Abstract
Although antibodies have attracted attention as next-generation biopharmaceuticals, the costs of purifying the products and of arranging the environment for cell cultivation are high. Therefore, there is a need to increase antibody efficacy and improve product quality as much as possible. Since antibodies are glycoproteins, their glycan structures have been found to affect the function of antibodies. Especially, afucosylation of the N-linked glycan in the Fc region is known to significantly increase antibody-dependent cellular cytotoxicity. In this study, we established a double-mutant ΔGMDΔGFT in which GDP-mannose 4,6-dehydratase and GDP-fucose transporter were knocked out in Chinese hamster ovary cells, a platform for biopharmaceutical protein production. By adapting ΔGMDΔGFT cells to serum-free medium and constructing suspension-cultured cells, we established host CHO cells with no detected fucosylated glycans and succeeded in production of afucosylated antibodies. We also demonstrated that, in culture in the presence of serum, fucosylation occurs due to contamination from serum components. Furthermore, we found that afucosylation of glycans does not affect cell growth after adaptation to serum-free medium as compared to wild-type CHO cells growth and does not significantly affect the expression levels of other endogenous fucose metabolism-related enzyme genes.
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Abbreviations
- GMD:
-
GDP-mannose 4,6-dehydratase
- GFT:
-
GDP-fucose transporter
- Man:
-
Mannose
- Fuc:
-
Fucose
- GlcNAc:
-
N-Acetylglucosamine
- Fmoc:
-
9-Fluorenylmethyloxycarbonyl
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Acknowledgements
This work was supported by the research project of Manufacturing Technology Association of Biologics and Japan Agency for Medical Research and Development (AMED; 17ae0101003h0005 and 19ae0101066h0002).
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Misaki, R., Iwasaki, M., Takechi, H. et al. Establishment of serum-free adapted Chinese hamster ovary cells with double knockout of GDP-mannose-4,6-dehydratase and GDP-fucose transporter. Cytotechnology 74, 163–179 (2022). https://doi.org/10.1007/s10616-021-00501-3
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DOI: https://doi.org/10.1007/s10616-021-00501-3