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Within today's downstream processing of therapeutic proteins, there is an imminent need for alternatives to chromatographic separations, such as aqueous two-phase extraction. Challenges in the development and performance of aqueous two-phase extraction include a number of experiments to define appropriate process windows as well as enable multistage extraction. To reduce experimental effort, a hybrid thermodynamic model is presented in this contribution which allows the extraction system to be optimized with a smaller number of experiments. Moreover, multistage applications of aqueous two-phase extraction are presented to purify enzymes. To purify the investigated enzymes, two different apparatuses were used: namely, a mixer–settler unit and a centrifugal partition chromatograph.

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