Rapamycin reduces hybridoma cell death and enhances monoclonal antibody production

Abstract

Rapamycin was used as a medium additive to slow the progression of CRL 1606 hybridomas through the cell cycle, under the hypothesis that such a modulation might reduce cell death. Cell cycle distributions for CRL hybridomas in the G1 phase of the cell cycle ranged from 20% to 35% during batch, fed-batch, and continuous culture experiments, independent of culture time, dilution rate, growth rates, or death rates. Rapamycin, an mTOR signaling inhibitor, immunosuppressant, and G1-phase arresting agent, was identified and tested for efficacy in restraining cell cycle progression in CRL 1606 hybridoma cultures. However, in the presence of 100 nM rapamycin, the percentage of cells in the G1 phase of the cell cycle during fed-batch cultures was only increased from 28% to 31% in control cultures to 37% to 48% for those with rapamycin. Accordingly, rapamycin only slightly reduced culture growth rate. Instead, the use of rapamycin more notably kept viability higher than that of control cultures by delaying cell death for 48 h, thereby enabling viable proliferation to higher maximum viable cell densities. Furthermore, rapamycin enhanced specific monoclonal antibody production by up to 100% during high-viability growth. Thus, over the course of 6-day fed-batch cultivations, the beneficial effects of rapamycin on viable cell density and specific productivity resulted in an increase in final monoclonal antibody titer from 0.25 to 0.56 g/L (124%). As rapamycin is reported to influence a much broader range of cellular functions than cell cycle alone, these findings are more illustrative of the influence that signal transduction pathways related to mTOR can have on overall cell physiology and culture productivity.

pH Condition in temperature shift cultivation enhances cell longevity and specific hMab productivity in CHO culture

“…The control of cell growth by the addition of rapamycin was effective in prolonging cell viability for CRL 1606 hybridoma (Balcarcel and Stephanopoulos 2001). To clarify the relationship between prolonging cell longevity and increasing specific hMab productivity, we selected rapamycin to control the cell cycle progression without shifting the temperature and compared the effect of the rapamycin addition with that of the temperature shift.…”

“…Strategies for controlling cell growth include the expression or addition of cell cycle inhibitors like p27 (Kaufmann et al 2001), p21cip1 (Watanabe et al 2002), and rapamycin (Balcarcel and Stephanopoulos 2001); or exposure to hyperosmotic pressure (Ryu et al 2000) or low culture temperature. Low temperature cultivation is a simple and very effective method of controlling cell proliferation (Reuveny et al 1993).…”

Cell cycle phase dependent productivity of a recombinant Chinese hamster ovary cell line

“…An additional optimization strategy would be prevention of the onset of apoptosis after cessation of growth, at which time cells are accumulating in the G1 cell cycle phase. Identification of regulatory mechanisms for cell cycle progression and of associated environmental manipulation are emerging areas of research (Balcarcel and Stephanopoulos 2001;Bjorklund et al 2006;Carvalhal et al 2003;deZengotita et al 2001;Ibarra et al 2003;Sun et al 2004;Sunstrom et al 2000).…”

Effects of hydroxyurea on monoclonal antibody production induced by anti-mIgG and LPS stimulation on murine B cell hybridomas

“…Although all these strategies may improve the protein production rate, addition of stimulatory agents is considered as a simple and low cost method, which could quickly impact on protein productivity. Chemical drugs as aphidicolin, mimose (Lalande 1990;Sukhorukov et al 1994), rapamycin (Balcarcel and Stephanopoulos 2001), doxorubicin (Sukhorukov et al 1994), staurosporine (Abe et al 1991), hydroxyurea (Fallon and Cox 1979), dimethyl sulfoxide (Fiore et al 2002;Ling et al 2003), and butyrate (Kruh 1982;Ganne et al 1991;Chen et al 1993;Cherlet and Marc 2000;Mimura et al 2001) are commonly used as substances able to generate a cell population that is enriched for a particular phase of the cell cycle, which can eventually increase the protein production.…”