A detailed understanding of the enhanced hypothermic productivity of interferon-gamma by Chinese-hamster ovary cells.

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.

Active hypothermic growth: a novel means for increasing total interferon-gamma production by Chinese-hamster ovary cells.

When grown under hypothermic conditions, CHO (Chinese-hamster ovary) cells become growth-arrested in the G0/G1 phase of the cell cycle and also often exhibit increased recombinant-protein production. We have shown in the accompanying paper [Fox, Tan, Tan, Wong, Yap and Wang (2005) Biotechnol. Appl. Biochem. 41, 255-264] that the positive effect of low temperature on recombinant-protein production is due to elevated mRNA levels and not due to G0/G1-phase growth arrest and that a cell line can still show growth-associated productivity at low temperature. This finding led to the hypothesis that improved total production of recombinant protein would be achieved by stimulating cells to actively grow at low temperature, a culture condition previously unreported in the literature. In the present study we have validated this hypothesis by stimulating hypothermic (32 degrees C) growth through the use of different growth factors. Hypothermic growth was stimulated in fetal-bovine-serum-supplemented adherent cultures using basic fibroblast growth factor or insulin. Hypothermic growth was also stimulated in suspension cultures normally grown in protein-free medium by using supplementation with fetal bovine serum. These methods resulted in up to 7.7- and 4.9-fold increases in total recombinant-protein production compared with the 37 and 32 degrees C control cultures respectively. This proof-of-concept study will motivate the creation of cell lines capable of growing at low temperatures for use in industrial processes.

Maximizing interferon-gamma production by Chinese hamster ovary cells through temperature shift optimization: experimental and modeling.

The Chinese hamster ovary (CHO) cell line producing interferon-gamma (IFN-gamma) exhibits a 2-fold increase in specific productivity when grown at 32 degrees C compared to 37 degrees C. Low temperature also causes growth arrest, meaning that the cell density is significantly lower at 32 degrees C, nutrients are consumed at a slower rate and the batch culture can be run for a longer period of time prior to the onset of cell death. At the end of the batch, product concentration is doubled at the low temperature. However, the batch time is nearly doubled as well, and this causes volumetric productivity to only marginally improve by using low temperature. One approach to alleviate the problem of slow growth at low temperature is to utilize a biphasic process, wherein cells are cultured at 37 degrees C for a period of time in order to obtain reasonably high cell density and then the temperature is shifted to 32 degrees C to achieve high specific productivity. Using this approach, it is hypothesized that IFN-gamma volumetric productivity would be maximized. We developed and validated a model for predicting the optimal point in time at which to shift the culture temperature from 37 degrees C to 32 degrees C. It was found that by shifting the temperature after 3 days of growth, the IFN-gamma volumetric productivity is increased by 40% compared to growth and production at 32 degrees C and by 90% compared to 37 degrees C, without any decrease in total production relative to culturing at 32 degrees C alone. The modeling framework presented here is applicable for optimizing controlled proliferation processes in general.