Downstream Processing

Successfully getting a host cell to take up a recombinant plasmid and express the gene of interest is a major milestone, but it is not, by itself, the finished product. The recombinant protein at this stage exists mixed together with thousands of the host cell's own native proteins, other cellular macromolecules, and cell debris, usually at a very low concentration, and it is often in a biologically inactive form. The set of further steps needed to convert this crude cellular material into a pure, stable, biologically active, and marketable product is collectively called downstream processing.

Downstream processing typically begins with separating the host cells (or, in cases where the protein is secreted, simply the cells) from the surrounding culture medium, followed by breaking open the cells if the desired protein has accumulated inside them rather than being secreted. The crude mixture is then subjected to a series of purification steps -- commonly including various forms of chromatography that separate proteins based on properties such as size, electrical charge, or specific binding affinity -- to isolate the target protein away from all the other cellular material, progressively increasing its purity at each step.

Purification alone is often not enough, because many recombinant proteins, especially complex ones, are not produced by the host cell in their final, biologically active three-dimensional shape. This is a particularly important issue for proteins produced in a bacterial host like E. coli, which lacks some of the more elaborate protein-folding and modification machinery found in animal or human cells; a protein may need to undergo additional processing, such as proper folding into its correct tertiary structure, or the formation of specific chemical bonds (such as the disulphide bonds needed to convert the two separately produced insulin chains into functional insulin), before it becomes biologically active. …