1896","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Production of a Dulaglutide Analogue by Apoptosis-Resistant Chinese Hamster Ovary Cells in a 3-Week Fed-Batch Process"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-5118-5067","authenticated-orcid":false,"given":"Rolan R.","family":"Shaifutdinov","sequence":"first","affiliation":[{"name":"Laboratory of Mammalian Cell Bioengineering, Skryabin Institute of Bioengineering, Federal State Institution, \u201cFederal Research Centre \u2018Fundamentals of Biotechnology\u2019 of the Russian Academy of Sciences\u201d, 60 let Oktjabrja pr-t 7, bld. 1, 117312 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8950-6061","authenticated-orcid":false,"given":"Maria V.","family":"Sinegubova","sequence":"additional","affiliation":[{"name":"Laboratory of Mammalian Cell Bioengineering, Skryabin Institute of Bioengineering, Federal State Institution, \u201cFederal Research Centre \u2018Fundamentals of Biotechnology\u2019 of the Russian Academy of Sciences\u201d, 60 let Oktjabrja pr-t 7, bld. 1, 117312 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0064-4458","authenticated-orcid":false,"given":"Ivan I.","family":"Vorobiev","sequence":"additional","affiliation":[{"name":"Laboratory of Mammalian Cell Bioengineering, Skryabin Institute of Bioengineering, Federal State Institution, \u201cFederal Research Centre \u2018Fundamentals of Biotechnology\u2019 of the Russian Academy of Sciences\u201d, 60 let Oktjabrja pr-t 7, bld. 1, 117312 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Polina E.","family":"Prokhorova","sequence":"additional","affiliation":[{"name":"Medsintez Plant LLC, Kirova 28, 620028 Ekaterinburg, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexey B.","family":"Podkorytov","sequence":"additional","affiliation":[{"name":"Medsintez Plant LLC, Kirova 28, 620028 Ekaterinburg, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9483-2892","authenticated-orcid":false,"given":"Nadezhda A.","family":"Orlova","sequence":"additional","affiliation":[{"name":"Laboratory of Mammalian Cell Bioengineering, Skryabin Institute of Bioengineering, Federal State Institution, \u201cFederal Research Centre \u2018Fundamentals of Biotechnology\u2019 of the Russian Academy of Sciences\u201d, 60 let Oktjabrja pr-t 7, bld. 1, 117312 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,16]]},"reference":[{"key":"ref_1","first-page":"3","article-title":"Overview of diabetes agents in cardiovascular disease: It takes an orchestra to play Tchaikovsky in symphony","volume":"32","author":"Loh","year":"2025","journal-title":"Curr. 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