Bioreactors, mammalian cells

PLATE 4 Bioreactors that use mammalian cells, like this tower fermentor, are on the cutting edge of new biotechnology manufacturing processes. Courtesy, Cetus Corporation. 

Insect cell systems represent multiple advantages compared with mammalian cell cultures (1) they are easier to handle (Table 2.1) (2) cultivation media are usually cheaper (3) they need only minimum safety precautions, as baculovirus is harmless for humans (4) they provide most higher eukaryotic posttranslational modifications and heterologous eukaryotic proteins are usually obtained in their native conformation (5) the baculovirus system is easily scalable to the bioreactor scale. However, because of the viral nature of the system, continuous fermentation for transient expression is not possible - the cells finally die. 

Mammalian cell suspension cultures are the preferred choice for large-scale recombinant protein production in stirred-tank bioreactors. The most widely used systems are Chinese hamster ovary (CHO) cells and the murine myeloma fines NSO and SP2/0. In half of the biological license approvals from 1996-2000, CHO cells were used for the production of monoclonal antibodies and other recombinant glycosylated proteins, including tPA (tissue plasminogen activator) and an IgGl fusion with the tumor necrosis factor (TNF) receptor, the latter marketed as Enbrel [7]. 

Etanercept is produced by recombinant technology in a Chinese hamster ovary mammalian cell expression system. The WCB is grown in a proprietary media system. The cells are cultured initially in flasks and then inoculated into the bioreactor vessel. The product is purified in a number of chromatographic steps, followed by viral inactivation and viral filtration steps. 

Generally, a distinction can be made between membrane bioreactors based on cells performing a desired conversion and processes based on enzymes. In ceU-based processes, bacteria, plant and mammalian cells are used for the production of (fine) chemicals, pharmaceuticals and food additives or for the treatment of waste streams. Enzyme-based membrane bioreactors are typically used for the degradation of natural polymeric materials Hke starch, cellulose or proteins or for the resolution of optically active components in the pharmaceutical, agrochemical, food and chemical industry [50, 51]. In general, only ultrafiltration (UF) or microfiltration (MF)-based processes have been reported and little is known on the application of reverse osmosis (RO) or nanofiltration (NF) in membrane bioreactors. Additionally, membrane contactor systems have been developed, based on micro-porous polyolefin or teflon membranes [52-55]. 

Commercial scale cultivation of mammalian cells is accompHshed using different technologies roller bottles, microcarriers, suspension (batch, fed-batch or perfusion mode) and hollow fiber bioreactors (Table 2). However, especially for products needed in large amounts, suspension cultivation seems to be the most effective system [4, 5]. Suspension-based systems are characterized by a homogeneous concentration of cells, nutrients, metabolites and product, thereby facilitating scale-up [6] and enabling an accurate monitoring and control of the culture [7]. 

The use of hydrocyclones for separating mammalian cells from the culture medium opens the possibility of using them to perform perfusion in bioreactors. As hydrocyclones have no moving parts, they are ideally suited for operation under aseptic conditions as required by the biotechnology industry. 

Himmelfarb et al. [80] introduced spin-filters as a cell retention device for high cell density perfusion cultivations of mammalian cells in suspension. Spin-filters are cylinders with a porous wall, which are placed inside stirred tank bioreactors, either mounted on the impeller shaft or driven by an independent motor (Fig. 5). Perfusate is pumped out from inside the spin-filter at the same... 

Tokashiki M, Yokoyama S (1997) Bioreactor designed for animal cells. In Hanser H, Wagner R (eds) Mammalian cell biotechnology in protein prodnction. Walter de Gruyter, Berlin, p 279... 

K Schugerl, J. Lticke, U Oels Bubble Column Bioreactors. Tower Bioreactors without Mechanical Agitation. - R. Acton, J.D.Lynn Description and Operation of a Large-Scale, Mammalian Cell, Suspensio Culture Facility. -S. Aiba, M. Okabe A Complementary Approach to Scale-Up Simulation and Optimization of Microbial Processes. - LKjaer-gaard The Redox Potential ItUseandControl in Biotechnology. 

A comparison between insect and mammalian cell culture technology shows various similarities and differences. Due to the fact that insect cell lines typically derive from specific organs or non-differentiated embryonic tissues, the same cell line can proliferate in suspension or in adherent manner (in monolayer). This versatility in growth mode, which contrasts with most mammalian cell lines, increases the choices of bioreactor types and culture strategies for production. Contact inhibition shown by various mammalian cell lines is low or absent for insect cells, and they tend to aggregate in suspension or in adherent cultures. 

Adherent insect cell release from solid surfaces generally does not require trypsinization, unlike anchorage-dependent mammalian cells. Insect cells, like mammalian cells, need rigorous aseptic manipulation during cell transfer, inoculation, and propagation in bioreactors. A minimal inoculum density is required for both cell types. Typically, insect and mammalian cell cultures are initiated with inocula of 1-2 X 105 cells per milliliter of liquid medium (Agathos, 1991). Table 2.3 summarizes the major differences and similarities between these cells. 

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Qi HN, Goudar CT, Michaels JD, Henzler H-J, Jovanovic GN, Konstantinov KB (2003), Experimental and theoretical analysis of tubular membrane aeration for mammalian cell bioreactors, Biotechnol. Prog. 19 1183-1189. 

Wu P, Ozturk SS, Blackie JD, Thrift JC, Figueroa C, Naveh D (1995), Evaluation and applications of optical cell density probes in mammalian cell bioreactors, Biotechnol. Bioeng. 45 495-502. 

Hydrocyclones are very simple devices and always operate with a flow ratio Rf > 0. They may be easily designed to give a desired separation efficiency (Castilho and Medronho, 2000), and their performance may also be easily predicted (Coelho and Medronho, 2001). In the last few years, it has been shown either theoretically or experimentally that hydrocyclones may be used in animal cell separations (Luebberstedt et al., 2000 Medronho et al., 2005 Elsayed et al., 2006 Pinto et al., 2007) aimed mainly at mammalian cell retention in perfusion bioreactors (Jockwer et al., 2001 Elsayed et al., 2005). 

Figure 2. H NMR spectra of mammalian cells growing in a hollow-fiber bioreactor without (left) and with (right) suppression of the water signal (4.7 ppm) using the CHESS pulse sequence (Haase et al., 1985). The spectra were acquired using the STEAM sequence with two, three-pulse CHESS cycles (Callies, Jackson, and Brindle, unpublished observations) (Moonen and Van Zijl, 1990).

In a number of different bioreactor cultivations with bacteria, yeasts, molds and mammalian cells, it was shown how the electronic nose can serve to visualize the course of the processes. The pattern recognition method that best manages to mirror the complex sensor array responses during extended cultivations is two- or three-dimensional PCA. Examples from such electronic nose applications are given below. 

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