Cell downstream process

In addition to protein impurities emanating directly from the source material, other proteins may be introduced during upstream or downstream processing. For example, animal cell culture media are typically supplemented with bovine serum/foetal calf serum (2-25 per cent), or with a defined cocktail of various regulatory proteins required to maintain and stimulate growth of these cells. Downstream processing of intracellular microbial proteins often requires the addition of... 

Traditionally, the upstream fermentation and cell culture processes have been viewed as being distinct from the subsequent downstream processing and purification steps, and the two different sets of processes nave been optimized individually. In some instances, careful consideration of the conditions used in the fermentation process, or manipulation of the genetic makeup of the host, can simplify and even... 

Biocatalysts in nature tend to be optimized to perform best in aqueous environments, at neutral pH, temperatures below 40 °C, and at low osmotic pressure. These conditions are sometimes in conflict with the need of the chemist or process engineer to optimize a reaction with respect to space-time yield or high product concentration in order to facilitate downstream processing. Furthermore, enzymes and whole cells are often inhibited by products or substrates. This might be overcome by the use of continuously operated stirred tank reactors, fed-batch reactors, or reactors with in situ product removal [14, 15]. The addition of organic solvents to increase the solubility of substrates and/or products is a common practice [16]. 

Product extraction Effluent and waste disposal Medium preparation Seed vessel Purification Cell free supernatant Cell biomass Production bioreactor Downstream processing Medium sterilisation Primary culture Upstream processing... 

The culture can be used directly for the conversion of phenylpyruvic add to resting cells L-phenylalanine. Therefore, a batch process with resting cells can be carried out, with some glucose added for maintenance (fed-batch fermentation). Another approach is to harvest the cells from the fermentation broth and to use them in a separate bioreactor in higher concentrations than the ones obtained in the cell cultivation. An advantage of the last method can be that the concentration of compounds other than L-phenylalanine is lower, so that downstream processing may be cheaper. 

Costs of downstream processing for bioprocesses are increased by 1) low concentrations of products, 2) numerous impurities at low concentration and 3) intracellular materials (if cell disruption is necessary). However, the high specificity of biocatalysts is a benefit to downstream processing since products closely related to the desired product are less likely to be present Waste products of bioprocesses are likely to be less environmentally damaging, which also reduces downstream processing costs. 

When we need to remove cells from fermentation broth, sedimentation is considered as a downstream processing method. Alum, lime, polyelectrolyte are commonly used. 

After fermentation, subsequent midstream to downstream processes such as cell disruption, centrifugation, extraction and drying will be carried on for product recovery. Fig. 9 shows a white sheet of PHB obtained from fermentation of sweet sorghum juice (SSJ) by Bacillus aryahhattai. 

The susceptibility of biological systems, including procaryotic and eucaryo-tic cultures and enzyme solutions, to the forces prevailing under normal processing conditions has been extensively studied and is the subject of comprehensive reviews [12-16], including other chapters in this volume. Downstream processing operations, as well as routine pumping, will expose cell suspensions... 

At harvest, the benzylpenicilhn is in solution extracellularly, together with a range of other metabolites and medium constituents. The first step in downstream processing is to remove the cells by filtration or centrifugation. This stage is carried out under conditions that avoid contamination with (3-lactamase-producing microorganisms which could lead to serious or total loss of product. 

Downstream Processing Microfiltration plays a significant role in downstream processing of fermentation products in the pharmaceutical and bioprocessing industry. Examples are clarification of fermentation broths, sterile filtration, cell recycle in continuous fermentation, harvesting mammahan cells, cell washing, mycelia recovery, lysate recovery, enzyme purification, vaccines, and so forth. 

Grandics, R, Szathmary, S., Szathmary, Z., and O Neill, T., Integration of cell culture with continuous, on-line sterile downstream processing, Ann. N. Y. Acad. Sci., 646, 322, 1991. 

Whole-cell biotransformations frequently showed insufficient stereoselectivities and/or undesired side reactions because of competing enzymatic activities present in the cells. These side reactions can modify the substrates and/or products. Furthermore, whole-cell biotransformations are limited due to the intrinsic need to grow biomass, which generates its own metabolites that are not related to the biotransformation reactions and, therefore, which need to be removed during the downstream process. Both the cells themselves and the unrelated metabolites produced are impurities that need to be removed after the biotransformation reaction. With isolated enzymes, there are no organism and unrelated metabolites to remove after the biotransformation processes. 

As summarized above, various approaches were taken to alter the PHA production or to facilitate downstream processing by employing recombinant pseudomonads. However, as in the case with recombinant R. eutropha, no study has been carried out on the high cell density culture of recombinant pseudomonads or on the scale-up of fermentation. 

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