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Product type Example Example producer
Simple organic molecules Ethanol Saccharomyces cerevisiae Pachysolen tannophilus, some
Butanol Clostridium acetobutylicum, C. saccharoacetobutylicum
Acetone Clostridium acetobutylicum, C. saccharoacetobutylicum
Acetic acid Various acetic acid bacteria
Lactic acid Lactobacillus spp.
Amino acids Lysine Corynebacterium glutamicum
Glutamic acid Corynebacterium glutamicum
Enzymes Proteases Various Bacilli, e.g. Bacillus licheniformis
Amylases Bacillus subtilis, Aspergillus oryzae
Cellulases Trichoderma viride, Penicillium pinophilum
Antibiotics Penicillin Penicillium chrysogenum
Bacitracin Bacillus licheniformis
THE DRUG MANUFACTURING PROCESS 133
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 3.13). During this process, biomass (i.e. cell mass) accumulates. In most cases, product accumulates intracellulary and the 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 25-37°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.
Mammalian cell culture systems
Mammalian cell culture is more technically complex and more expensive than microbial cell fermentation. It is, therefore, usually only used in the manufacture of therapeutic proteins that show extensive and essential post-translational modifications. In practice, this usually refers to glycosylation and the use of animal cell culture would be appropriate where the carbohydrate content and pattern is essential either to the protein’s biological activity, stability or serum halflife. Therapeutic proteins falling into this category include erythropoietin (Chapter 6), the gonadotrophins (Chapter 8), some cytokines (Chapters 4-7) and intact monoclonal antibodies (Chapter 10).
The culture of animal cells differs from that of microbial cells in several generalized respects, including:
• they require more complex media;
• extended duration of fermentation due to slow growth of animal cells;
• they are more fragile than microbial cells due to the absence of an outer cell wall.
Basic animal cell culture media generally contains:
• most L-amino acids;
• many/most vitamins;
• salts (e.g. NaCl, KCl, CaCl2);
• carbon source (often glucose);
• antibiotics (e.g. penicillin or streptomycin);
• supplemental serum;
• buffering agent (often CO2-based).
Antibiotics are required to prevent microbial growth consequent to accidental microbial contamination. Supplemental serum (often bovine or fetal calf serum, or synthetic serum composed of a mixture of growth factors, hormones and metabolites typically found in serum) is required as a source of the often ill-defined growth factors required by some animal cell lines.
The media constituents, several of which are heat-labile, are generally dissolved in WFI and filter-sterilized into the pre-sterile animal cell reactor. Reactor design (and operation) differs somewhat from microbial fermentations, mainly with a view to minimizing damage to the more fragile cells during cell culture (Figure 3.15). Although the generalized reactor design presented in Figure 3.15 is commonly employed on industrial scale, alternative reactor configurations are also available. These include hollow-fibre systems as well as the classical roller-bottle systems. Roller bottles are still used in the industrial production of some vaccines, erythropoietin and growth hormone-based products. Roller bottles are cylindrical bottles which are partially filled with media, placed on their sides and mechanically rolled during cell culture. This system is gentle on the cells and the rolling action ensures homogeneity in the culture media and efficient oxygen transfer. The major disadvantage associated with applying roller-bottle technology on an industrial scale is that many thousands of bottles are required to produce a single batch of product.