Upstream fermentation

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... 

The complexity of biological processes generally requires many stages to produce a final, purified product from a particular composition of raw materials. Although a typical bioprocess consists of two main parts, upstream fermentation and downstream product recovery, it is not unusual to have between 10 and 20 steps in the overall process. This reflects the complex nature of a typical fermentation broth, which will consist of an aqueous mixture of cells, intracellular or extracellular products, unreacted substrates, and by-products of the fermentation process. From this mixture, the desired... 

In the development of new products, optimization of the fermentation medium for titer only often ignores the consequences of the medium properties on subsequent downstream processing steps such as filtration and chromatography. It is imperative, therefore, that there be effective communication and understanding between workers on the upstream and downstream phases of the produc t development if rational trade-offs are to be made to ensure overall optimahty of the process. One example is to make the conscious decision, in collaboration with those responsible for the downstream operations, whether to produce a protein in an unfolded form or in its native folded form the purification of the aggregated unfolded proteins is simpler than that of the native protein, but the refolding process itself to obtain the product in its final form may lack scalabihty. 

There is much current interest aimed at the implementation of processes that integrate the upstream and downstream operation for protein recovery.131419 Although adsorption in fluidised beds provides a considerable saving in cost and time over conventional purification techniques, it still deploys a discrete operation with which the desired protein is captured at termination of fermentation or once a cell suspension has been disrupted. The main... 

Biopharm production can be divided into upstream and downstream processing (Figure 5.5). Upstream processing refers to the initial fermentation process that results in the initial generation of product, i.e. the product biosynthesis phase. Downstream processing refers to the actual purification of the protein product and generation of finished product format (i.e. filling into its final product containers,... 

The upstream processing element of the manufacture of a batch of biopharmaceutical product begins with the removal of a single ampoule of the working cell bank. This vial is used to inoculate a small volume of sterile media, with subsequent incubation under appropriate conditions. This describes the growth of laboratory-scale starter cultures of the producer cell line. This starter culture is, in turn, used to inoculate a production-scale starter culture that is used to inoculate the production-scale bioreactor (Figure 5.7). The media composition and fermentation conditions required to... 

Upstream processing (fermentation) and downstream processing (purification and formulation) are physically separated, by being undertaken in separate buildings. Fermentation is generally undertaken in specially designed 42 000 1 stainless steel vessels. After recovery of the product from the cells, a number of chromatographic purification steps are undertaken, essentially within... 

Simultaneous saccharification and co-fermentation (SSCF) one-stage enzymatic hydrolysis of cellulose and fermentation of pentoses and hexoses all in one process step. The upstream hydrolysis of the hemicellulose takes place in a separate process step. 

Upstream processing The fermentation process that initially generates the product. 

When production volume is sufficient, it is economical to build one plant for one product. Batch production in a single unit may be limited by maximum reactor size. Holdups of greater than 20,000 gal are handled in separate parallel reactors. To use common upstream and downstream facilities, the reactors may not be operated simultaneously but on overlapping schedules. When long reaction times cannot be avoided, the reaction sections operate batch wise however, feeding reactants and recovering products may be continuous for economic reasons. This practice is typical of many processes, such as the saponification of natural fats in intermediate quantities. In the production of ethanol by fermentation, two reactions (saccharification and fermentation) are operated on a batch basis, while hydrolysis (conversion of starch to dextrin) and product recovery by distillation are continuous. 

The basis utilized by Petrides et al.15 is 1500 kg of purified BHI per year. They indicate that this represents 10-15% of the world demand.17 In essence, the following downstream steps are involved in sequence during synthetic BHI production. The fermentation step (not a downstream step) is also included to provide some continuity. The steps are fermentation, cell harvesting, cell disruption, inclusion body recovery, inclusion body solubilization, enzymatic conversion, refolding, sulfitolysis, CNBr cleavage, final purification steps, and crystallization. In the flow chart provided by Petrides et al.15, a surge tank separates the upstream from the downstream processes. This tank is in between the fermentor and the downstream processing steps. 

Decolorization and Finishing. Decolor-ization is sometimes required for certain applications, mostly as an aesthetic preference. It is always desirable to solve these issues upstream. For example, color can be minimized by choice of fermentation medium components and control of the sterilization cycle so as to lessen the Maillard reactions between nitrogen and sugars and carameliza-tion. Color can also be reduced by treatment with activated carbon, use of antioxidants, and by diafiltration with membranes. Carbon-impregnated filter pads can be used to combine polish filtration with a decolorization step. 

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