Glycosylation analysis of an aggregated antibody produced by Chinese hamster ovary cells in bioreactor culture

doi:10.1016/j.jbiosc.2013.11.001

Journal of Bioscience and Bioengineering

Volume 117, Issue 5, May 2014, Pages 639-644

https://doi.org/10.1016/j.jbiosc.2013.11.001 Get rights and content

N-Glycosylation of therapeutic antibodies contributes not only to their biological function, but also to their stability and tendency to aggregate. Here, we investigated the impact of the glycosylation status of an aggregated antibody that accumulated during the bioreactor culture of Chinese hamster ovary cells. High-performance liquid chromatography analysis showed that there was no apparent difference in the glycosylation patterns of monomeric, dimeric, and large aggregated forms of the antibody. In contrast, lectin binding assays, which enable the total amounts of specific sugar residues to be detected, showed that both galactose and fucose residues in dimers and large aggregates were reduced to 70–80% of the amount in monomers. These results strongly suggest that the lack of N-linked oligosaccharides, a result of deglycosylation or aglycosylation, occurred in a proportion of the dimeric and large aggregated components. The present study demonstrates that glycosylation heterogeneities are a potential cause of antibody aggregation in cell culture of Chinese hamster ovary cells, and that the lack of N-glycosylation promotes the formation of dimers and finally results in large aggregates.

Section snippets

Cell culture, antibody production, and purification

The CHO Top-H cell line producing the Ex3-scDb-Fc bispecific diabody 23, 24 was cultivated in suspension culture using serum-free ExCD medium [a mixture of ExCell 302 (SAFC Bioscience, St. Louis, MO, USA) and IS CHO-CD (Irvine Scientific, Santa Ana, CA, USA) supplemented with 1000 nM MTX and 1 mM G418]. The cells were cultivated in a 1-L glass bioreactor (Biott, Tokyo, Japan) containing 750 mL of the medium for approximately 2 weeks. The temperature was maintained at 37°C during cultivation.

Characterization of the aggregated bispecific diabody that accumulated during the CHO cell culture process

The CHO Top-H cell line producing Ex3-scDb-Fc was cultivated in a 1-L glass bioreactor. The cells were inoculated at 3 × 10⁵ cells/mL from the preculture in the mid-exponential growth phase. The maximum cell density reached 50 × 10⁵ cells/mL. The culture medium was harvested after 15 days when the cell viability reached 60%. The final concentration of Ex3-scDb-Fc was approximately 40 mg/L (Supplementary Fig. S1). Ex3-scDb-Fc was purified by protein A affinity chromatography. In the affinity

Acknowledgments

This study was supported by the Advanced Research for Medical Products Mining Programme of the National Institute of Biomedical Innovation (NIBIO).

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Glycosylation analysis of an aggregated antibody produced by Chinese hamster ovary cells in bioreactor culture

Section snippets

Cell culture, antibody production, and purification

Characterization of the aggregated bispecific diabody that accumulated during the CHO cell culture process

Acknowledgments

Immunol. Lett.

Mol. Immunol.

J. Biol. Chem.

J. Biosci. Bioeng.

J. Biosci. Bioeng.

J. Biosci. Bioeng.

J. Biosci. Bioeng.

Curr. Opin. Biotechnol.

Trends Pharmacol. Sci.

J. Biosci. Bioeng.

Protein Expr. Purif.

J. Mol. Biol.

Biochim. Biophys. Acta

Methods

J. Biosci. Bioeng.

J. Biosci. Bioeng.

J. Biosci. Bioeng.

Chem. Biol.

The impact of glycosylation on the biological function and structure of human immunoglobulins

Annu. Rev. Immunol.