Bioreactor operation mode

The bioreactor operation mode is normally defined at the outset of process configuration. Insect cells have been cultured in almost all known cultivation modes batch [10], repeated-batch [70], perfusion [71-74], fed-batch [75, 76], semi-continuous [77,78] and continuous [79]. In spite of this multitude of different strategies, the batch or, eventually, fed-batch mode is normally preferred due to the lytic infection cycle of the baculovirus. 

FIGURE 22 Bioreactor operational modes (a) a well-mixed batch bioreactor, (b) a well-mixed fed-batch bioreactor. 

FIGURE 24 Bioreactor operational modes (a) a series of continuous flow well-mixed bioreactors, (b) use of different feeds at different stages in a series of continuous flow well-mixed bioreactors, (c) a series of continuous flow well-mixed reactors with biomass recycle. 

There are very limited information on this topic, since most of the experiments on bio-isobutene production are lab scale and did not reach the phase of commercial productivity. This is why there is almost no data on isobutene fermentation in different bioreactor operation modes. Still, some considerations can be made theoretically. 

Bioprocess Technology Modelling and Transport Phenomena Operational Modes of Bioreactors... 

Batch mixed reactor There are three principal modes of bioreactor operation (a) batch (b) fed batch (c) continuous. 

The typical bioreactor is a two-phase stirred tank. It is a three-phase stirred tank if the cells are counted as a separate phase, but they are usually lumped with the aqueous phase that contains the microbes, dissolved nutrients, and soluble products. The gas phase supplies oxygen and removes by-product CO2. The most common operating mode is batch with respect to biomass, batch or fed-batch with respect to nutrients, and fed-batch with respect to oxygen. Reactor aeration is discussed in Chapter 11. This present section concentrates on reaction models for the liquid phase. 

Suspension systems can be operated in different modes batch, fed-batch, chemostat, and perfusion (Fig. 1). These operation modes differ basically in the way nutrient supply and metabolite removal are accomplished, which in turn determines cell concentration, product titer and volumetric productivity that can be achieved [8]. The intrinsic limitation of batch processes, where cells are exposed to a constantly changing environment, limits full expression of growth and metabolic potentials. This aspect is partially overcome in fed-batch cultures, where a special feeding strategy prolonges the culture and allows an increase in cell concentration to be achieved. In perfusion and chemostat processes nutrients are continuously fed to the bioreactor, while the same amount of spent medium is withdrawn. However, in perfusion cultures the cells are retained within the bioreactor, as opposed to continuous-flow culture (chemostat), which washes cells out with the withdrawn medium [9]. 

Bioreactors are operated in discontinuous mode, with a sequential or continuous feed of the substrate (fed-batch operation) or in continuous mode. The choice of the operating mode depends mainly on the reaction characteristics ... 

In order to make a decision on the most appropriate operation mode for a bioreactor, several factors must be taken into consideration. At industrial scale, the most important factors are ... 

The low volumetric productivities that characterize batch cultivation processes are a disadvantage for the use of this operation mode for production. However, a variant known as repeated batches is an interesting alternative. It consists of initially carrying out a batch cultivation for the time needed to attain the desired product concentration. At that moment, just a part of the bioreactor contents is harvested. The remaining cell suspension inside the bioreactor is then used as inoculum for a new batch, by filling the vessel with fresh medium. This procedure can be repeated several times, until a decrease in cell growth or product formation is observed. The use of repeated batches allows a decrease of the time the bioreactor is non-productive. This eliminates the time periods that would be necessary for cleaning and sterilization between each batch. 

The continuous mode allows the establishment of well-defined steady states, so that the relationship between the concentration of substances inside the bioreactor and different biological reaction rates can be studied. Therefore, this operation mode is a powerful tool for cell characterization (Altamirano et al., 2001). 

In this operation mode, it is possible to mitigate the major limitation of continuous cultures, that is, the low productivity due to the loss of cells in the bioreactor outlet. In perfusion, this issue is overcome by using a cell retention device to maintain cells inside the bioreactor. Figure 9.17 shows a scheme of a stirred-tank bioreactor operating in perfusion mode, as well as the kinetic behavior of a perfusion run. 

In the case of homogeneous bioreactors, the maximum cell concentration in perfusion cultures can attain 107-108 cells mL-1. When this operation mode is used in heterogeneous bioreactors, cell concentration in the cell compartment can approach the packing limit of tissues, which is in the order of 109 cells mL. Product concentrations reported for these processes vary considerably, but are most commonly in the range of 100— 500mgL 1. Culture duration can be in the range of several days up to several months (Bodeker, 1994). 

It is important to emphasize the general character of this definition for the volumetric productivity (Py), since it includes all phases of a production cycle in a bioreactor, allowing an evaluation of the impact of bioreactor preparation time and duration of growth and production phases on productivity. As can be observed from Equations 19 to 22, for an industrial bioreactor with a given volume and operation mode, the volumetric productivity depends basically on cell concentration in the production phase and on the specific product formation rate (qp). 

Figure 9.19 shows typical cell concentrations reached in the main industrial bioreactors and a comparison of these values with those found in microbial fermentations. As can be observed, batch and fed-batch cultivations attain dry biomass values comparable to those of continuous cultures of microorganisms, so that mass and heat transfer capacities are not limited for these operation modes. However, high cell density cultivation in heterogeneous bioreactors, such as hollow-fiber devices, reaches dry biomass values similar to the maxima observed in microbial cultures. 

Parameter Stirred-tank bioreactors operated in batch and fed-batch mode Stirred-tank bioreactors operated in perfusion mode Heterogeneous bioreactors (packed-bed or hollow-fiber) operated in perfusion mode... 

Results for a 300 L bioreactor operating in perfusion mode using a specially designed hydrocyclone as a cell retention device. 

Recombinate is produced in CHO cells cultivated in 2500 L bioreactors, operated in fed-batch mode. The purification process starts by removing cells by a filtration step, followed by three chromatographic steps immunoaffinity, anion exchange, and cation exchange (Bhattacharyya et al., 2003). 

Various combinations of bioreactors and operation mode have been used for the production of mAbs in several systems of expression, as shown in Chapter 9. All cells utilized for the production of mAbs grow in suspension. Those that did not initially have this capacity have been adapted (as is the case for CHO and BHK) (Butler, 2005). This results in a large number of options for production systems. Cells with this characteristic are easily cultivated in stirred-tank reactors, which have been scaled up to a volume of 10 000 L (Chu and Robinson, 2001 Kretzmer, 2002). This kind of bioreactor provides excellent homogeneity, facility for the implementation of control techniques, and the principles of scaling up are relatively well known. Other kinds of bioreactors for the production of mAbs are also available, such as air-lift, with volumes up to 1000 L, and also fixed-bed bioreactors (Moro et al., 1994 Irving et al., 1996 Kretzmer, 2002). 

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