Molecular downstream processing

Because of the differences existing between the quality of different distillation cuts and those resulting from their downstream processing, it is useful to group them according to a major characteristic. That is, they are grouped into the three principal chemical families which constitute them paraffins, naphthenes and aromatics. From a molecular point of view, their chemical reactivities follow this order ... 

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. 

Diafiltration is a process whereby an ultrafiltration system is utilized to reduce or eliminate low molecular mass molecules from a solution and is sometimes employed as part of biopharmaceuti-cal downstream processing. In practice, this normally entails the removal of, for example, salts, ethanol and other solvents, buffer components, amino acids, peptides, added protein stabilizers or other molecules from a protein solution. Diafiltration is generally preceded by an ultrafiltration step to reduce process volumes initially. The actual diafiltration process is identical to that of ultrafiltration, except for the fact that the level of reservoir is maintained at a constant volume. This is achieved by the continual addition of solvent lacking the low molecular mass molecules that are to be removed. By recycling the concentrated material and adding sufficient fresh solvent to the system such that five times the original volume has emerged from the system as permeate, over 99... 

For more comprehensive validation studies, the molecular mass profile of the DNA spike should roughly approximate to the molecular mass range of endogenous contaminant DNA in the crude product. Obviously, the true DNA clearance rate attained by downstream processing procedures (e.g. gel filtration) will depend to some extent on the molecular mass characteristics of the contaminant DNA. 

The third major Hmitation of bioprocesses is the quite low product concentration compared with chemical processes, resulting in high downstream processing costs. This is mainly caused by product inhibition of cell growth and biosynthesis. Physiological improvements in cell growth and product formation only have a limited impact on this aspect. Chemical or directed mutagenesis may provide better chances for improvement. Unfortunately, the molecular mechanisms of product inhibition are not well understood. 

Molecular cloning is now a standard procedure to overproduce specific enzymes of use in biocatalysis in a host microorganism that is suited for the process. The host strain should fulfil the objectives of downstream processing, which are high recovery, high purity, reproducibility and low cost scale-up. 

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