Reviewing Current Approaches for Optimizing Separations in Aqueous Two-Phase Systems

Seth Kriz and Caryn Heldt, researchers from Michigan Technological University in Houghton, Michigan, recently published a review in the Journal of Chromatography A that focused on how biomolecule separations could be optimized when using aqueous two-phase systems (ATPS) (1).

Houghton and It's Lift Bridge and located in the Upper Peninsula of Michigan | Image Credit: © Jacob - stock.adobe.com

Aqueous two-phase systems (ATPS) are a type of liquid-liquid fractionation technique that have gained interest for its use in the extraction, separation, purification, and enrichment of proteins and other biomolecules (2). ATPS are formed by two solutions, the former formed by dissolving a polymer in water above its critical concentration and the latter by either dissolving a second polymer or an inorganic salt (3). ATPS created two semi-miscible aqueous phases, which resulted in a low-shear environment friendly to biomolecules. Different phase forming components create unique environments that selectively partition biomolecules, creating a controllable separation process. Using polymers and salts that are generally considered safe and environmentally friendly makes ATPS an economical and sustainable alternative to chromatography, according to the scientists.

As biomanufacturing attempts to adopt continuous processing, attention has been shifted to ATPS for its ability to fully operate continuously while incurring lower costs than many chromatographic methods. However, while chromatography exhibits high specificity and is moving toward continuous loading, elution still occurs discontinuously in many instances. Moreover, despite 60 years of exploration and development in these fields, robust development of ATPS process understanding has been prevented, mainly due to the separations being controlled by complex networks of interlinked driving forces behind ATPS. Additionally, most ATPS separations continue to be optimized using slow and costly manual screening methods.

There is a growing body of literature centered around developing statistical and mechanistic models of ATPS to predict liquid-liquid equilibria and separations with less experimental burden. In this review, these models’ application to ATPS were surveyed, comparing their progress and potential to promote rapid short-term and long-term bioseparation development. The approaches were explored in terms of their mechanisms, advantages, and challenges, and how they can each optimize ATPS. From there, they were categorized into experimental screening approaches, statistical models, and mechanistic models. Their relative strengths and weaknesses were discussed, followed by strategies for integrating the models into flowsheets. This allows for systematic identification of optimal extraction conditions based on key criteria such as purity, yield, activity, and cost. Finally, the researchers proposed future directions that can help enable ATPS to become industrially relevant.

The discussion evaluated how statistical tools, such as response surface methodology and artificial neural networks, can be adapted to ATPS technology. This was contrasted with the process understanding generated through applying semi-empirical thermodynamic models. Strategies were also explored to automate separation optimization for new biomolecules using these models, which would allow for the creation of artificial data. Overall, the scientists hoped to expand our understanding of the landscape of models applied to ATPS, potentially starting discussions on how to bring ATPS technology closer to commercialization and enabling continuous processing on a broader scale.

References

(1) Kriz, S. A.; Heldt, C. L. Current Experimental, Statistical, and Mechanistic Approaches to Optimizing Biomolecule Separations in Aqueous Two-Phase Systems. J. Chromatogr. A 2025, 1749, 465881. DOI: 10.1016/j.chroma.2025.465881

(2) Iqbal, M.; Tao, Y.; Xie, S.; et al. Aqueous Two-Phase System (ATPS): An Overview and Advances in its Applications. Biol. Proced. Online 2016, 18, 18. DOI: 10.1186/s12575-016-0048-8

(3) Aqueous Two Phase. ScienceDirect 2020. https://www.sciencedirect.com/topics/engineering/aqueous-two-phase (accessed 2025-4-23)