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Please use this identifier to cite or link to this item: http://hdl.handle.net/10087/6623

Title: Process Development for Efficient Production of Antibodies with High Antibody-Dependent Cellular Cytotoxicity Activity from a YB2/0 Cell Line
Other Titles: 高い細胞傷害活性を有する抗体医薬品のY B 2 / 0 細胞を用いた効率的な物質生産研究
Authors: 今野, 由信
コンノ, ヨシノブ
Konno, Yoshinobu
Keywords: Antioxidant
CoenzymeQ10
Antibody
YB2/0
CHO
Glycosylation
Fucosylation
Osmolality
8-hydroxy-2'-deoxyguanosine
Antibody-dependent cellular cytotoxicity
ADCC
Intracellular calcium level
fed-batch
perfusion
cell culture
Issue Date: 30-Sep-2011
Publisher: 群馬大学工学部
Abstract: The contribution of biopharmaceutical industries to general healthcare is rapidly increasing with over 165 products having been approved globally since 1982. Within the therapeutic applications of biopharmaceuticals, monoclonal antibodies (MAbs) are of growing interest. Recently, more than twenty therapeutic MAbs and related proteins have been launched in the market. This situation is a double-edged sword because it leads to pressure on pharmaceutical economy. Minimizing the cost of goods (COGS) and maximizing antibody activity are therefore active areas of research in the development of MAbs for therapeutic use. We have screened several enhancers of specific MAb production rate (SPR) using the rat hybridoma YB2/0 cell line and found that coenzyme-Q10 (CoQ10) is a promising enhancer candidate. CoQ10 is well known as a strong antioxidant in the respiratory chain and is used for healthcare and other applications. Because CoQ10 is negligibly water soluble, most studies are limited by low concentrations. We added CoQ10 to a culture media using dispersion of nano-particles (Q-Media) at several concentrations and conducted a fed-batch culture. Although the Q-Media had no effect on cumulative viable cell density, it enhanced the SPR by 66%. In addition, the Q-Media had no effect on the binding or cytotoxic activity of MAbs. Q-Media also enhanced SPR with CHO and NS0 cell lines by 30%. On the other hand, the Q-Media did not alter the concentration of the oxidative stress marker 8-hydroxy-2'-deoxyguanosine in the culture supernatant. Furthermore, Q-Media decreased the ratio of lactate production to glucose consumption only slightly, and CoQ10 (232 μM) elevated intracellular Ca2+ concentration, as did ATP (10 μM). These observations suggest that CoQ10 serves as a powerful aid in the production of MAbs by enhancing SPR without changing the character of cell growth, or adversely affecting quality or biological activity of MAbs. Antibody-dependent cellular cytotoxicity (ADCC) is dependent on the fucose content of oligosaccharides bound to MAbs. As MAbs with a low fucose content exhibit high ADCC activity, it is important to control the defucosylation levels (deFuc%) of MAbs and to analyze the factors that affect deFuc%. In this study, we observed that the deFuc% was inversely related to culture medium osmolality for the MAbs produced in the YB2/0 cell line, with the r2 value as high as 0.92. Moreover, deFuc% exhibited the same correlation irrespective of the type of compound used for regulating osmolality (NaCl, KCl, fucose, fructose, creatine, or mannitol) or culture scale (1–400 L). We succeeded in controlling MAb deFuc% by maintaining a constant medium osmolality constant in both perfusion and fed-batch cultures. The regulation of medium osmolality with glucose is, however, sufficient for designing the deFuc% desired for efficacious ADCC in YB2/0 cell culture. In agreement with these observations, real-time PCR analyses revealed decreased transcription of genes involved in the glycolysis, GDP-fucose supply, and fucose transfer. In this sutudy, both methods to enhance the efficiency of the production are achieved as an extension to existing processes. The present method to control deFuc% with medium osmolality will open the way to use those mammalian cells, for glycoprotein production, that could not be employed because of unwantedly high and/or uncontrollable fucose content in the oligosaccharides attached to the protein. These findings will enable the use of the defucosylated IgG1 at lower doses with no reduction in efficacy without restart such as chainging the cell bank.
Description: 学位記番号:工博甲428
URI: http://hdl.handle.net/10087/6623
Academic Degrees and number: 12301甲第428号
Degree-granting date: 2011-09-30
Degree name: 博士(工学)
Degree-granting institutions: 群馬大学
Appears in Collections:学位論文

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