Academic Journal
Comparison of knockdown approaches for the generation of stable cell populations expressing afucosylated antibodies.
| Τίτλος: | Comparison of knockdown approaches for the generation of stable cell populations expressing afucosylated antibodies. |
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| Συγγραφείς: | Tsachaki M; Drug Substance Development, IGI Therapeutics SA, La Chaux-de-Fonds, Switzerland., Samy A; Drug Substance Development, IGI Therapeutics SA, La Chaux-de-Fonds, Switzerland., Peltret M; Drug Substance Development, IGI Therapeutics SA, La Chaux-de-Fonds, Switzerland., Brunstein Laplace T; Drug Substance Development, IGI Therapeutics SA, La Chaux-de-Fonds, Switzerland., Vaxelaire-Hily E; Drug Substance Development, IGI Therapeutics SA, La Chaux-de-Fonds, Switzerland., Mosbaoui F; Drug Substance Development, IGI Therapeutics SA, La Chaux-de-Fonds, Switzerland., Galea A; Drug Substance Development, IGI Therapeutics SA, La Chaux-de-Fonds, Switzerland., Contarin F; Drug Substance Development, IGI Therapeutics SA, La Chaux-de-Fonds, Switzerland., Duarte L; Drug Substance Development, IGI Therapeutics SA, La Chaux-de-Fonds, Switzerland., Giovannini R; Process Development, IGI Therapeutics SA, La Chaux-de-Fonds, Switzerland., Bertschinger M; Drug Substance Development, IGI Therapeutics SA, La Chaux-de-Fonds, Switzerland. |
| Πηγή: | MAbs [MAbs] 2026 Dec; Vol. 18 (1), pp. 2684393. Date of Electronic Publication: 2026 Jun 12. |
| Τύπος έκδοσης: | Journal Article; Comparative Study |
| Γλώσσα: | English |
| Στοιχεία περιοδικού: | Publisher: Taylor & Francis Country of Publication: United States NLM ID: 101479829 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1942-0870 (Electronic) Linking ISSN: 19420862 NLM ISO Abbreviation: MAbs Subsets: MEDLINE |
| Imprint Name(s): | Publication: 2015- : Philadelphia, PA : Taylor & Francis Original Publication: Austin, TX : Landes Bioscience |
| Ιατρικοί όροι (MeSH): | Fucosyltransferases*/genetics , Fucosyltransferases*/metabolism , Gene Knockdown Techniques*/methods , Antibodies, Monoclonal*/genetics , Antibodies, Monoclonal*/biosynthesis , Antibodies, Monoclonal*/metabolism , Fucose*/metabolism, RNA, Small Interfering/genetics ; MicroRNAs/genetics ; Recombinant Proteins/genetics ; Recombinant Proteins/biosynthesis ; Animals ; CHO Cells ; Cricetulus ; Cricetinae ; Glycosylation ; Humans |
| Περίληψη: | Targeted silencing of cellular genes in cell lines stably expressing a secreted recombinant protein offers a flexible and convenient strategy for modulating product quality attributes. However, unlike genetic knockout of a cellular gene or the use of potent inhibitors to eliminate cellular enzyme activity, gene knockdown approaches using microRNAs (miRNA) or short hairpin RNAs (shRNA) may result in only partial reduction of enzyme activity and only partial change of the desired product quality attribute. Moreover, through saturation of the Dicer pathway, shRNA may introduce cytotoxic effects that affect product yield. In this study, we demonstrate that miRNAs are a more suitable tool than shRNAs for α1,6-fucosyltransferase (FUT8) knockdown in Chinese hamster ovary (CHO) cells, when expressed under the control of an RNA-Polymerase II promoter. In the presence of miRNAs targeted to the FUT8 mRNA, the fucosylation levels of the N-glycan linked to Asn297 (EU numbering) of a recombinant monoclonal antibody (mAb) were considerably reduced (>90% afucosylation by hydrophilic interaction liquid chromatography - high‑performance liquid chromatography; no detectable fucosylated product by intact mass spectrometry), without a relevant impact on mAb expression. By comparison, overexpression of shRNAs targeted to the FUT8 mRNA resulted in higher levels of residual fucosylation of the mAb and a larger reduction in mAb titers. miRNAs overcame a limitation of the knockdown approach with shRNAs, making this approach a valuable tool for biopharmaceutical manufacturing. |
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| Contributed Indexing: | Keywords: CHO cells; Knockdown; afucosylation; antibody expression Local Abstract: [plain-language-summary] miRNA driven by a Pol2 promoter is a more suitable tool than shRNA for knock down of enzymes in mAb producing Chinese hamster cellsmiRNA expression cassettes allow specific knockdown without impacting the protein production machinerymiRNA based knock down approach is favorable for drug production, as it does not rely on inhibitor molecules lacking safety evaluation or pre-selection of host cells. |
| Substance Nomenclature: | EC 2.4.1.- (Fucosyltransferases) EC 2.4.1.68 (Glycoprotein 6-alpha-L-fucosyltransferase) 0 (Antibodies, Monoclonal) 28RYY2IV3F (Fucose) 0 (RNA, Small Interfering) 0 (MicroRNAs) 0 (Recombinant Proteins) |
| Entry Date(s): | Date Created: 20260612 Date Completed: 20260614 Latest Revision: 20260813 |
| Update Code: | 20260813 |
| PubMed Central ID: | PMC13274123 |
| DOI: | 10.1080/19420862.2026.2684393 |
| PMID: | 42285912 |
| Βάση Δεδομένων: | MEDLINE |
| ISSN: | 1942-0870 |
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| DOI: | 10.1080/19420862.2026.2684393 |