Integration of aqueous (micellar) two-phase systems on the proteins separation

A two-step approach combining an aqueous two-phase system (ATPS) and an aqueous micellar two-phase system (AMTPS), both based on the thermo-responsive copolymer Pluronic L-35, is here proposed for the purification of proteins and tested on the sequential separation of three model proteins, cytochrome c, ovalbumin and azocasein. Phase diagrams were established for the ATPS, as well as co-existence curves for the AMTPS. Then, by scanning and choosing the most promising systems, the separation of the three model proteins was performed. The aqueous systems based on Pluronic L-35 and potassium phosphate buffer (pH?=?6.6) proved to be the most selective platform to separate the proteins (S_Azo/Cyt?=?1667; S_Ova/Cyt?=?5.33 e S_Azo/Ova?=?1676). The consecutive fractionation of these proteins as well as their isolation from the aqueous phases was proposed, envisaging the industrial application of this downstream strategy. The environmental impact of this downstream process was studied, considering the carbon footprint as the final output. The main contribution to the total carbon footprint comes from the ultrafiltration (~?49%) and the acid precipitation (~?33%) due to the energy consumption in the centrifugation. The ATPS step contributes to ~?17% while the AMTPS only accounts for 0.30% of the total carbon footprint.