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Comparison of knockdown approaches for the generation of stable cell populations expressing afucosylated antibodies
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DOI：10.1080/19420862.2026.2684393.png)
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Abstract
En 中文
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.
Keywords:
Knockdown
antibody expression
afucosylation
CHO cells
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