Biomolecule Separation Using Temperature-Induced Phase Separation with Recycling of Phase-Forming Polymers

We review our recent work on the thermoseparating polymers in aqueous two-phase extractions of biomolecules. Random copolymers of ethylene oxide and propylene oxide (EOPO copolymers) are thermoseparating in water solution, i.e., at temperatures above the cloud point a concentrated copolymer phase and a water phase almost free of copolymer are formed. This phase behavior makes it possible to recycle copolymers after biomolecule extraction. A system for protein purification has been developed based on an EOPO copolymer/hydroxypropyl starch aqueous two-phase system. The target protein is extracted into the EOPO phase. Nonionic surfactants have been added to improve the partitioning to the copolymer phase. The EOPO phase is removed after extraction, and the temperature is increased above the cloud point. The target protein is recovered in the water phase, and the copolymer and surfactant is obtained as a concentrated phase free from protein. Copolymer and surfactant can be recovered after thermoseparation to 85-90% and four recycle steps have been accomplished. Covalent binding of an affinity ligand to the EOPO copolymer can improve the partitioning of a target protein to the EOPO phase. The EOPO ligand can be recycled after thermal separation. A novel one-polymer aqueous two-phase system containing only thermoseparating EOPO copolymer and water has been developed for smaller biomolecules, e.g., peptides. Above the copolymer's cloud point, hydrophobic peptides will partition to the EOPO-enriched phase and hydrophilic peptides to the water phase. Proteins can be extracted in a one-polymer phase system containing a hydrophobically modified EOPO copolymer (HM-EOPO). The target protein is partitioned to the HM-EOPO phase. The copolymer can be recycled after back-extraction of the protein to a new water phase.