Perfusion cultivation

Himmelfarb et al. [80] introduced spin-filters as a cell retention device for high cell density perfusion cultivations of mammalian cells in suspension. Spin-filters are cylinders with a porous wall, which are placed inside stirred tank bioreactors, either mounted on the impeller shaft or driven by an independent motor (Fig. 5). Perfusate is pumped out from inside the spin-filter at the same... 

Mercille et al. [141] compared TFF and VFF for the perfusion cultivation of a hybridoma cell line producing an IgM, either using serum-supplemented or protein-free medium, with and without the addition of DNAse. They observed... 

Castilho LR, Anspach FB, Deckwer W-D (2002), An integrated process for mammalian cell perfusion cultivation and product purification using a novel dynamic filter, Biotechnol. Progr. 18 776-781. 

Iding K, Liitkemeyer D, Fraune E, Gerlach K, Lehmann J (2000), Influence of alterations in culture condition and changes in perfusion parameters on the retention performance of a 20 pm spinfilter during a perfusion cultivation of a recombinant CHO cell line in pilot scale, Cytotechnology 34 141-150. 

Castilho LR (2001), Development of a dynamic filter for integrated perfusion cultivation and purification of recombinant proteins from mammalian cells, Fortschritt-Berichte Series - Reihe 17 (Biotechnologie/Medizintechnik), VDI-Verlag, Diissel-dorf. 

Until now, bioreactors of various types have been developed. These include loop-fluidized bed [14], spin filter, continuously stirred turbine, hollow fiber, stirred tank, airlift, rotating drum, and photo bioreactors [1]. Bioreactor modifications include the substitution of a marine impeller in place of a flat-bladed turbine, and the use of a single, large, flat paddle or blade, and a newly designed membrane stirrer for bubble-free aeration [13, 15-18]. Kim et al. [19] developed a hybrid reactor with a cell-lift impeller and a sintered stainless steel sparger for Thalictrum rugosum cell cultures, and cell densities of up to 31 g L1 were obtained by perfusion without any problems with mixing or loss of cell viability the specific berberine productivity was comparable to that in shake flasks. Su and Humphrey [20] conducted a perfusion cultivation in a stirred tank bio-... 

Fig. 16.1 General schematic for fed-batch and perfusion cultivation. X=biomass ...

The arguments to move to fed-batch, and to abandon perfusion cultivation - as given by various companies (e.g., Medarex and Serono) - included ... 

However, there are arguments not to abandon perfusion cultivation ... 

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. 

Several innovative membrane-based bioreactor designs have recently been proposed, including that by Sussman et al. [10], which involves the cultivation of hepatoma cells on the exterior surfaces of semipermeable capillary hollow fiber membranes which are bundled together with an enclosing plastic shell (Fig. 2). Required nutrient medium is circulated within the capillaries. After the hepa-tocytes have attached and formed a mass of liver tissue, the capillary membranes are perfused with the media. 

Fig. 4. Primary liver cells are cultivated on the exterior surfaces of semipermeable capillary hollow fiber membranes. Medium is perfused through the extrafiber space. Through the fibers oxygen supply is supported...

To avoid inhibition of proUferation and differentiation the pH in hematopoietic cell cultures has to be controlled carefully. Especially in non-perfused culture the pH shifts during the cultivation due to secreted acidic metaboUtes like lactate. However is has been reported recently that pH control alone is not sufficient to eliminate inhibition of cell growth and metaboUsm as other inhibitory factors also accumulate in un-fed cultures [51]. 

Commercial scale cultivation of mammalian cells is accompHshed using different technologies roller bottles, microcarriers, suspension (batch, fed-batch or perfusion mode) and hollow fiber bioreactors (Table 2). However, especially for products needed in large amounts, suspension cultivation seems to be the most effective system [4, 5]. Suspension-based systems are characterized by a homogeneous concentration of cells, nutrients, metabolites and product, thereby facilitating scale-up [6] and enabling an accurate monitoring and control of the culture [7]. 

Batch, fed-batch and perfusion processes are commonly used in large-scale cultivation of mammalian cells. Chemostat cultures are a valuable tool for cell physiology studies but not a serious contender as a production process [3]. 

Cell Line Product Reactor Volume (L) Max. Perfusion Rate (d ) Cultivation Time (d) Centrifuge Type g-factor " Feed Flow Rate " (L min ) Max. Viable Cell Cone. (10 mL- ) Separation Efficiency (%) Reference... 

Combining these techniques, they carried out cultivations for 250-350 h, and were able to repeatedly use the same cartridge (four times at least) without measurable deterioration in filtration efficiency. However, when perfusion rate and cell concentration in the bioreactor increased, fouling eventually occurred. Van Reis et al. [92] provided backpressure on the filtrate line to control filtrate rates and so to avoid too high initial filtration rates, which can cause rapid fouling. De la Broise et al. [99] compared the filter performance using membranes of different pore sizes (2 and 10 pm). In both cases partial retention of the produced IgM was observed and membranes had to be changed every 5 days, the... 

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. 

Mammalian cells were cultivated in high cell density perfusion operation mode for the production of therapeutically important proteins. Material and energy balances were determined and cell regulation was investigated. Methods were developed for the recovery and purification of the products with chromatography and expanded bed chromatography. 

Fermentation and alternative production techniques, such as roller bottles, can be carried out in four different ways. They are (1) batch process, (2) fed-batch process, (3) chemostat process, and (4) perfusion process. Batch and fed-batch processes require termination of cell growth while chemostat and perfusion processes allow continuous cell cultivation. 

When anchorage-dependent cells are immobilized by microcarriers they can be cultivated in homogeneous bioreactors (Jo et al., 1998). Furthermore, the immobilization on the carriers allows the use of other bioreactors that will be discussed in the following sections, such as fluidized-bed and packed-bed bioreactors. The microcarriers can be separated from the supernatant by sedimentation, facilitating downstream processing or cell retention in perfusion processes. 

The different operation modes used in microbial fermentations are employed also in animal cell cultivation. Although many different classifications can be adopted, the most general is the one that considers the following operation modes batch, fed-batch, continuous, and perfusion, which is a continuous mode with cell recycle/retention (Castilho and Medronho, 2002). 

Continuous cultivation with cell retention (perfusion)... 

Dalm MCF, Jansen M, Keijzer, TMP, Grunsven WMJ, Oudshoorn A, Tramper J, Martens DE (2005), Stable hybridoma cultivation in a pilot-scale acoustic perfusion system long-term process performance and effect of recirculation rate, Biotechnol. Bioeng. 91 894-900. 

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