Production of a Dulaglutide Analogue by Apoptosis-Resistant Chinese Hamster Ovary Cells in a 3-Week Fed-Batch Process

Shaifutdinov, R.R.; Sinegubova, M.V.; Vorobiev, I.I.; Prokhorova, P.E.; Podkorytov, A.B.; Orlova, N.A.

Авторы: Shaifutdinov Rolan R., Sinegubova Maria V., Vorobiev Ivan I., Prokhorova Polina E., Podkorytov Alexey B., Orlova Nadezhda A.
Журнал: Pharmaceuticals
Том: 18
Номер: 12
Год издания: 2025
Издательство: MDPI
Местоположение издательства: Basel, Switzerland
Первая страница: 1896
Последняя страница: 1896
DOI: 10.3390/ph18121896
Аннотация: first_pagesettingsOrder Article ReprintsOpen AccessArticleProduction of a Dulaglutide Analogue by Apoptosis-Resistant Chinese Hamster Ovary Cells in a 3-Week Fed-Batch Processby Rolan R. Shaifutdinov 1ORCID,Maria V. Sinegubova 1ORCID,Ivan I. Vorobiev 1ORCID,Polina E. Prokhorova 2,Alexey B. Podkorytov 2 andNadezhda A. Orlova 1,*ORCID1Laboratory of Mammalian Cell Bioengineering, Skryabin Institute of Bioengineering, Federal State Institution, “Federal Research Centre ‘Fundamentals of Biotechnology’ of the Russian Academy of Sciences”, 60 let Oktjabrja pr-t 7, bld. 1, 117312 Moscow, Russia2Medsintez Plant LLC, Kirova 28, 620028 Ekaterinburg, Russia*Author to whom correspondence should be addressed.Pharmaceuticals 2025, 18(12), 1896; https://doi.org/10.3390/ph18121896Submission received: 18 November 2025 / Revised: 8 December 2025 / Accepted: 12 December 2025 / Published: 16 December 2025(This article belongs to the Section Pharmaceutical Technology)Downloadkeyboard_arrow_down Browse Figures Review Reports Versions NotesAbstractBackground: 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·cell−1·day−1) and remained stable for 69 days in medium without MTX/MSX. Size exclusion-high-performance liquid chromatography showed ≥95% 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.
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Production of a Dulaglutide Analogue by Apoptosis-Resistant Chinese Hamster Ovary Cells in a 3-Week Fed-Batch Processстатья

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