Very limited information is available in the public domain regarding the general design and sizing of an upstream processing line for pilot- or large-scale production. Chu and Robinson (2001) present an overview of the technologies (bioreactor type and mode, medium type) for the various commercial products recently approved (recombinant proteins, vaccines, diagnostics and tissue culture), but few details on the scale of the cultivation vessels are actually available. In the following, typical approaches based upon our experience are discussed. Large-scale production of recombinant proteins has been selected as the example here, since it is currently the field of animal cell-derived products that is growing fastest (in terms of new molecules launched on the market), and that requires the largest amounts of drug substance (up to several hundred kilograms per year for monoclonal antibodies). Similar design principles can be applied for other smaller-volume products (vaccines, gene-therapy products), although with a simpler inoculum expansion train.
For commercial recombinant proteins, the production step is commonly run, at least for the processes developed during the last decade, in stirred-tank bioreactors, with a working volume of 10 000-15 000 litres in the fed-batch mode and 300-2000 litres in the perfusion mode. In the latter case, size is limited by the difficulties in scaling-up the cell-retention device.
The inoculum required for the production stage is prepared in a stepwise mode, using a series of cultivation systems of increasing size. The maximum allowable expansion factor between two steps of the inoculum expansion train is strongly process- and cell-line dependent; the goal is to keep cells at high viability, in the exponential growth phase. This typically occurs in the concentration range of 105-106 cells/ml, although much higher densities, up to 107 cells/ml, can be reached in optimized fed-batch processes. In practice, a conservative expansion factor of approximately 5 or 6 between two cultivation vessels is normally applied; each culture, if it is run in a batch mode, will last 3-4 days, including a possible lag phase after each transfer. At laboratory scale, cells can be harvested by centrifugation and resuspended in fresh medium to ensure optimal growth performance at each step. Above the litre scale, this becomes very difficult to perform while maintaining sterile conditions. For this reason, cells are normally transferred together with the spent medium into the next-step bioreactor. At laboratory scale, the inoculum is normally transferred by pipetting in a Class II (micro)biological safety cabinet. Transfers to and from small bioreactors (2-20 litres) are typically performed via flexible tubing that is connected by sterile welding; at larger scale, all transfers are performed via hard pipes, using transfer panels (see Section 188.8.131.52).
In the example of a fed-batch process with a production step of 10 000 litres, typically 1-2 ml of cell suspension at about 106 cell/ml from cryovials are expanded first in T and/or spinner flasks and then in a train of bioreactors with working volumes of, for instance, 20 litres, 80 litres, 400 litres and 2000 litres; the whole content of this last step can then serve as the inoculum for the production stage. A more efficient approach for inoculum expansion in the laboratory is to start with large cryobags (50-100 ml), containing frozen cells at high densities (20-40 X 106 cells/ml); the first bioreactor can then be inoculated in one step and in a much shorter time (Heidemann et al. 2002). Bioreactors can also be run in a fed-batch mode, during inoculum expansion, with the addition of a large amount of fresh medium over several days, leading to a significant volume increase during the culture; this allows a reduction in the number of bioreactor steps required for inoculum expansion. In this case, the vessel must be designed such that it can operate over a broad range of liquid volumes (Heidemann et al. 2002). Bioreactors of 50 litres and larger are commonly made of stainless steel; below this, both glass and stainless steel are used although the latter is recommended above 20 litres for its better mechanical properties. Recently, disposable plastic bags have become available as an alternative to spinner flasks and tanks, with volumes of 100 ml to 500 litres (Wave Bioreactor®, Wave Biotech, Bridgewater, NJ, USA; Singh 1999) (see also Chapter 10); they can also be run in a perfusion mode. These bags have the typical advantages of disposable systems (minimal investment, no cleaning and sterilization, including validation); additionally, since stirring is achieved via a rocking movement, the range of working volume is much broader than in a stirred vessel. The culture volume can thus be increased by a factor of 5-10 in the same bag, via gradual feeding of fresh medium, thereby reducing the number of inoculum expansion steps. The drawbacks are relatively high running costs and the fact that pH and pO2 cannot be controlled as accurately as in a stirred-tank bioreactor.
In a fed-batch process, the production step is typically extended to 10-15 days, in order to maximize accumulation of the recombinant product. For optimum utilization of the equipment, more than one production bioreactor per inoculum train should be installed. A ratio of 2 is typically applied; in this case, however, the production step is still the bottleneck if the culture lasts more than 8 days. Figure 14.11 illustrates the example discussed here, with four production bioreactors for a fed-batch process, two inoculum expansion trains and the associated media preparation tanks.
In perfusion processes, the same aforementioned principles can be applied to the inoculum expansion train. In this case, however, only one or two bioreactor steps are usually required to inoculate a production bioreactor, due to the lower working volume at this stage. In the inoculum train, cells can be grown in batch, fed-batch or perfusion mode. For instance for the ReFacto® process, a seed bioreactor is inoculated from the content of spinner flasks and is first run in fed-batch mode, with continuous addition of fresh medium (Eriksson et al. 2001). Once the desired working volume is reached, the perfusion mode is switched on and when a sufficient number of cells is available, the content of the seed bioreactor can be transferred to the production bioreactor. The production bioreactor is also operated first in a fed-batch mode until the desired working volume (500 litres) and cell population density are achieved; the perfusion mode is then switched on. Similar examples are given in Boedeker (2001) and Harrison (1998) where the production processes for the various forms of recombinant Factor VIII and IX are reviewed. For immobilized cells on microcarriers, inoculum expansion is difficult to achieve; Durrschmidt et al. (1999) have reported strategies where they could expand the working volume in a fluidized-bed bioreactor by a factor of 6 via sequential addition of microcarriers and recolonization.
Since a perfusion culture usually lasts several weeks or months, one expansion train is sufficient to inoculate several production bioreactors, provided they are started sequentially. However it is common practice to install an excess of expansion trains (e.g. one for each three or four perfusion bioreactors); this ensures that a production bioreactor can be rapidly re-inoculated at any time, as needed. The additional capital costs are offset by a more efficient use of the production capacity.
In a pilot plant, similar principles can be applied; since the production scale is typically lower (1000-5000 litres for a fed-batch process), the inoculum expansion train should only contain two or three bioreactor steps. By definition, higher flexibility is required compared with manufacturing facilities for commercial products; consequently, the use of flexible tubing and disposable equipment (storage bags for solutions, disposable bioreactors) is recommended. An interesting concept of a flexible and modular pilot plant is discussed in Bardone et al. (1994).
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