TABLE 49 Batch size of cell cultures and estimated time required for fermentation

Description Batch Size (liters) Time (days)"

Laboratory shake flask 0.1 1-2

Bottles or large flask 1-2 2-4

Batch fermenter 50 4-6

Batch fermenter 2500 6-8

Batch fermenter 25,000 to 100,000 10-16

"Estimated based on using E. coli as host cells for producing recombinant proteins.

A. Batch

Fermenter

Fermenter

Cell inoculum is added to a defined volume of medium in either fermenter or roller bottles, and allowed to grow and Roller bottle be harvested at predetermined optimal time point

B. Fed-batch

Similar to batch cultivation except nutrients, O2 and pH are maintained by feeding appropriate supplements at predetermined intervals to increase product yield

C. Chemostat

At predetermined intervals, nutritional supplements are added and culture fluid is withdrawn.

D. Perfusion

Inline cell separator

Figure 4.12. Configuration of large-scale, cell cultivation methods for pharmaceutical scale production of recombinant proteins.

Inline cell separator

Hollow fiber reactor with adherent cells

Similar in principle to chemostat configuration except the cells are retained within the fermenter and roller bottles using inline or external cell separation devices. The hollow fiber filter allows continuous separation of cells from tissue culture fluid containing products. In this configuration, the recombinant product is often designed to be excreted into medium allowing it to be collected in perfusate.

Figure 4.12. Configuration of large-scale, cell cultivation methods for pharmaceutical scale production of recombinant proteins.

often grown in roller bottles (Figure 4.12). Recombinant granulocyte-colony stimulating factor (G-CSF) is produced by growing recombinant Chinese hamster ovary cells using roller bottles. Alternatively, spherical support particles of 1 to 200 mm diameters (microcarriers) as well as porous microcarriers are used to increase the surface area to volume ratio.

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.

Batch Process

In a batch configuration, host cells that contain an expression vector for the recombinant product are added to a predetermined volume of growth medium (Figure 4.12A). The cells are allowed to grow until the nutrients in the medium are depleted or the excreted by-products reach inhibitory levels. At that time, the cells are harvested, and recombinant protein, found in inclusion bodies, cell-membrane fractions, or cytoplasm, are isolated after disruption of the harvested cells. Because the host cells are destroyed at the end of each run, they must be replaced every three to seven days for fermentation or every two weeks for roller-bottle or microcarrier-support production of adherent cells. To ensure uniformity and reproducibility, the FDA requires manufacturers of recombinant proteins to establish and validate a seed stock of recombinant host cells that are validated to contain the characterized expression vector and to be free of contaminants.

Fed-Batch Process

For cell culture or fermentation processes, where the growth rate of cells and thereby recombinant product formation is limited by the availability of nutrients, replenishment can be carried out in a fed-batch manner to improve product yield (Figure 4.12B). By providing a balanced mixture of nutrients, such as oxygen and amino acids required for cell growth, and chemicals to neutralize accumulating growth inhibitors, the product yield or titer and the cell density can be improved by as much as 10fold over that of batch configuration. Fed-batch cultivation may last up to 30 days with cell densities as high as 1.4 x 107 cells/ml. At the end of the run, the cells are harvested and the recombinant products are isolated. This configuration is essential, and often used, for mass production of recombinant proteins that are localized in cellular fractions, requiring disruption of cells to isolate the target proteins.

Chemostat Process

Continuous mass production of cells by addition of nutrients essential to promote cell growth and removal of medium and product at a predetermined time or rate is known as the chemostat process (Figure 4.12C). This strategy works best for recombinant proteins that are secreted from microbial cells and can be collected in the culture media.Typically the growth of these microbial cells is limited to a defined single substrate that can be easily replenished. Because the recombinant product can be collected, thereby preserving the cells, and because the essential substrate can be consistently replenished, production efficiency is high. However, product titer may not be as high as that obtained with fed-batch configuration but production costs overall may be lower. With the added risks of contamination due to prolonged culture, this process is not used routinely for preparation of recombinant proteins.The cells used in this cell culture configuration must be monitored to ensure their genetic and functional stability for the cultivation period, which can last up to several months.

Perfusion Configuration

The basic principle of this configuration is to retain the growth of cells in culture through removal of spent medium containing product through a filtration device and replacement with fresh medium (Figure 4.12D). In this case the cells can continue to grow under optimal conditions for long periods of time while the product is continuously harvested. Because the exchange of fresh and spent medium is facilitated through a perfusion process, this config uration is known as perfusion culture. However, the added complexity and cost of the perfusion configurations limit its use to the production of more expensive recombinant proteins such as mammalian cell products.

The medium exchange can be facilitated within the bioreactor or configured as an external attachment. A number of filter designs have been described and used for this purpose. They are the spin filter, acoustic filter, and hollow fiber culture system. The acoustic filter system is a relatively new method that uses static acoustic waves (nodes of the wave) to concentrate the cells in the effluent stream leading to sedimentation and retention in culture, while allowing the fluid to pass through. This method has been successfully tested in processing of large-scale cell cultures. The other two filtration systems rely on passage of effluent stream through porous filters that are subject to clogging. Modulating the flow rate and pattern of the effluent fluid through the system can reduce clogging.

Perfusion configurations can grow cells for up to two months, without having to reseed the cells. Consequently the process poses a risk of contamination. However, it has been shown that the preparation of some antibodies by means of hybridoma cell culture, which may last up to 30 days, can be realized with little or no contamination.

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