Continuous Mode Perfusion

The advantages of sanibatch processing do not imply that continuous processing has been neglected as a means of achieving higher production rates at lower costs. On the other hand, considerable research is being devoted to make continuous operation feasible in the near future. 

The major drawbacks of continuous operation are (i) the use of pumps for circulating the cell broths (in suspension systans) that can damage sensitive cells and (ii) elaborate process control for dissolved oxygen, CO, pH, and temperature that is more crucial as compared to the relatively flexible fed-batch systems (Catapano et al. 2009). 

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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). 

Butler (1987) has considered the physics of gaseous exchange and rate of nutrient supply and concludes that a 100 1 culture running in the continuous mode may be more efficient than a 1000 1 batch system. Different systems for large scale production have been compared by van Wezel (1985)who concludes that continuous perfusion systems are the most efficient for production of cellular components. 

New cell culture techniques, which may improve the applicability of renal epithelial cultures, are also required. Currenfly there exist two commercially available cell culture perfusion systems, which allow the continuous perfusion of culture media and optimized oxygenation [243]. These systems allow stable longterm culture of quiescent adherent cells [244]. Continuous medium perfusion furthermore may lead to the re-expression of lost functions in continuous cell hues and the maintenance of differentiated properties in primary cells. Recently our laboratory has demonstrated that LLC-PKj cells maintained in a newly developed perfusion system (EpiFlow ) changed from a glycolytic to a more oxidative phenotype [72]. Evidence is also available from experiments in our laboratory that this mode of cultivation helps to prolong the lifetime of primary cultures of proximal tubular cells. Combining perfusion culture with co-culture of a cell type that is an anatomical neighbour in vivo (e.g. epithelial with endothelial, interstitial or immune cells) may improve the state of differentiation of both partner cells and increase the complexity of autocrine and paracrine interaction [73]. 

A high-performance model has to be validated for different procedures batch, fed-batch, continuous and perfused modes. Often, the extrapolation of the model to another mode needs the adjustment of different parameters. 

The most common cell culture systems developed for pilot- and commercial-scale production of monoclonal antibodies (MAbs) are hollow-fibre and ceramic matrix modules, stirred bioreactors and airlift fermenters. These systems allow cultivation of cells in batch, fed-batch, continuous or perfusion mode. The selection of a culture system and culture mode for the large-scale production of a particular MAb should take into account the growth and antibody-production characteristics of the particular hybridoma line. This module therefore presents an overview of the important characteristics of these systems. Detailed descriptions with accompanying results and a large collection of cited literature are given elsewhere (Seaver, 1987 Mizrahi, 1989 sections 5.1 and 5.9). 

Operating mode such as batch, fed-batch, continuous, or perfusion. 

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]. 

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. 

Fig. 24.4. Study of the biosensor stability with biological sample. Arrows indicate where (i.e. biological sample) a solution obtained by dialysing a human serum with the microdialysis probe was flowed in the biosensor cell. At the beginning a perfusion solution and control solution (glucose 5 mmol l-1) were used instead of the serum to test the biosensor response. Control solution of glucose was also used during and at the end of the experiment to evaluate the stability of the biosensor. Continuous flow mode 10 pi min-1). Applied potential —50 mV vs. int. ref. Reprinted from Ref. [59] with permission from Elsevier.

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. 

Fig. 4. Two spin-filter bioreactors configured in series for the growth of embryogenic cells and the promotion of somatic embryo maturation on a large scale. The stage 1 bioreactor is for cell proliferation and operates on a continuous culture mode the stage 2 bioreactor is for somatic embryo maturation and operates in a perfusion mode...

The following cultivation modes have been described for production of MAbs in in vitro large-scale systems batch, fed-batch, chemostat and perfusion (Reuveny Lazar, 1989). Batch and fed-batch processes are the most common methods. More demanding are chemostat and perfusion modes, which allow a continuous production of MAbs. All methods are applicable in homogeneous and, with the exception of chemostat, non-homogeneous systems. 

Operation in a perfusion mode differs from fed batch operation in that the former not only involves a continuous supply of fresh substrate in the feed stream, but also continuous removal of soluble metabolic products and waste components present in the process fluid. Such removal minimizes the potential for inhibition of the biochemical reaction by soluble products. Perfusion systems are most frequently employed in the cultivation of mammalian cells (see Section 13.3.2.2). However, readers should note that diffusional or mass transfer limitations on biochemical reactions may be imposed by the use of permselective membranes. 

A SPECT system consists of one or more scintillation cameras mounted to a gantry that can revolve about a fixed horizontal axis (the axis of rotation) " (Fig. 27.13). SPECT studies are usually acquired over a full 360° arc, although myocardial perfusion studies typically use only data from the 180° arc that minimizes tissue attenuation. SPECT acquisitions are performed with the scintillation camera located at preselected angular locations (step-and-shoot mode), or in a continuous rotation mode. In the step-and-shoot mode, the detector rotates to each angular position and collects data in a preselected frame duration while the detector is motionless. In the continuous-rotation mode, the smdy duration is selected and the rotation speed is adjusted to complete the orbit during this time. Projections are collected as the detector rotates and are binned into 60 to 120 frames over 360°. 

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. 

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