Downstream processing intracellular products

Downstream processing Intracellular products have to be extracted from the cells (by sonication, freeze thawing, and/or homogenization), and separated from the cells (centrifugation or filtration). Extracellular products require concentration and separation from the bulk supernatant. 

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. 

The vast bulk of proteins synthesized naturally by E. coli (i.e. its homologous proteins) are intracellular. Few are exported to the periplasmic space or released as true extracellular proteins. Heterologous proteins expressed in E. coli thus invariably accumulate in the cell cytoplasm. Intracellular protein production complicates downstream processing (relative to extracellular production) as ... 

Figure 8.7 Overview of the manufacture of Betaferon, a recombinant human IFN-(3 produced in E. coli. The product differs from native human IFN-(3 in that it is unglycosylated and cysteine residue 17 had been replaced by a serine residue. E. coli fermentation is achieved using minimal salts/glucose media and product accumulates intracellularly in inclusion body (IB) form. During downstream processing, the lbs are solubilized in butanol, with subseguent removal of this denaturant to facilitate product refolding. After two consecutive gel-filtration steps, excipients are added, the product is filled into glass vials and freeze-dried. It exhibits a shelf life of 18 months when stored at 2-8 °C...

The separation schemes vary with the state of the products. For example, intracellular products must first be released by disrupting the cells, while those products bound to cell membranes must be solubilized. As the concentrations of products secreted into the fermentation media are generally very low, the recovery and concentration of such products from dilute media represent the most important steps in downstream processing. In this chapter, several cell-liquid separation methods and cell disruption techniques are discussed. 

The fermentation products can be the cells themselves (biomass), components within the fermentation broth (extracellular), or those trapped in cells (intracellular), examples of which are listed in Table 10.1. As shown in Figure 10.1, if the product of our interest is the cell, cells are separated from the fermentation broth and then washed and dried. In the case of extracellular products, after the cells are separated, products in the dilute aqueous medium need to be recovered and purified. The intracellular products can be released by rupturing the cells and then they can be recovered and purified. The downstream processing for enzyme reactions will be similar to the process for extracellular products. 

Huddleston, J. G., and Lyddiatt, A. (1996). Two-phase aqueous systems for the recovery of intracellular microbial products. In Downstream Processing of Natural Products (M. S. Verrall, ed.), pp. 53-69. Wiley, New York. 

Where the product must be captured from the intracellular environment, the first downstream processing steps are cell disruption and the removal of debris. Several methods have been used for disruption, including sonication, pressure homogenization, enzymatic treatment, and wet... 

In very basic terms, whether for intracellular or extracellular products, downstream processing usually involves a clarification step followed either by adsorption onto a resin, or solvent extraction followed by concentration. In... 

---