Since the mechanism governing the partitioning behavior of biomolecules, such as proteins and enzymes, in polyethylene glycol (PEG)-salt aqueous two-phase systems (ATPS) is complex and not easily predictable, many laborious experiments have to be performed for an optimization of these systems, causing increased overall cost. However, the multivariate statistical design of experiments (DoE) methodology is representing a promising and efficient optimization technique which can overcome the limitations of traditional optimization methods. Therefore, DoE has emerged as a powerful and efficient optimization tool for PEGsalt ATPS, since it is faster, more efficient and cost-effective, allowing a simultaneous and rigorous evaluation of process/system parameters. In the present review, different DoE process steps are represented to highlight the feasibility of this approach to operate as a promising and efficient optimization tool, thus facilitating the evaluation of the partitioning behavior, recovery and purification of different proteins and enzymes in PEG-salt ATPS. In this context, several experimental designs, such as factorial and response surface designs, have been discussed and evaluated by statistical regression analysis and analysis of variance (ANOVA), as well as various applications of PEG-salt ATPS using DoE have been outlined which may further promote the optimization of these systems.

Nowadays, there is an increasing demand to establish new feasible, efficient downstream processing (DSP) techniques in biotechnology and related fields. Although several conventional DSP technologies have been widely employed, they are usually expensive and time-consuming and often provide only low recovery yields. Hence, the DSP is one major bottleneck for the commercialization of biological products. In this context, polyethylene glycol (PEG)-salt aqueous two-phase systems (ATPS) represent a promising, efficient liquid-liquid extraction technology for the DSP of various biomolecules, such as proteins and enzymes. Furthermore, ATPS can overcome the limitations of traditional DSP techniques and have gained importance for applications in several fields of biotechnology due to versatile advantages over conventional DSP methods, such as biocompatibility, technical simplicity, and easy scale-up potential. In the present review, various practical applications of PEG-salt ATPS are presented to highlight their feasibility to operate as an attractive and versatile liquid-liquid extraction technology for the DSP of proteins and enzymes, thus facilitating the approach of new researchers to this technique. Thereby, single- and multi-stage extraction, several process integration methods, as well as large-scale extraction and purification of proteins regarding technical aspects, scale-up, recycling of process chemicals, and economic aspects are discussed.

Different poly(ethylene glycol) (PEG) + potassium phosphate or sodium citrate aqueous two-phase systems (ATPS) were investigated at 23 °C, containing different PEG types (molecular weights 2000 g•mol −1 to 8000 g•mol −1 ) and pH values (5 to 9). Furthermore, the effect of the added salt NaCl (0 wt % to 8 wt %) on the PEG + potassium phosphate/sodium citrate ATPS was studied at 23 °C. The experimental binodal data were successfully correlated with the empirical nonlinear equation proposed by Hu. The effects of increasing molecular weight of PEG, pH, NaCl, and salt type on the obtained binodal curves were determined, resulting in a binodal curve shift toward the origin. Thus, an expansion of the two-phase region occurred by increasing molecular weight of the PEG, pH, and NaCl and due to the Gibbs free energy of hydration of ions of phosphate. Furthermore, the phase equilibrium compositions, tie-line lengths, slopes of tie-lines, critical points, and effective excluded volumes were obtained for all studied systems. Finally, the experimental tie-line compositions were successfully correlated by using the Othmer−Tobias and Bancroft equations, and linear dependency was confirmed.

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