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appropriate conditions. This describes the growth of lab-scale starter cultures of the producer cell line. This starter culture is in turn used to inoculate a production-scale starter culture, which is used to inoculate the production-scale bioreactor (Figure 3.13). The medium composition and fermentation conditions required to promote optimal cell growth/product production will have been established during initial product development, and routine batch production is a highly repetitive, highly automated process. Bioreactors are generally manufactured from high-grade stainless steel, and can vary in size from a few tens of litres to several tens of thousands of litres (Figure 3.14). At the end of the production-scale fermentation process, the crude product is harvested, which signals commencement of downstream processing.
Microbial cell fermentation
Over half of all biopharmacuticals 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 the case of some monoclonal antibodies; Appendix 1). While industrial-scale animal cell culture shares many common principles with microbial fermentation systems, it also differs in several respects, as subsequently described. 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 3.17). As a result, a wealth of technical data and experience has accumulated in the area. A generalized microbial fermenter design is presented in Figure 3.15. 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 side walls) serve to enhance impeller mixing by preventing vortex formation. Various ports are also present through which probes are inserted which monitor pH, temperature and sometimes the concentration of a critical metabolite (e.g. the carbon source). Additional ports serve to
THE DRUG MANUFACTURING PROCESS 131
Figure 3.14. (a) Typical industrial-scale fermentation equipment as employed in the biopharmaceutical
sector. (b) Control of the fermentation process is highly automated, with all fermentation parameters being adjusted by computer. (b). Photos (a) and (b) courtesy of SmithKline Beecham Biological Services, s.a., Belgium. Photo (c) illustrates the inoculation of a lab-scale fermenter with recombinant microorganisms used in the production of a commercial interferon preparation. Photo (c) courtesy of Pall Life Sciences, Ireland
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 WFI. Heat-stable nutrients required for producer cell growth are then added and the resultant media is sterilized in situ. This can be achieved by heat and many fermenters have inbuilt heating elements or, alternatively, outer jackets (see also Figure 3.4) 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 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;
• economics (total media cost);
• extracellular or intracellular nature of product. If the biopharmaceutical is an extracellular
Figure 3.15. Design of a generalized microbial cell fermentation vessel (a) and an animal cell bioreactor (b). Animal cell bioreactors display several structural differences as compared to 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 and; (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 by permission of John Wiley & Sons Ltd from Walsh (2002)
Table 3.17. Various products (non-biopharmaceutical) of commercial significance manufactured industrially using microbial fermentation systems