Cells fermenting

PVDF-based microporous filters are in use at wineries, dairies, and electrocoating plants, as well as in water purification, biochemistry, and medical devices. Recently developed nanoselective filtration using PVDF membranes is 10 times more effective than conventional ultrafiltration (UF) for removing vimses from protein products of human or animal cell fermentations (218). PVDF protein-sequencing membranes are suitable for electroblotting procedures in protein research, or for analyzing the phosphoamino content in proteins under acidic and basic conditions or in solvents (219). 

We can also use microbial cells (fermentation) containing the desired catalytic activity without isolating the enzymes responsible. 

Mass transfer considerations are critical in any bioprocess. In typical, aerobic, suspended cell fermentations, the major concern is the oxygen transfer rate, determined by the overall mass transfer coefficient, kft, and the driving force. In three-phase biofluidization, in which the cells are immobilized as a biofilm or within carrier particles, the situation is further complicated by possible intraparticle diffusion limitations. Numerous recent studies have addressed these issues. 

The above definition of classical biotechnological processes can easily be adapted to the concept of molecular farming. With plant cell fermentation the analogy is ob-... 

Figure 5.9 Design of a generalized microbial cell fermentation vessel (a) and an animal cell bioreactor (b). Animal cell bioreactors display several structural differences compared with microbial fermentation vessels. Note in particular (i) the use of a marine-type impeller (some animal cell bioreactors-air lift fermenters-are devoid of impellers and use sparging of air-gas as the only means of media agitation) (ii) the absence of baffles (iii) curved internal surfaces at the bioreactor base. These modifications aim to minimize damage to the fragile animal cells during culture. Note that various additional bioreactor configurations are also commercially available. Reprinted with permission from Proteins Biochemistry and Biotechnology (2002), J. Wiley Sons...

Mammalian cell culture is more technically complex and more expensive than microbial cell fermentation. Therefore, it is usually only used in the manufacture of therapeutic proteins that show extensive and essential post-translational modifications. In practice, this usually refers to glycosylation, and the use of animal cell culture would be appropriate where the carbohydrate content and pattern are essential to the protein s biological activity, its stability or serum half-life. Therapeutic proteins falling into this category include EPO (Chapter 10), the gonadotrophins (Chapter 11), some cytokines (Chapters 8-10) and intact monoclonal antibodies (Chapter 13). 

After initial cell fermentation and product extraction from the producer cells, the crude preparation is subject to multiple chromatographic steps, including ion-exchange, hydrophobic interaction chromatography and gel-filtration chromatography. The purified product is presented in lyophilized form in vials (1 mg active/vial) and excipients include a phosphate buffer, sodium chloride and serum albumin. 

A brilliant example for the industrial-scale application of plant cell fermentation is the new process for the production of the anticancer drug paclitaxel developed by Bristol-Myers Squibb (see Figure 15.1). It starts with clusters of paclitaxel producing cells from the needles of the Chinese yew, T. chinensis, and was introduced in 2002. The API is isolated from the fermentation broth and is purified by chromatography and crystallization. The new process substitutes the previously used semisynthetic route. It started with lO-deacetylbaccatin(III), a compound that contains most of the structural complexity of paclitaxel and can be extracted from leaves and twigs of the European yew, T. baccata. The chemical process to convert 10-deacetylbaccatin(III) to paclitaxel is complex. It includes 11 synthetic steps and has a modest yield. 

Figure 15.1 Plant cell fermentation process for paclitaxel. Source Bristol-Myers Squibb.

Ariff and Webb studied production of glucoamylase using freely suspended cells of Aspergillus awamori in batch and continuous fermentations. Glucoamylase yields based on glucose consumed were 900 and 1080 U/g for batch and continuous fermentations, respectively. The immobilization of viable cells was achieved by adsorption to cubes of reticulated polyurethane foam. In comp uison with freely suspended cell fermentations, neither batch nor continuous fermentations of immobilized cells improved glucoamylase production significantly in tenns of yield or productivity. 

The Taxol Story-Development of a Green Synthesis via Plant Cell Fermentation ... 

Figure 7.7 Plant cell fermentation and extraction process.

In the cell fermentation stage of the process, calluses are propagated in a wholly aqueous medium in large fermenters under controlled conditions at ambient temperature and pressure. The feedstock for cell growth consists of renewable nutrients, sugars, amino acids, vitamins, and trace elements. 

Ritter, S.K. (2004) Chem. Eng. News, 82 (27), 4. Also Development of Green Synthesis for Taxol Manufacture via Plant Cell Fermentation and Extraction submitted for 2004 EPA Greener Synthetic Chemistry award, http //www. epa.gov/greenchemistry/pubs/pgcc/ winners/gspa04.html (accessed May 2009). 

Mountford, P.G. (2006) Development of a Green Synthesis for Taxol Manufac-mre via Plant Cell Fermentation and Extraction. Hosted by Colegio de Quimicos de Puerto Rico, Weshn RioMar, PR, Aug. 15-18. 

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