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Abstract

Directed control of cell metabolism by a modification of the physicochemical conditions (presence of Na-butyrate and modification of the temperature) was used to modulate the productivity of human recombinant tissular plasminogen activator (t-PA) expressed under control of SV40 promoter in Chinese Hamster Ovary (CHO) cell lines. We showed that both by adding Na-butyrate or lowering temperature from 37 degrees C to 32 degrees C there is an increase in the amount of t-PA excreted, while cell growth is significantly reduced. The treatments also increased the intracellular amount of t-PA. We measured the distribution of cell cycle phases by cytometry and used a modification of the equations of Kromenaker and Srienc (1991, 1994 a, b) to analyse the intracellular t-PA production rate in the different cell cycle phases. Intracellular t-PA was shown to accumulate in G1 phase in all conditions (at 37 degrees C, at 32 degrees C and in presence of butyrate). Moreover, we have shown that the distribution of the time cells treated by butyrate are maintained in the G1cell cycle phase is significantly increased. t-PA produced in the different cell culture conditions tested was analysed by zymogram and western blotting: neither butyrate, neither the shift of temperature changed significantly the overall quality of the protein. The N-glycan patterns of recombinant human t-PA was also analysed with carbohydrate-specific lectins. Butyrate caused a transitory increase in N-linked complex high-mannose oligosaccharides without any effect on the sialic acid content of t-PA.

Model‐based identification of cell‐cycle‐dependent metabolism and putative autocrine effects in antibody producing CHO cell culture

“…An increase of the antibody production has been mostly described during the G0 or G1 phase for hybridoma or CHO cells ( Kim, & Lee, 2016;Suzuki & Ollis, 1989). In contrast to our study, temperature shifts or butyrate treatment were used to arrest the cell cycle in the G1 phase and therefore enrich G0 cells with assumed higher productivity in the stationary phase, in which most of the product is formed (Du et al, 2015;Hendrick et al, 2001;Münzer et al, 2015;Wippermann et al, 2017). Since no net mitotic growth and no progress of the cells throughout the cell cycle takes place in the stationary phase, this phase was not considered in this study, which focuses on the influence of cell cycle and growthassociated autocrine factors.…”

Evaluation of butyrate‐induced production of a mannose‐6‐phosphorylated therapeutic enzyme using parallel bioreactors

“…Toward this end, an adherent CHO‐K1‐derived cell line that secretes recombinant human ‐glucuronidase was adapted for growth in serum‐free chemically defined medium , and a cell bank was established. As butyrate has been previously shown to increase protein production in CHO cells in a number of bioproduction models , we evaluated its utility to increase production of human ‐glucuronidase.…”

The effects of microcarrier culture on recombinant CHO cells under biphasic hypothermic culture conditions

“…Mild hypothermia has been reported to increase the specific productivity of a number of recombinant proteins (Furukawa and Ohsuye 1998;Kaufmann et al 1999;Hendrick et al 2001;Ducommun et al 2002;Yoon et al 2003;Fox et al 2004). In our studies, the specific productivity of SEAP at 33°C was higher than the productivity before the temperature shift or the productivity of the singletemperature culture, regardless of culture condition (i.e., suspension or microcarrier).…”