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THE DRUG MANUFACTURING PROCESS 125
Figure 3.9. Generalized overview of the industrial-scale manufacture of recombinant E2 classical swine fever-based vaccine, using insect cell culture production systems. Clean (uninfected) cells are initially cultured in 500-1000 litre bioreactors for several days, followed by viral addition. Upon product recovery, viral inactivating agents such as b-propiolactone or 2-bromoethyl-iminebromide are added in order to destroy any free viral particles in the product stream. No chromatographic purification is generally undertaken as the product is substantially pure; the cell culture media is protein-free and the recombinant product is the only protein exported in any quantity by the producer cells. Excipients added can include liquid paraffin and polysorbate 80 (required to generate an emulsion). Thiomersal may also be added as a preservative. The final product generally displays a shelf-life of 18 months when stored refrigerated
product and generation of finished product format (i.e. filling into its final product containers, freeze-drying if a dried product format is required), followed by sealing of the final product containers. Subsequent labelling and packaging steps represent the very final steps of finished product manufacture.
Figure 3.10. Overview of the industrial manufacture of the interferon-co product ‘Vibragen Omega’. Refer to text for details
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Figure 3.11. Overview of the production process for a biopharmaceutical product. Refer to text for specific details
Cell banking systems
Recombinant biopharmaceutical production cell lines are most often initially constructed by the introduction into these cells of a plasmid housing a nucleotide sequence coding for the protein of interest. After culture, the resultant product-producing cell line is generally aliquoted into small amounts, which are placed in ampoules and subsequently immersed in liquid nitrogen. The content of all the ampoules is therefore identical, and the cells are effectively preserved for indefinite periods when stored under liquid nitrogen. This batch of cryopreserved ampoules form a ‘cell bank’ system, whereby one ampoule is thawed and the cells therein cultured in order to seed, for example, a single production run. This concept is applied to both prokaryotic and eukaryotic biopharmaceutical-producing cells.
The cell bank’s construction design is normally two-tiered, consisting of a ‘master cell bank’ and a ‘working cell bank’ (Figure 3.12). The master cell bank is constructed first, directly from a culture of the newly constructed production cell line. It can consist of several hundred individually stored ampoules. These ampoules are not used to directly seed a production batch. Instead, they are used, as required, to generate a working cell bank. The generation of a single working cell bank normally entails thawing a single master cell bank ampoule, culturing of the cells therein, with their subsequent aliquoting into multiple ampoules. These ampoules are then cryopreserved and form the working cell bank. When a single batch of new product is required, one ampoule from the working cell bank is thawed and used to seed that batch. When all the
Figure 3.12. The master cell bank/working cell bank system. For simplicity each bank shown above contains only five ampoules. In reality, each bank would likely consist of several hundred ampoules. Working cell bank number 2 will be generated from master cell bank vial number 2 only when working cell bank number 1 is exhausted, and so on
vials composing the first working cell bank are exhausted, a second vial of the master cell bank is used to generate a second working cell bank, and so on.
The rationale behind this master cell bank/working cell bank system is to ensure an essentially indefinite supply of the originally developed production cells for manufacturing purposes. This is more easily understood by example. If only a single-tier cell-bank system existed, containing 250 ampoules, and 10 ampoules were used per year to manufacture 10 batches of product, the cell bank would be exhausted after 25 years. However, if a two-tier system exists, where a single master cell bank ampoule is expanded as required, to generate a further 250 ampoule working cell bank, the entire master cell bank would not be exhausted for 6250 years.
The upstream processing element of the manufacture of a batch of biopharmaceutical product begins with the removal of a single ampoule of the working cell bank. This vial is used to inoculate a small volume of sterile growth medium, with subsequent incubation under
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Figure 3.13. Outline of the upstream processing stages involved in the production of a single batch of product. Initially, the contents of a single ampoule of the working cell bank (a) is used to inoculate a few hundred ml of medium (b). After growth, this lab-scale starter culture is used to inoculate several litres/tens of litres of medium present in a small bioreactor (c). This production scale starter culture is used to inoculate the production-scale bioreactor (d), which often contains several thousands/tens of thousands litres of medium. This process is equally applicable to prokaryotic or eukaryotic-based producer cell lines, although the bioreactor design, conditions of growth, etc. will differ in these two instances