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The cell line D29, which was easily and rapidly established by the promoter-activated production and glutamine synthetase hybrid system, secreted recombinant human interleukin-6 (hIL-6) at a productivity rate of 39.5 mug 10(-6) cells day(-1), one of the highest reported levels worldwide. The productivity rate was about 130-fold higher than that of the cell line A7, which was established without both promoter activation and gene amplification. Although D29 cells had a high copy number and high mRNA level of the hIL-6 gene as well as a high secretion rate of hIL-6, large amounts of intracellular hIL-6 protein accumulated in D29 cells compared to A7 cells. Northern blotting analysis showed no change in the GRP78/BiP expression level in D29 cells. In contrast, an electrophoresis mobility shift assay revealed strong activation of NF-kappaB in D29 cells. These results suggest that large amounts of hIL-6 translated from large amounts of hIL-6 mRNA cause excess accumulation of intact hIL-6 in the endoplasmic reticulum (ER), and that subsequent negative feedback signals via the ER overload response inhibit hIL-6 protein secretion. To enhance the hIL-6 productivity rate of D29 cells by releasing the negative feedback signals, the effect of pyrrolidinedithiocarbamate, an inhibitor of NF-kappaB activation, was examined. Suppression of NF-kappaB activation in D29 cells produced a 25% augmentation of the hIL-6 productivity rate. Therefore, in highly productive cells like D29 cells, the release of negative feedback signals could increase the total amount of recombinant protein secretion.

We previously developed a promoter-activated production (PAP) system using amplified ras oncogene to activate the cytomegalovirus (CMV) promoter controlling the foreign gene in mammalian cells. CHO cells were demonstrated to be suitable for the PAP system. Here, we show that very high-level production of a recombinant protein was achieved when the human CMV promoter was inserted into a glutamine synthetase (GS) minigene expression plasmid, pEE14. A highly productive host CHO cell line, ras clone I containing amplified ras oncogene, was further transfected with the plasmid expressing both hIL-6 gene and GS minigene, and selected with methionine sulphoximine. We were able to establish a hIL-6 hyper-producing cell line, D29, which exhibited a peak productivity rate of approximately 40 mug hIL-6 10(-6) cells day(-1) through a combination of the PAP system and the GS gene amplification system. The cellular productivity of D29 cells was about 13-fold higher than control hIL-6-producing cells derived from CHO cells whose hIL-6 gene was amplified by the GS gene amplification system, and about 5-fold higher than the I13 cells established by the PAP system, which contains amplified ras oncogene and non-amplified hIL-6 gene. When D29 cells were cultured for a month, an accumulation rate of approximately 80 mug hIL-6 ml(-1) per 3 days was achieved on the 9th day. These results indicate that this PAP and GS hybrid system enables the efficient and rapid establishment of recombinant protein hyper-producing cell lines.

We have developed an oncogene activated production (OAP) system using amplified ras oncogene to activate the CMV promoter controlling the foreign gene in mammalian cells (Yano, T. et al., 1994). CHO cells were demonstrated to be suited for the OAP system (Perry, S. et al., 1996). Here we show that very high level production of recombinant protein can be achieved by combining the OAP system with the glutamine synthetase (GS) gene amplification system. The human interleukin 6 (hIL-6) gene regulated by the human CMV promoter was inserted into a GS-mini-gene expression plasmid pEE14. A highly productive host CHO cell line, ras-clone I containing amplified ras oncogene was further transfected with the plasmid expressing both hIL-6 and GS mini-gene, and selected with methionine sulphoximine (MSX) in G-DME medium supplemented with 10% dialyzed fetal bovine serum. We could establish a hEL-6 hyper-producing cell line DRGI-29 derived from ras-clone I, which exhibited a peak productivity value of about 40 ¼:g hIL-6/106 cells/day through a combination of the OAP system and GS gene amplification system. The cellular productivity of DRGI-29 cells was about 13 times higher than the control hIL-6-producing cells derived from normal CHO cells whose hIL-6 gene was amplified by GS gene amplification system, and about 5 times higher than I13 cells established by the OAP system, which contains amplified ras oncogene and non-amplified hIL-6 gene (Perry, S. et al., 1996). When DRGI-29 cells were cultivated for 30 days, the accumulation rate of about 80 μg hIL-6/ml/3days on 9th day. Whereas, the maximum productivity was achieved after long-term culture in the case of I13 cells (Perry, S. et al., 1996). These results indicate that this OAP and GS hybrid system enables the efficient and rapid establishment of recombinant protein hyper-producing cell lines.