Bioreactors, production scales

Bubble behaviour was studied in the pilot-scale bioreactor so that a complete model of flow, OTR, mixing, cooling, energy requirement and disengagement could be developed for this system and larger production-scale vessels of similar type. 

The production-scale fermentation unit, with a projected annual capacity of over50,000 tonnes was fully commissioned in 1980. The bioreactor (Figure 4.8) is 60 m high, with a 7 m base diameter and working volume 1,500 m3. There are two downcomers and cooling bundles at the base. Initial sterilisation is with saturated steam at 140°C followed by displacement with heat sterilised water. Air and ammonia are filter sterilised as a mixture, methanol filter sterilised and other nutrients heat sterilised. Methanol is added through many nozzles, placed two per square metre. For start-up, 20 litres of inoculum is used and the system is operated as a batch culture for about 30 h. After this time the system is operated as a chemostat continuous culture, with methanol limitation, at 37°C and pH 6.7. Run lengths are normally 100 days, with contamination the usual cause of failure. 

BPD is a contract research facility, which started operations in 1996. From the storage of frozen materials, the customized laboratory services to the consulting in process development and scaling up, BPD has been accumulating useful experiences in the biotechnological scenario. Very flexible set of fermenters are used to satisfy customer needs, through very small and fast adaptation and modifications. Bioreactors productivity varies from 100 mg to 1000 mg scale. 

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 media, with subsequent incubation under appropriate conditions. This describes the growth of laboratory-scale starter cultures of the producer cell line. This starter culture is, in turn, used to inoculate a production-scale starter culture that is used to inoculate the production-scale bioreactor (Figure 5.7). The media composition and fermentation conditions required to... 

Figure 5.7 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) are used to inoculate a few hundred millilitres of media (b). After growth, this laboratory-scale starter culture is used to inoculate several litres/tens of litres of media 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 media. 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...

Membrane bioreactors have been reported for the production of diltiazem chiral intermediate with a multiphase/extractive enzyme membrane reactor [15, 16]. The reaction was carried out in a two-separate phase reactor. Here, the membrane had the double role of confining the enzyme and keeping the two phases in contact while maintaining them in two different compartments. This is the case of the multiphase/ extractive membrane reactor developed on a productive scale for the production of a chiral intermediate of diltiazem ((2R,3S)-methylmethoxyphenylglycidate), a drug used in the treatment of hypertension and angina [15]. The principle is illustrated in... 

Figures 9.2 and 9.3 show schemes that illustrate inoculum development from the cryotubes to production scale for suspension and adherent cells, respectively. In these hypothetical process schemes, the expression production bioreactor is used arbitrarily for any of the types of bioreactor presented in the next section of this chapter. In general, different flasks and several intermediate bioreactors are used for cell propagation to reach the quantity of cells necessary to inoculate the production bioreactor. The number of propagation steps is a function of the final scale of the production bioreactor.

The plastic bag is disposable and is provided sterile by the manufacturer. This makes it a very attractive bioreactor type for production purposes, especially if the production scale is not very large. Since it is not sterilized in-house, there is no need for validation of the sterilization procedure. The validation of bag sterilization is the responsibility of its manufacturer. Since it is not reused, there is no need for cleaning and validation of cleaning procedures. Figure 9.6 (see color section) shows a photograph and a schematic representation of a wave bioreactor. 

The design of bioreactors for perfusion operation is more sophisticated, which makes the equipment more expensive. However, the productivity increases obtained by perfusion operation allow the use of much more compact systems than those operated under batch or fed-batch mode. In this way, perfusion bioreactors can be up to 10-fold smaller for a given production scale (Bibila and Robinson, 1995), decreasing the costs not only of the bioreactors themselves, but also of storage tanks and downstream processing equipment. 

The manufacturer must establish the maximum cultivation span for a particular cell in vitro (passage number). The characterization must include the growth rate, morphology, specific yield, and quality of the molecule of interest (Wiebe and May, 1990). The post-production cells (PPCs) must be removed from the bioreactor and tested at least once to evaluate whether new contaminants were introduced or induced by the cultivation conditions. Changes in the culture medium or production scale require new evaluation of the PPCs to determine any effects on the yield and product consistency (Levine and Castillo, 1999). 

As mentioned in Chapter 9, since production scale-up is related to the increase of cell culture surface for adherent cells, consideration must be given to the relationship between the surface area available for cell growth and the bioreactor volume (Kent and Mutharasani, 1992). 

Sivakumar, G. Bacchetta, L. Gatti, R. Zappa, G. 2005. FIPLC screening of natural vitamin E from mediterranean plant biofactories - a basic tool for pilot-scale bioreactors production of alpha-tocopherot. J. Plant Physiol. 162 1280-1283. 

In this overview an explanation has been given for the wide and complex range of cell culture bioreactor systems that are available. This is to put some perspective on the ones that are described in more detail in the following chapters. The selection of a suitable system follows the adage horses for courses , i.e. a process is selected that fulfils the particular criteria for the cell, product, scale and quantity required, plus the resources in facilities, manpower and experience that are available. 

Doran outlines several of the key parameters involved in the scale-up of a biological process from laboratory-scale shake flasks to production-scale bioreactors. In the first stage of studies a bench-top bioreactor, typically 1-2 L, is used to determine the oxygen requirements of the cells, their shear sensitivity, foaming characteristics, and any limitations that the reactor imposes on the organism. The results of these early studies enable decisions regarding operation in the batch, fed-batch, or continuous mode. A pilot-scale... 

The yield of secondary metabolites in a large-scale fermenter typically ranges from 0.1 to lOg/L of broth. Such poor yield leads to cumbersome and expensive processes for both product separation and broth disposal. Within the last decade, several novel bioreactors have been developed for the intensification of fermentation processes. Examples include a centrifugal bioreactor, a rotating packed bed fermenter, and a sonobioreactor. Most of these, however, are yet to be implemented on a production scale because they generally lack practicality and well-defined scale-up criteria. 

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