Effect of shear stress on expression of a recombinant protein by Chinese hamster ovary cells

Abstract

A flow chamber was used to impart a steady laminar shear stress on a recombinant Chinese hamster ovary (CHO) cell line expressing human growth hormone (hGH). The cells were subjected to shear stress ranging from 0.005 to 0.80 N m(-2). The effect of shear stress on the cell specific glucose uptake, cell specific hGH, and lactate productivity rates were calculated. No morphological changes to the cells were observed over the range of shear stresses examined. When the cells were subjected to 0.10 N m(-2) shear in protein-free media without Pluronic F-68, recombinant protein production ceased with no change in cell morphology, whereas control cultures were expressing hGH at 0.35 microg/10(6 )cells/h. Upon addition of the shear protectants, Pluronic F-68 (0.2% [w/v]) or fetal bovine serum (1.0% [v/v] FBS), the productivity of the cells was restored. The effect of increasing shear stress on the cells in protein-free medium containing Pluronic F-68 was also investigated. Cell specific metabolic rates were calculated for cells under shear stress and for no-shear control cultures performed in parallel, with shear stress rates expressed as a percentage of those obtained for control cultures. Upon increasing shear from 0.005 to 0.80 N m(-2), the cell specific hGH productivity decreased from 100% at 0.005 N m(-2) to 49% at 0.80 N m(-2) relative to the no-shear control. A concurrent increase in the glucose uptake rate from 115% at 0.01 N m(-2) to 142% at 0.80 N m(-2), and decreased lactate productivity from 92% to 50%, revealed a change in the yield of products from glucose compared with the static control. It was shown that shear stress, at sublytic levels in medium containing Pluronic F-68, could decrease hGH specific productivity.

The fate of Pluronic F‐68 in chondrocytes and CHO cells

“…As the shear protective effect of PF-68 has been reported to be proportional to its concentration (Ma et al, 2004;Zhang et al, 1992), it is important to know if this uptake phenomenon can significantly affect the Pluronic content in the culture medium. For CHO cells, a 0.2% (w/v) PF-68 concentration is commonly used (Keane et al, 2003;Schroder et al, 2004) and we showed that the presence of cells only slightly affects this concentration. At 0.2% (w/v) with 4 Â 10 6 cells/mL, only a 2.4% decrease of the initial concentration was observed, after 18 h. At the end of a classical bioreactor culture, cell densities can reach up to 10 Â 10 6 cells.…”

“…Each Pluronic fits for a specific application, from drug solubilization to bioprocess applications. For instance, Pluronic F-68 (PF-68, (EO) 75 -(PO) 30 -(EO) 75 ) is used as a shear protective agent which limits cell death in mechanically agitated gas-sparged bioreactors (Murhammer and Goochee, 1990) and is particularly useful in serum-free medium culture (Keane et al, 2003;Schroder et al, 2004). The protective effect of PF-68 has been demonstrated for suspension culture of mammalian cells (Jordan et al, 1994;Ramirez and Mutharasan, 1990;Zhang et al, 1992), insect cells (Goldblum et al, 1990;Murhammer and Goochee, 1990), and plant cells (Sowana et al, 2002).…”

Production of a self-activating CBM-factor X fusion protein in a stable transformed Sf9 insect cell line using high cell density perfusion culture

“…However, Sf9 perfusion cultures up to 30 Â 10 6 cells ml À1 with a viability maintained at $90% have been reported despite using the ultrasonic separator in the recirculation mode (Zhang et al 1998). The cultivation medium used in that study contained 1% serum (FBS), which can be an effective protecting agent against hydrodynamic stress (McQueen and Bailey 1989;Kunas and Papoutsakis 1990;Chisti 2001;Ma et al 2002;Keane et al 2003). In Figure 4, serum free medium was used, which could explain the greater sensitivity of the insect cell culture to shear stress created by recirculation pumping.…”

Microplatforms for Gradient Field Generation of Various Properties and Biological Applications

“…For some cells, shear stress does not affect phenotype; however, other cells have mechanisms to sense and react to shear stress. 32 It was reported that a very low shear stress level (10 neutrophil cell movements. 35 Therefore, it is important to check the shear stress level of the fluid flow while designing a microsystem.…”