高い細胞傷害活性を有する抗体医薬品のYB2/0細胞を用いた効率的な物質生産研究 Process development for efficient production of antibodies with high antibody-dependent cellular cytotoxicity activity from a YB2/0 cell line

博士論文

Thesis or Dissertation

The contribution of biopharmaceutical industries to general healthcare is rapidly increasing\nwith over 165 products having been approved globally since 1982. Within the therapeutic\napplications of biopharmaceuticals, monoclonal antibodies (MAbs) are of growing interest.\nRecently, more than twenty therapeutic MAbs and related proteins have been launched in the\nmarket. This situation is a double-edged sword because it leads to pressure on pharmaceutical\neconomy. Minimizing the cost of goods (COGS) and maximizing antibody activity are therefore\nactive areas of research in the development of MAbs for therapeutic use.\nWe have screened several enhancers of specific MAb production rate (SPR) using the rat\nhybridoma YB2/0 cell line and found that coenzyme-Q10 (CoQ10) is a promising enhancer\ncandidate. CoQ10 is well known as a strong antioxidant in the respiratory chain and is used for\nhealthcare and other applications. Because CoQ10 is negligibly water soluble, most studies are\nlimited by low concentrations. We added CoQ10 to a culture media using dispersion of\nnano-particles (Q-Media) at several concentrations and conducted a fed-batch culture. Although\nthe Q-Media had no effect on cumulative viable cell density, it enhanced the SPR by 66%. In\naddition, the Q-Media had no effect on the binding or cytotoxic activity of MAbs. Q-Media also\nenhanced SPR with CHO and NS0 cell lines by 30%. On the other hand, the Q-Media did not\nalter the concentration of the oxidative stress marker 8-hydroxy-2'-deoxyguanosine in the culture\nsupernatant. Furthermore, Q-Media decreased the ratio of lactate production to glucose\nconsumption only slightly, and CoQ10 (232 μM) elevated intracellular Ca2+ concentration, as did\nATP (10 μM). These observations suggest that CoQ10 serves as a powerful aid in the production\nof MAbs by enhancing SPR without changing the character of cell growth, or adversely affecting\nquality or biological activity of MAbs.\nAntibody-dependent cellular cytotoxicity (ADCC) is dependent on the fucose content of\noligosaccharides bound to MAbs. As MAbs with a low fucose content exhibit high ADCC\nactivity, it is important to control the defucosylation levels (deFuc%) of MAbs and to analyze the\nfactors that affect deFuc%. In this study, we observed that the deFuc% was inversely related to\nculture medium osmolality for the MAbs produced in the YB2/0 cell line, with the r2 value as\nhigh as 0.92. Moreover, deFuc% exhibited the same correlation irrespective of the type of\ncompound used for regulating osmolality (NaCl, KCl, fucose, fructose, creatine, or mannitol) or\nculture scale (1–400 L). We succeeded in controlling MAb deFuc% by maintaining a constant\nmedium osmolality constant in both perfusion and fed-batch cultures. The regulation of medium\nosmolality with glucose is, however, sufficient for designing the deFuc% desired for efficacious\nADCC in YB2/0 cell culture. In agreement with these observations, real-time PCR analyses\nrevealed decreased transcription of genes involved in the glycolysis, GDP-fucose supply, and\nfucose transfer.\nIn this sutudy, both methods to enhance the efficiency of the production are achieved as an\nextension to existing processes. The present method to control deFuc% with medium osmolality\nwill open the way to use those mammalian cells, for glycoprotein production, that could not be\nemployed because of unwantedly high and/or uncontrollable fucose content in the\noligosaccharides attached to the protein. These findings will enable the use of the defucosylated\nIgG1 at lower doses with no reduction in efficacy without restart such as chainging the cell bank.

学位記番号：工博甲428