11.22 Fractionation of cell homogenate Recombinant insulin is produced using E.
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11.22 Fractionation of cell homogenate Recombinant insulin is produced using E. coli fermentation. The harvested cells are homogenised using a bead mill to release the protein inclusion bodies. The cell homogenate is then treated with CNBr to cleave the cell proteins into small fragments, leaving the proinsulin as a large, uncleaved molecule. The CNBr-treated homogenate contains several components, including 5% w/w cell debris, 8% w/w proinsulin, 5% w/w cleaved proteins, and 0.5% w/w sugar. The mixture is treated using membrane filtration to remove the smaller molecules and fragments. The membrane chosen for this application gives retention coefficients of 0.95 for the cell debris, 0.90 for proinsulin, 0.25 for cleaved protein, and 0 for sugar. The effect of fouling on solute retention is negligible. The cell debris is concentrated fivefold by this process. (a) What is the composition of the retentate? (b) What is the composition of the permeate?Explanation / Answer
a) The retentate is composed by 4.75 g/100g of cell debris, 7.2 g/100g of proinsulin, 1.25g/100g of cleaved protein and 0 g/100g of sugar. This is obtained from the retention coefficient. These coefficient tell us the percentage of a component that is retained. In the case of cell debris, the coefficient is of 0.95, which means that 95% of the debris is retained. We only need to perform a simple arithmetic equation to know how much was retained. 5% w/w means that, of 100g, 5g correspond to the component we are talking about. So we calculate what is the 95% of 5g and that is the concentracion that remains in the retentate. This procedure is repeated with the rest of the components.
b) We apply the same procedure here. In the case of the cell debris, only 5% passes trhough the membrane, so we calculate which is the 5% of 5g and we know the concentration of the permeate, which is 0.25g/100g. For the proinsulin, 0.8g/100g. For the cleaved protein, 3.75 g/100g. For the sugar, 100% is filtrated, so the concnetracion in the permeate is the same we had in the beginning: 0.5% w/w.
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