RESUMEN
Culturing recombinant CHO (Chinese-hamster ovary) cells at low temperatures (30-33 degrees C) increases specific recombinant protein productivity by 2-5-fold. However, even though the specific productivity is increased, cell growth is decreased in low-temperature culture such that the final recombinant protein titre remains unchanged or is even diminished, owing to the lower cell density. Exposing mammalian cells to low temperatures results in a change in the expression of many 'cold-stress' genes. CIRP (cold-inducible RNA-binding protein) is a cold-stress protein that is highly expressed at 32 degrees C, but not at 37 degrees C. In the present study we demonstrated that overexpression of CIRP at 37 degrees C can increase the recombinant-protein titre in CHO cells. Stable overexpression of CIRP at 37 degrees C improved the final titre of CHO IFN-gamma, a recombinant CHO cell line producing human IFN-gamma (interferon-gamma), by 25% in adherent culture and up to 40% in suspension culture. Real-time PCR analysis showed that the increase in the recombinant IFN-gamma titre could be attributed to increased recombinant IFN-gamma mRNA levels, while growth data showed that CIRP overexpression did not result in growth arrest in CHO IFN-gamma cells. Glycan analysis showed that the increase in IFN-gamma titre as a result of CIRP overexpression did not affect the site occupancy, glycan structures or sialic acid content of IFN-gamma. Using this strategy, the final IFN-gamma titre was increased by 40% compared with current temperature-based strategies. Furthermore, there is no decrease in cell growth or recombinant-protein glycosylation quality, as previously observed in low-temperature culture.
Asunto(s)
Mejoramiento Genético/métodos , Interferón gamma/metabolismo , Ingeniería de Proteínas/métodos , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/biosíntesis , Animales , Células CHO , Frío , Cricetinae , Cricetulus , Humanos , Interferón gamma/genética , Regulación hacia ArribaRESUMEN
Culturing CHO (Chinese-hamster ovary) cells at low temperature leads to growth arrest in the G0/G1 phase of the cell cycle and, in many cases, causes an increase in the specific productivity of recombinant protein. Controlled proliferation is often used to increase CHO specific productivity, and thus there is speculation that the enhanced productivity at low temperature is due to G0/G1-phase growth arrest. However, we show that the positive effect of low temperature on recombinant protein production is due to elevated mRNA levels and not due to growth arrest and that a cell line can still exhibit growth-associated productivity at low temperatures. Using a CHO cell producing recombinant human IFN-gamma (interferon-gamma), we show that productivity increases as the percentage of cells in the S phase of the cell cycle increases, at both 32 and 37 degrees C. The increased productivity is due to higher recombinant IFN-gamma mRNA levels. We also show that, for a given cell-cycle distribution, specific productivity increases as the temperature is lowered from 37 to 32 degrees C. Thus specific productivity is maximized when cells are actively growing (high percentage of S-phase cells) and also exposed to low temperature. These findings have important implications for cell-culture optimization and cell-line engineering, providing evidence that a CHO cell line capable of actively growing at low temperature would provide improved total protein production relative to the current growth strategies, namely 37 degrees C active growth or low-temperature growth arrest.