Cell culture process synthesis

Wilson, S.A. and Roberts, S.C. (2012) Recent advances towards development and commercialization of plant cell culture processes for the synthesis of biomolecules. Plant Biotechnol. J., 10, 249 - 268. 

The discovery of Zanamivir as a potent and selective inhibitor of influenza virus sialidase prompted several researchers to investigate the synthesis and structure-activity relationship studies of Neu5Ac2en-based compounds as potential sialidase inhibitors. Exploration of these SAR studies were undertaken to optimize inhibitory activity and to improve the physicochemical properties of the sialic acid-based influenza virus sialidase inhibitor. A few in vitro assays are commonly employed to measure the effectiveness of influenza virus sialidase inhibitors. The first involves a fluorometric assay that measures release of a synthetic fluorophore following its cleavage from Neu5Ac by sialidase. Dye-uptake assay, such as the Neutral Red uptake assay, measures the uptake of a vital stain, Neutral Red in cell culture. The process requires intact membranes and active metabolism in the cell, and is expressed as percent protective rate against virus infection. The plaque-reduction assay is used to measure sialidase inhibition indirectly in cell culture, and provides some measure of the inhibitor s effect on the viability of the influenza virus. In vitro and in vivo systems for analysis of inhibitors of influenza virus enzymes have been reviewed.71... 

Before implantation several in vitro tests were performed. For evaluation of a possible toxic reaction, we investigated the material and the whole devices in vitro with cell culture methods. Direct contact and extraction tests with a mouse fibroblasts cell line (L 929) and a neuroblastoma cell line (neuro-2-a) were performed according to the international standard ISO 10993 ( Biological Evaluation of Medical Devices ). The materials and devices showed no toxicity, i.e. no significant differences in membrane integrity of the cell membranes, mitochondrial activity and DNA synthesis rate. The neuro-2-a cell line is so sensitive that even small changes in process technology are detectable. The flexible polyimide structures proved to be non toxic. 

The quantitative extent of CYP induction depends on the dosage (concentration) of the inducer and on the duration of exposure. However, the induction process, in contrast to inhibition, is not as straightforward to study in vitro, since induction requires intact cellular protein synthesis mechanisms as available in cell culture models (57-62). 

Nonstatic cultures of animal cells are common with agitation and aeration performed as associated operations. These processes, allied to the fact that animal cells do not possess a cell wall, can result in cell damage if they are intense, provoking effects that alter cell metabolism, cell cycle, DNA synthesis, and protein expression, and induce cell death by apoptosis or necrosis. 

Microbial polysaccharides have been shown to stress plant cells, resulting in elicitation (induction) and increased metabolite synthesis. Induction of various enzymes has been reported. Chitosan successfully elicited chitinase production in carrot (Daucus carota) cell cultures and elicitation of desired food ingredients and processing aids via chitosan has been attempted. 

This book is based predominantly on the patent literature and provides how to data regarding the production, purification, and application of commercial enzymes. Coverage is not limited to food applications, and 70 subjects are grouped as Enzymes, Enzymatic Processing, Enzyme Stabilization, Polymer-Enzyme Products, Cell Culture, Protein Analysis, Nucleic Acids etc.. Amino Acids, Peptide Synthesis, and Applications. Indexing includes U.S. patent number, company and patent assignee, inventor, and subject. 

The ionophores and several other specialty products are included in Table 13.8 for comparison purposes. Products of mammalian cell culture such as plasminogen activator and erythropoietin are included as fermentation products in this listing because they are normally manufactured by cellular processes in bioreactors. Aside from the five commodity chemicals in this table, the most dramatic change in the commercial chemicals produced by fermentation results from the impact of genetic engineering and recombinant DNA methods on the specialty products. Antibiotics and biopolymers (hormones, enzymes, etc.) with molecular structures too complex for conventional chemical synthesis will continue to be manufactured by microbial processes (Hinman, 1993). 

The production of flavour substances by cell or tissue cultures is still a dream for the future in most cases. Today the extraction of product from intact living plants is still less expensive than the production by isolated cells and tissues. On the other hand, it is very attractive to make use of the secondary metabolism of plant cells for the synthesis of natural flavours in a controlled way to avoid contaminating by-products and thus considerably simplify downstream processing. Further advantages of such cell culture systems would be the independence from agriculture combined with the risk for possible shortage and variances in product quality, the ability to scale-up the process to create an inexhaustible source of well-defined product. 

Efforts to get access to improved manufacturing processes or improved application properties of paclitaxel resulted in two different pathways for the total synthesis of the natural product, as well as analogs. Furthermore, paclitaxel and/or precursors can be obtained under optimized conditions from cell cultures of Taxus media generated by hybridizing Taxus baccata and Taxus cuspidata in overall yields of about 130 mg 11 within two weeks [61]. Paclitaxel can also be obtained by the culture of appropriate microbial strains isolated from paclitaxel producing yew trees [62 - 64]. 

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