Aqueous Two-Phase System (ATPS) Technique

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Aqueous two-phase system (ATPS), also known as all-aqueous system, is formed by phase separation of an aqueous mixture containing two incompatible polymers, or one polymer and other salts above critical concentrations. It shows great potential on the extraction, recovery and purification of various types of bioproducts, particularly proteins. The advantages of this technique over other conventional purification methods are the simply setting process, high purification efficiency, capacity of continuous operation and high customizability.

How Does It Work?

Phase diagram of ATPS and images of polymer mixtures under different states Fig. 1 (a) Phase diagram of ATPS: The part below the binodal curve is the single-phase area, and the upper part is the two-phase area. (b) Images of polymer mixtures under different states: (b1) Supercritical (two phases), (b2) critical, and (b3) subcritical (one phase). (Chemical Society Reviews, 2020, 49(1): 114-142)

In ATPS, the aqueous solution containing some hydrophilic high-molecular polymers can form two phases at an appropriate concentration. According to the second law of thermodynamics, when there is mutual repulsion between two high molecular polymers, the repulsive effect between molecules with larger relative molecular weight will be dominant compared with the mixing entropy, thus, two phases are formed when equilibrium is reached. At lower concentrations, the aqueous mixture forms a single phase, while at higher concentrations, the mixture spontaneously forms two immiscible phases, as shown in the Fig. 1.

Fig. 2 Schematic illustration of purification of target bioproduct based on ATPS. (Journal of bioscience and bioengineering, 2018, 126(3): 273-281)

The ability of ATPS on the purification of biomolecules was not applied until 1950s. As regards to the purification of PEGylated proteins, the interaction of protein with polymers like hydrophobic ligand can easily concentration protein in the polymer phase. However, the case varies to the PEGylated protein as the hydrophilia has increased after the chemical modification of PEG on protein. It is more likely to be dissolve in traditional aqueous solutions, which raises the problems of separation. The addition of salt to decrease the solubility of PEGylated protein and the choose of higher molecular weight PEG to have better miscibility with modified protein can solve this issue. More factors like pH, temperature, salt concentration, separation time and amount of protein can all be manipulated to promote the separation and purification of PEGylated protein.

Different Types of APTS

Polymer-polymer system

Polymer-polymer systems are preferably used for the separation, recovery and purification solutes sensitive to the ionic environment as these systems pose low ionic strength. In this system, equilibrium constant (Kp) value will be higher if the ratio of grafted PEG chains in the conjugate is higher. Most widely used polymers are PEG and polymers such as dextran, starch, polyvinyl alcohol, etc.

Polymer-salt system

In addition, the mixed solution of polymer and inorganic salt can also form aqueous two-phase, for example, PEG/potassium phosphate, PEG/ammonium phosphate, PEG/sodium sulfate, etc. The upper phase of PEG/inorganic salt system is rich in PEG, and the lower phase is rich in inorganic salt. For the separation of PEGylated conjugates from unreacted species, it can be achieved with a single PEG chain bound to a protein.

Thermo-separating polymer system

Thermo-separating polymer system is consisted of ethylene oxide (EO)-co-propylene oxide (PO) copolymers (EOPO) with dextran or salts. By heating up the aqueous solution above the lower critical solution temperature (LCST), the linear and non-ionic EOPO random copolymers will be thermo-separated into two phases from aqueous solution.

Alcohol-salt system

This kind of ATPS is formed by mixing two immiscible phases which are composed of alcohol and salt solutions. By evaporating the alcohol, the target proteins or other analytes can be easily extracted and recovered. The alcohol-salt ATPS has less toxicity impact to the environment and is cheaper compared to the conventional ATPSs.

What Can ATPS Be Used For?

Proteins & PEGylated proteins
Enzymes
Monoclonal Antibodies (mAbs)
DNA and nucleic acids

Virus & virus like particles
Cells and organelles
Low molecular weight biomolecules
And many more

Strengths & Weaknesses of ATPS

Strengths and Weaknesses of ATPS

References

Phong W N, Show P L, et al. Recovery of biotechnological products using aqueous two phase systems. Journal of bioscience and bioengineering, 2018, 126(3): 273-281.
Chao Y, Shum H C. Emerging aqueous two-phase systems: from fundamentals of interfaces to biomedical applications. Chemical Society Reviews, 2020, 49(1): 114-142.

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