Over half of all biopharmaceuticals thus far approved are produced in recombinant E. coli or S. cerevisiae. Industrial-scale bacterial and yeast fermentation systems share many common features, an overview of which is provided below. Most remaining biopharmaceuticals are produced using animal cell culture, mainly by recombinant BHK or CHO cells (or hybridoma cells in
Table 5.10 Various products (non-biopharmaceutical) of commercial significance manufactured industrially using microbial fermentation systems
Simple organic molecules Ethanol
Acetic acid Lactic acid Lysine
Glutamic acid Proteases
S. cerevisiae Pachysolen tannophilus Some clostridia Clostridium acetobutylicum Clostridium saccharoacetobutylicum C. acetobutylicum C. saccharoacetobutylicum Various acetic acid bacteria Lactobacilli
Various bacilli, e.g. Bacillus licheniformis Bacillus subtilis Aspergillus oryzae Trichoderma viride Penicillium pinophilum Penicillium chrysogenum Bacillus licheniformis the case of some monoclonal antibodies; Chapter 13). While industrial-scale animal cell culture shares many common principles with microbial fermentation systems, it also differs in several respects, as described subsequently. Microbial fermentation/animal cell culture is a vast speciality area in its own right. As such, only a summary overview can be provided below and the interested reader is referred to the 'Further reading' section.
Microbial cell fermentation has a long history of use in the production of various biological products of commercial significance (Table 5.10). As a result, a wealth of technical data and experience have accumulated in the area. A generalized microbial fermenter design is presented in Figure 5.9. The impeller, driven by an external motor, serves to ensure even distribution of nutrients and cells in the tank. The baffles (stainless steel plates attached to the sidewalls) serve to enhance impeller mixing by preventing vortex formation. Various ports are also present through which probes are inserted to monitor pH, temperature and sometimes the concentration of a critical metabolite (e.g. the carbon source). Additional ports serve to facilitate addition of acid/base (pH adjustment) or, if required, addition of nutrients during the fermentation process.
Typically, the manufacture of a batch of biopharmaceutical product entails filling the production vessel with the appropriate quantity of purified water. Heat-stable nutrients required for producer cell growth are then added and the resultant medium is sterilized in situ. This can be achieved by heat, and many fermenters have inbuilt heating elements or, alternatively, outer jackets through which steam can be passed in order to heat the vessel contents. Heat-labile ingredients can be sterilized by filtration and added to the fermenter after the heat step. Media composition can vary
Ports, for e.g. pH probes, thermometer, etc.
Marine type impeller
Figure 5.9 Design of a generalized microbial cell fermentation vessel (a) and an animal cell bioreactor (b). Animal cell bioreactors display several structural differences compared with microbial fermentation vessels. Note in particular: (i) the use of a marine-type impeller (some animal cell bioreactors-air lift fermenters-are devoid of impellers and use sparging of air-gas as the only means of media agitation); (ii) the absence of baffles; (iii) curved internal surfaces at the bioreactor base. These modifications aim to minimize damage to the fragile animal cells during culture. Note that various additional bioreactor configurations are also commercially available. Reprinted with permission from Proteins; Biochemistry and Biotechnology (2002), J. Wiley & Sons from simple defined media (usually glucose and some mineral salts) to more complex media using yeast extract and peptone. Choice of media depends upon factors such as:
• Exact nutrient requirements of producer cell line to maximize cell growth and product production.
• Extracellular or intracellular nature of product. If the biopharmaceutical is an extracellular product then the less complex the media composition the better, in order to render subsequent product purification as straightforward as possible.
Fermentation follows for several days subsequent to inoculation with the production-scale starter culture (Figure 5.7). During this process, biomass (i.e. cell mass) accumulates. In most cases, product accumulates intracellularly and cells are harvested when maximum biomass yields are achieved. This 'feed batch' approach is the one normally taken during biopharmaceutical manufacture, although reactors can also be operated on a continuous basis, where fresh nutrient media is continually added and a fraction of the media/biomass continually removed and processed. During fermentation, air (sterilized by filtration) is sparged into the tank to supply oxygen, and the fermenter is also operated at a temperature appropriate to optimal cell growth (usually between 25 and 37 °C, depending upon the producer cell type). In order to maintain this temperature, cooling rather than heating is required in some cases. Large-scale fermentations, in which cells grow rapidly and to a high cell density, can generate considerable heat due to (a) microbial metabolism and (b) mechanical activity, e.g. stirring. Cooling is achieved by passing the coolant (cold water or glycol) through a circulating system associated with the vessel jacket or sometimes via internal vessel coils.
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