For antibody purification using partitioning see “antibody purification”

Many thermoseparating polymers contain ethylene oxide groups. PEG is a thermospearating polymer but its cloud point is too high (above 100C) for use in separating bioaterials. Ethylene oxide (EO-propylene oxide (PO) random copolymers have lower cloud points and have been used in two stage separation system for protein purification. In the first stage, the target protein is partitioned to the EOPO rich phase. In the seocnd stage, the EOPO rich phase is thermoseparated. A new two phase system is formed with a EOPO-rich bottom phase and a water rich top phase. In this system, proteins are partitioned to the water phase and the copolyer can be recycled. (Johansson, “Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification” Biotechnology and bioengineering, 1999, 66(4), 247-257). 

Examples of polymers which display thermoseparation in water are poly(vinylcaprolactam), poly(N–isopropylacrylamide), EHEC (ethyl-hydroxyethyl-cellulose) and random or block copolymers of ehtylene oxide (EO) and propylene oxide (PO). The random copolymers of EO and PO segments EOPO polyers) are seaprated as a liquid phase pon temperature increase. Most studies of biomolecule partitioning in thermoseparated systems have been done in EOPO-polymer containing systems. In these systems the concentraiton of EOPO-polymer in the bottom phase is usually 40-60 %, while the top phase contains almost 100% water. Thus these systems are called water/EOPO systems. (Hans-Olof Johansson, Bioseparation 7: 259-267, 1999).

EOPO/Salts –

A thermoseparating random copolymer (Ucon 50-HB-5100) composed of  50% ethylene oxide and 50% propylene oxide has been used to form an aqueous two-phase system by heating the polymer water solution above the cloud point of the copolymer. In the formed two phase system a water rich top phase is in equilibrium with an aqueous polymer rich bottom phase. Hydrophobic peptides (containing aromatic amino acids) were strongly partitioned to the polymer rich phase, while hydrophilic peptides were enriched in the water rich phase. A decreased partitioning to the polymer rich phase was obtained upon temperature increase. With the salt NaCLO4, the polypeptide was partitioned to the polymer rich phase while with Na2SO4 the polypeptide was partitioned to the water rich phase. (Hans-Olof Johansson, Biochimica et Biophysica Acta 1335 (1997), 315-325.

Hallgren (US2008/0293926) disclose a method of separating a target from a liquid by providing a polymer in an aqueous liquid such as water which the polymer comprises at least one hydrophobic portion, contacting the polymer-containing liquid with the liquid comprising the target, applying a stimulus such as temperature or pH change to the resulting mixture and maintaining it until a reversible phase separation is obtained. One phase is polymer rich and contains target(s) and another phase is polymer poor. In an advantageous embodiment, the polymer present has a predominating hydrophobic character but also comprises one or more hydrophilic porition. Useful polymers include a block copolymer cased on ethylene oxide and proylene oxide. In one embodiment, Hallgren discloses isolating an antibody from an unclarified CHO brother by combining in a containiner the broth with ethylene oxide propylene oxide (EOPO) copolymer and at least one added salt selected from NaCl, Na2PO4, KPO4, NaSO4, potassium citrate, (NH4) SO4, sodium citrate, sodium acetate and ammonium acetate in a concentraiton of 100-300 mM and gently mixing the liuqid under conditions where the polymer is above its cloud point so that it forms a one thermoresponsive polymer, two phase system wehre the antibody partitiontions into the phase not enriched in the thermoresponsive polymer while nontarget compounds paritition to varying degrees to the phase interface or the thermoresponsive polymer enriched phase. 

EOPO/Dextran- WO2004/020629 discloss EOPO polyers to further facilitate the separation of plasmids which have already partitioned in a two polymer phase system. At room temperature the two-polymer, two phase system formed with EOPO and dextran polyers forms in same manner as PEG and dextran system. The less dense EOPO enriched upper phase is isolated from the EOPO and dextran polymer aqueous two phase system. The temperature of the EOPO enriched phase is then raised so that the upper phase undergoes further phase separation into a water enriched phase and a self associated EOPO polymer enriched phase. Advantageously, the water enriched phase should contain the desired target. 

Van Alstine (WO 2010/080062) disclose enriching one target protein from a liquid by differentially partitioning between two aqeuous phases. The phases are formed by adding a single type of responsive, self associating hydrophilic polymer and optionally use additional salts to an aqeuous biological solution (such as a fermentation sample) under thermal conditions where the solution separates into a one polymer, two phase system with one phase enriched in the polymer. The polymers used are aqueous in the sense that they form aqueous phases when combined with water. In one embodiment, the hydrophilic poly(ether) is a syntehtic polyer comprising ethylene oxide units such as poley(ethylene)glycol (PEG), ethylene oxide propylene oxide (EOPO) in either random copolymer form (e.g., Breox® or UCON polymers) or block polymers (e.g., Pluronic® polymers) ethoxy-containing polysaccharides and isopropylacrylamide modified polyers. EOPO for example separates into two phases when above its cloud temperature (Tc) and is consequently regarded a thermoseparating polymer. 

Tjerneld (US6,454,950) disclose a method for partitioning a compound in a two phase system in which one of the phases is rich and the other poor in a  thermoseparating polymer, collecting one of the phases and if desired further working up said compound from the collected phase. In one embodiment the polymer is HM-EOPO and in the other Breox copolymer (random copolymer with equal amount of ethylene oxide and propylene oxide. Partitioning of various proteins are illustration including BSA, lysozyme and apolipoprotein A-1. The technique of back extraction is also illustration from the HM_EOPO polymer phase to the water phase (first protein is partitioned in a 4% HM-EOPO polymer water system wehre the apolipoportein A1 is partitioned to the HM-EOPO polymer, the top water phase removed and exchange for buffer and the system mixed to allow for separation again (protein now back extracted into the new water phase). 

—EOPO (UCON) – Dextran – triethylene glycol-diglutaric acid (TEG-COOH): Ferreira (J. Chromatography A, 1195 (2008) 94-100 disclose using a thermosenstitive polymer of UCON (ethylene oxide/propylene oxide 50:50) with Dextran and TEG-COOH for the purificaiton of IgG from a CHO cell supernatant. Without TEG-COOH, IgG preferentally partitioned to the less hydrophobic dextran-rich phase (the recvoery yield of IgG in the top UCON-rich pahse was lower than 50%).  However, with the adding a free ligand containing carboxylic functionalities (TEG-COOH), a significant increase in the partitione of IgG from the bottom phase to the top phase was observed. 

–Poly(acrylic acid) (PAA) + EOPO + Salt: Hjorth (WO2008/156409 and US2010/0174052) dicloses partitioning in a multiphase system with a  1) first polymer which is synthetic poly(acid) such as poly(acrylic acid) (PAA) or polyacrylate, 2) a second synthetic polymer which ispoly(ether) such as poly(ethylene)glycol (PEG)  or ethylene oxide properylene oxide (EOPO) and 3. at least one salt. 

Johansson (US8,268,915) also teaches using a first polymer which is a poly(acid) such as poly(acrylic acid) (PAA) or poly(methacrylic acid) and a second polymer is is a poly(ether) such as a synthetic polymer comprising ethylene oxide units which includes poly(ethylene)glycol )PEG), ethylene oxide proylene oxide (EOPO) in either random copolymer form (e.g., Breox polymers) or block copolymers 9e.g., Puronic polymers) and at least one salt for the separation of biomolcules