Efficient downstream process

When sufficiently high levels of expression and protein accumulation are achieved, efficient downstream processing protocols must be developed to insure product quality and the economic feasibility of production. As the demand for safe, recombinant pharmaceutical proteins continues to expand, the market potential of plant-produced recombinant proteins is considerable. Molecular farming can produce recombinant proteins at a lower cost than traditional expression systems based on microbial or animal cell culture, and without the risk of contamination with human pathogens. 

Role of Statistically Designed Experiments in the Development of Efficient Downstream Processes... 

Formation of IBs during protein expression poses a bottleneck in the efficient downstream processing of therapeutic proteins. The reasons for IB formation are not fully known. Because translation is a slower process than protein folding, it is likely that the misfolding of translation intermediates plays some role. Further, since post-translational modifications, such as glycosylation and lyposylation, which are known to affect the secondary structure of proteins, are absent in bacteria, the non-modified protein structure may cause misfolding. The recovery of soluble... 

Efficient downstream processing of products is facilitated when the product can be recovered from the medium. Besides by elicitation and immobilization, excretion of the product can be obtained by permeabilization of the cells. Various strategies have been used chemical permeabilization, electrical permeabilization, ultrasonic permeabilization, and ion-tophoretic permeabilization 111). Chemical permeabilization comprises the use of, among others, organic solvents as DMSO and chloroform and surface active chemicals (as Triton X-100). Results with the various tech-... 

The hberation of a valuable constituent does not necessarily translate direclly into recoveiy in downstream processes. For example, flotation tends to be more efficient in intermediate sizes than at coarse or fine sizes [Mclvor and Finch, Minerals Engineeiing, 4(1), 9-23 (1991)]. For coarser sizes, failure to liberate may be the hmitation finer sizes that are liberated may still be carried through by the water flow. A conclusion is that overgrinding should be avoided by judicious use of size classifiers with recycle grinding. 

To meet sales specifications, gas produced at the wellheads must be free of water and hydrocarbon liquids. Twin turboexpanders are a key component in this process, providing dewpoint control with optimal efficiency. Initial processing takes place at the wellhead platforms, where methanol is injected to inhibit hydrate formation. A corrosion inhibitor is also added to prevent gas from damaging downstream equipment. 

Numerous biocatalytic routes to this challenging intermediate have been reported. " For example. Fox et al. have recently developed an efficient regioselective cyanation starting from low-cost epichlorohydrin (Scheme 1.26). Initial experiments found that halohydrin dehydrogenase from Agrobacterium radiobacter expressed in E. coli produced the desired product, but inefficiently. To meet the projected cost requirements for economic viability, the product needed to be produced at 100 g L with complete conversion and a 4000-fold increase in volumetric productivity. The biocatalyst needed to function under neutral conditions to avoid by-product formation, which causes downstream processing issues. 

The use of both hydrophilic [85, 87] and hydrophobic [84, 86] membranes has proven to be efficient in binding the enzyme. The main advantage of this system over emulsion systems lies in the ease of the downstream processing, as no enzyme-stabilized emulsion has to be broken. 

This paper will focus on the use of statistically designed experiments to develop effective purification processes in the most time and cost efficient fashion. Downstream processing and the recovery of proteins by severd different techniques have been discussed in other articles (1-3) and will not be discussed here. 

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