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.学位記番号：工博甲42
