Bioreactor operation batch cultivation

Batch cultivation (A) operational configuration and typical viable cell (Xv) and substrate (S) concentration profiles (B) concentration of cells, product and two substrates in a batch bioreactor culture of a myeloid transfectoma producing a humanized monoclonal antibody (Center of Molecular Immunology - CIM, Cuba). 

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. 

Immobilisation of an Acetobacter aceti strain in calcium alginate resulted in improvement of the operational stability, substrate tolerance and specific activity of the cells and 23 g phenylacetic acid was produced within 9 days of fed-batch cultivation in an airlift bioreactor [133]. Lyophilised mycelia of Aspergillus oryzae and Rhizopus oryzae have been shown to efficiently catalyse ester formation with phenylacetic acid and phenylpropanoic acid and different short-chain alkanols in organic solvent media owing to their carboxylesterase activities [134, 135] (Scheme 23.8). For instance, in n-heptane with 35 mM acid and 70 mM alcohol, the formation of ethyl acetate and propylphenyl acetate was less effective (60 and 65% conversion yield) than if alcohols with increased chain lengths were used (1-butanol 85%, 3-methyl-l-butanol 86%, 1-pentanol 91%, 1-hexanol 100%). This effect was explained by a higher chemical affinity of the longer-chain alcohols, which are more hydrophobic, to the solvent. 

Batch cultivation is perhaps the simplest way to operate a fermentor or bioreactor. It is easy to scale up, easy to operate, quick to turn around, and reliable for scale-up. Batch sizes of 15,000 L have been reported for animal cell cultivation [2], and vessels of over 100,000 L for fermentation are also available. Continuous processes can be classified into cell retention and non-cell retention. The devices typically used for cell retention are spin filters, hollow fibers, and decanters. Large-scale operation of continuous processes can reach up to 2,000 L of bioreactor volume. Typically, the process is operated at 1-2 bioreactor volumes... 

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. 

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. 

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). 

Submerged fermentation can be conducted in different modes of operation. The most traditional is batch fermentation, in which the bioreactor is filled with medium, inoculated and incubated under controlled conditions to the point in which the product (enzyme) has been synthesized to (or nearly to) its maximum level then the cells are harvested for enzyme recovery, if intracellular, or else discarded to recover the medium containing the enzyme, if extracellular. Fed-batch fermentation is a variant of the former in which, after certain time of batch cultivation, the bioreactor is fed with nutrients according to a controlled rate profile and up to a final volume and the product is then recovered as above. This mode of cultivation is particularly appealing for the production of enzymes because it allows the control of the metabolic... 

The fed-batch and batch cultivation systems share the same cleaning and sterilization process in which the bioreactor operation is stopped and the bioreactor is emptied. This stoppage creates considerable costs and operational downtime. The repeated or cyclic system, which can be applied to both batch and fed-batch cultivation systems, may be installed in order to maximize the productivity. The cyclic cultivation system does not enter the cleaning and sterilization process, but rather empties a portion of the bioreactor while preserving part of the batch for the next cycle. Another method to increase productivity is cell retention techniques such as fluidized beds, membranes, or external separators. These options allow multiple cycles without cleaning and sterilization, which is initiated only if it is deemed that mutation risks exceed tolerable levels (Bellgardt, 2000b). 

Bioreactor operation is either batch-wise or continuous. Batch-wise operation or batch cultures include aU the nutrients required for the growth of cells prior to cultivation of the oi anisms. After inoculation, cell growth commences and ceases once the organisms have exhausted all the available nutrients in the culture medium. A modification of this type of operation is a fed-batch or semi-batch operation in which the reactants are continuously fed into the bioreactor, and the reaction is allowed to go to completion, after which the products are recovered. Continuously operated hioreactors, use continuous culture systems that continuously feed culture medium into... 

Batch fermentation means the cultivation of microorganisms, where the sterile growth medium in desired volume is inoculated with the microorganisms into the bioreactor and no additional growth medium is added during the fermentation. The product will be harvested at the end of the process. Typically, PHA s production is performed using batch fermentation because of low cost for investment and no special control. In addition, sterilization of the feed stock is easier than other fermentation processes, and operation is flexible. 

The cultivation of cells and adenovirus propagation in suspension bioreactors has been performed in batch, fed-batch, and perfusion modes. In batch operation, no nutrients are added during the course of growth or infection. Although batch cultures are simple to perform and contamination risks are low, essential nutrients are rapidly depleted from the medium and cell viability decreases [110,121]. 

Operation in a perfusion mode can be viewed as a form of recycle of biomass that permits one to substantially increase the amount of biomass in the bioreactor. In a batch reactor used for cultivation of mammalian cells, the maximum cell concentration is roughly 1 million cells/mL. By... 

Scale and Mode of Operation Scale and also the mode of operation (Figure 1.2, batch, fed-batch, or continuous/perfusion cultivation) have an impact on the design and the interface to the peripheral units. A perfusion cell culture facility may have a relatively small bioreactor vessel but a rather large media preparation area and holding tanks for the perfused medium. 

Great progress was achieved in the past decades regarding the development of mammalian cell culture technology [2,34,98-102]. Nevertheless, the design and layout of cultivation systems and bioreactors have to meet cell-specific demands due to the special characteristics of these cells. Selection of a suitable type of cell culture bioreactor system and/or an appropriate operation mode (batch, fed-batch, or perfusion) is affected by technical, biological, economical, and regulatory considerations. 

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