PROBLEMS OF CULTIVATION OF MAMMALIAN CELLS IN FERMENTORS

In the past few decades there have been great interests in the cultivation of mammalian cells, including human cells in fermentors for the production of biologics for diagnostic and therapeutics used in human health care.
This is not a new direction in the progression of fermentation technology but a natural anticipated progress in the field of fermentation technology.

Historically speaking the field of fermentation technology has been based on the cultivation of microorganisms such as bacteria to carry out the transformation and production of fermentation products of great economic and industrial importance. It seems then that the power of the microbes are limitless in producing any kind of metabolites. Great strides in the development in microbial fermentation technology were made with parallel development in the study of microbiology, physiology, biochemistry and genetics with similar development in the design and construction of fermentors by bioprocess engineers.

The culmination of this progress is in a nutshell the fourth definition of fermentation by the engineers which defined fermentation being the study of the cultivation of high concentration of microorganisms in bioreactors microorganisms. The fermentor is seen as a special vessel which provides all the optimum conditions necessary to support the growth of high concentration of microorganisms and allow the transformations to occur.

With the development in plant biotechnology especially in the generation of tissue cultures, it then becomes a natural extension or progression to culture single plant cells in fermentors as there is really not much difference between microorganisms and plant cells when they occur as single plant cells.

Much knowledge, experience and breakthroughs in microbial cultivation in fermentors could be similarly applied to single plant cells. Well, at least superficially they seems similar, but in reality there are many problems faced in the cultivation of single plant and mammalian cells compared with the cultivation of microbial cells

THE PROBLEMS..
----------------------------

As we have said earlier the common factor shared between microorganisms and single plant and mammalian cells is that all the cells for the cultivation in the fermentors are SINGLE cells. Beyond it are more differences than similarities.

We can see the main differences between the cultivation of microbial cells and mammalian cells in the following:

1 Larger size
2 Longer time to grow
3 Fragility of cells
4 Lower oxygen consumption
5 Tendency to form multicellular aggregates
5 Complex medium requirements
6 The cells might end up not producing the desired products efficiently

The above factors are crucial when one consider cultivating the mammalian cells in fermentor.

The fragility of mammalian cells means that these cells are easily broken up by the various shear forces generated in the fermention broth such as effect of mixing by impellers, liquid shears and bubbles cavitation

Due to the long growth time means that it takes very long period for the mammalian cells to reach optimal concentration in the fermentors. This situation would make the fermentation process more susceptible to microbial contaminations. There is a stringent requirement to prepare clean room standards for mammalian cell cultivation.

The cultivation of mammalian cells require complex media which is more costly adding to the costs of the fermentation

In mammalian cell cultivation the issue is not so much about using cell substrate for the highest yield or productivity but more of:
1 instability protein expressions which might deteriorate with time
2 protein produced require extensive post translational modifications especially glycosylation of protein such as in production of monoclonal antibodies

Bacterial systems are poor choice in the production of the biologics because bacteria cannot carry out complex post translational modifications of proteins.They can only produce simple proteins such as insulins.

These proteins formed too are not easily secreted by the bacterial system and tend to accumulate inside the bacterial cells as crystalline deposits. Their release from the cells would enticed complicated downstream processing which are costly and there is also the danger of endotoxins being produced if gram negative bacteria are involved

The released proteins might require resolubilization and refolding to bring it to its effective state. This does not consider percentage losses of products during downstream activities

FERMENTOR REQUIREMENTS FOR MAMMALIAN CELL CULTIVATION
------------------------------------------------------------------------------
In view of the above considerations, any fermentor to be used for mammalian cell cultivation must be:

1 Exhibit minimum shearing forces either liquid to liquid shearing, liquid to impeller shearing or
bubbles shearing
2 Provide durable aseptic integrity throughout the fermentation duration

As we already know the CSTR is the most widely used fermentor in industrial fermentations and its engineering principles and reliability well established. Modifications can be made to the CSTR to make it able to cultivate mammalian cells

The fermentation vessel should be rounded at the bottom and equipped with baffles to prevent vortex which could damage the cells.

Strong air sparging is avoided and laminar hydrodynamic flow is encouraged. Micro bubbles are generated in a laminar flow mixing for oxygenation. Since the mammalian cells require low amount of oxygen supply of oxygen minimum aeration to maintain DOT values of about 20% saturation

Strong aseptic adaptations might be included with increasing the number of seals to prevent contamination. The sampling port should be equipped with direct steam sterilization to prevent contaminations during samplings







to be continued....