Sparging - shear sensitivity of animal cells

Abstract

Biopharmaceuticals are increasingly produced by modern biotechnological techniques. The in-vitro culture of animal cells in stirred tanks is one of the feasible systems, especially for proteins that require specific post-tanslational modifications to evoke a desired respons in patients. Animal cell are usually capable to perform these modifications in contrast to bacteria and yeast. Another advantage of animal cells is that they secrete their product into the culture medium, which is greatly facilitating the purification of the product.As a disadvantage of animal cells is mentioned their sensitivity for shear stress. The high energy containing regions of liquid flow that can develop in a stirred tank can damage the cells because of their relative large size compared to yeast cells and bacteria and their lack of a protective cell wall. As a result of the scale-up of stirred tank cultures with animal cells the first detrimental shear phenomenon to become manifest is the shear stress associated with the sparging of the cell suspension. Sparging is the most simple and efficient method to provide cells in large-scale stirred tanks with sufficient oxygen. The cell killing event of sparging is the bursting of bubbles at the surface of the suspension.In chapter 1 an overview is given of the status of the research on the shear sensitivity of animal cells untill about 1990, the starting point of the work on this thesis. The major conclusion of this overview is that because of a lack of a generic approach many fragmented data were gathered that allowed few generalized conclusions. The culture media used for shear testing was highly variable and often considered as of moderate importance. Later the central item of this thesis, the composition and features of the culture medium, emerged as the key factor to control shear sensitivity of animal cells.Chapters 2 and 3 describe the effect of a reduced serum content of the culture medium on the shear sensitivity of animal cells for sparging. The use of culture media without serum has become a requirement for the production of biopharmaceuticals. The protective effect of serum was quantified in a bubble column under standardized conditions and the protection by serum was characterized as an immediate non-metabolic effect. In addition, in these chapters, the "killing volume" hypothesis as developed for insect cells, is validated for hybridoma cells.In chapter 4 the effects of silicone antifoam are described. Just like serum, antifoam is preferably omitted during animal cell fermentations. However, for some sparging experiments in the bubble column the addition of antifoam was necessary and therefore its effects needed to be quantified.The protective effect of the polymer polyethylene-glycol (PEG) against cell death caused by sparging is described in chapter 5. The protection by PEG is correlated with the surface tension of the culture medium. It seems that the presence of PEG in the boundary layer of bubbles is essential for its protective effect.The subject of chapter 6 is the protective effect of dextran. This polymer provides protection for animal cells against the bursting of bubbles by increasing the viscosity of the liquid. To establish the protective effect, high concentrations of high molecular weight dextran are required.In chapter 7 an overview is given of the status of the research on the shear sensitivity of animal cells. Just like in this thesis, the focus in this trend article is determined by the composition and features of the culture medium. Trends in the production of biopharmaceuticals with animal cells, like the use of culture media without components from human or animal source, are discussed in this last chapter.</p