Culture systems perfusion high cell density

A problem with in vitro cultured hybridomas is low antibody 3held, typically in the range 20-100 mg/litre compared to the ascites in vivo route of 5-10 g/litre. Regulation in the use of experimental animals in many countries now means that the ascitic route is not an option, but to put matters in perspective 1 g of mAb obtainable from 100 mice can be produced in 10-20 litres of culture. This difference can be dramatically reduced if perfusion systems with high cell densities (over 5 X lO ml) are used. When mAbs are needed for research or diagnostic purposes then batches up to only 200 mg are needed and this can be achieved in 2-5 litres of culture. However for in vivo diagnostics and therapeutics where batch sizes of 10-100 g are required then the scale-up to fermenter/airlift reactor or perfusion systems is a necessity. 

The aim of most technologies is to generate a population of cells each of which secretes a desired product into the medium. For this to occur the cells do not need to be actively dividing and, in fact, this is sometimes counterproductive. Cells can remain viable at high cell densities using perfusion systems (Chapter 3) and the product purified from the spent medium (Spier, 1988). Furthermore, the simpler the growth medium (i.e. the fewer protein factors required) then the easier is the downstream processing and this is one reason for the development of serum-free media especially for the culture of hybridoma cells used for monoclonal antibody production ( 5.8). 

Perfusion systems have also been used for successful scale-up of MoAb production. During the culture period, cell growth occurs exponentially until the cell density reaches a maximum. At that point, the medium needs a continuous supplementation of fresh nutrients and elimination of waste. In perfusion systems, fresh nutrients are supplied and wastes are removed continuously so that the medium meets the physiological needs of the cells. At steady state, the cell concentration is determined by space and other limitations. High cell densities have been achieved by immobilizing the cells in porous ceramic matrices or hollow fiber devices. Intermediate cell densities have been achieved by perfusion reactors with a spin filter, or in a fluidized bed reactor in which the cells are embedded in sponge-like... 

Avgerinos GC, Drapeau D, Socolow JS, Mao J, Hsiao K, Broeze RJ (1990), Spin filter perfusion system for high density cell culture production of recombinant urinary type plasminogen activator in CHO cells, Bio/Technol. 8 54-58. 

A great deal of effort has gone into the development of high-density immobilized perfusion culture systems, which can be operated continuously. The limitation with many of these systems (e.g. hollow fibre) is that, whilst they achieve very high unit cell density (typically 10 mH), they do not scale up well volumetrically. This limitation has been overcome by the use of porous carrier immobilization techniques, where high unit cell density can be combined with good volumetric scale-up potential and long-term continuous operation. 

Culture procedures have been described from the simple flask and spinner cultures through increasing degrees of sophistication to the highly productive, but complex and expensive, perfusion bioreactors. The choice of culture system depends upon how much mAb is needed, the quality needed (i.e. for what purpose is it intended), and the expertise and cell culture facilities available. In order to choose on the basis of mAb yield the data in Table 4 summarizes each system described in this chapter with values for cell density and mAb production. Although somewhat hypothetical the values are based on an average... 

Caron AW, Tom RL, Kamen AA, Massie B. (1994) Baculovirus expression system scale up by perfusion of high-density Sf-9 cell-cultures. Biotechnol. Bioeng., 43 881-891. 

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