title: Glycosylation analysis of an aggregated antibody produced by Chinese hamster ovary cells in bioreactor culture.
abstract: 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.
authors: Institute of Technology and Science, The University of Tokushima, Minamijosanjima-cho 2-1, Tokushima 770-8506, Japan. Electronic address: onitsuka@bio.tokushima-u.ac.jp.
authors: Masayoshi
authors: M
authors: Onitsuka
authors: Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka 2-1, Suita, Osaka 565-0871, Japan.
authors: Akira
authors: A
authors: Kawaguchi
authors: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 6-6-11-606 Aoba-yama, Aramaki, Aoba-ku, Sendai 980-8579, Japan.
authors: Ryutaro
authors: R
authors: Asano
authors: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 6-6-11-606 Aoba-yama, Aramaki, Aoba-ku, Sendai 980-8579, Japan.
authors: Izumi
authors: I
authors: Kumagai
authors: Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka 2-1, Suita, Osaka 565-0871, Japan.
authors: Kohsuke
authors: K
authors: Honda
authors: Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka 2-1, Suita, Osaka 565-0871, Japan.
authors: Hisao
authors: H
authors: Ohtake
authors: Institute of Technology and Science, The University of Tokushima, Minamijosanjima-cho 2-1, Tokushima 770-8506, Japan; Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka 2-1, Suita, Osaka 565-0871, Japan.
authors: Takeshi
authors: T
authors: Omasa
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D019149:Bioreactors; D016466:CHO Cells; D018929:Cell Culture Techniques; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D005643:Fucose / Q000032:analysis / Q000378:metabolism; D005690:Galactose / Q000032:analysis / Q000378:metabolism; D006031:Glycosylation; D037102:Lectins / Q000737:chemistry / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D020836:Protein Structure, Quaternary
chemical_list: D018033:Antibodies, Bispecific; D037102:Lectins; D009844:Oligosaccharides; D005643:Fucose; D005690:Galactose
keywords: Antibody aggregation; Antibody production; Bispecific diabody; Cell culture; Chinese hamster ovary cell; Glycosylation heterogeneity