As to partitioning of antibodies see “purification of antibodies”

Extraction is the prcoess of moving one or mroe components of a mixture form one phase to another (compare to precipitation which is the process of removing one or mroe components from solution to form a solid phase, the precipitate). Liquid-liquid extraction using organic and aqueous extraction media is a traditional separation operation that can be applied to the purificaiton of biopharmaceuticals. In three-phase partitioning, proteins can be purified directly form cell homoenates by partitioning between a layer of butanol and a strong aqueous salt solution. Under these conditions, cell debris tends to separate into the organic phase and nucleic acids preciptiate at the interphase while proteins remain in solution. Aqueous to-phase extraction systems are the most widely used extraction operation, empoloying a mixture of aqueous polymers and/or salts. One phase generally contains polyethylene glycol (PEG) and the other contains a different polymer, such as dextran, or the salt potassium phospahte. Under ideal conditions, the desired protein can be separated into the PeG phase while the majority of contaminating proteins as well as other contaminants are trapped in the second phase, o in the interphase., and can be removed by centrifugation. (Uwe Gottschalk, Sartorius Biotech GmbH, “Downstream Processing” Chapter 18 in Filtration and Purificaiton in the Biopharmaceutical Industry, Second Edition. Informa healthcare 2008). 

Two-polymer-Two Phase systems: Generally

Aqueous two-phase systems (ATPS) are formed when aqueous solutions of two mutually incompatible components separate into two phases of different densities under the force of gravity. This technique allows clarification, concentration and partial purificaion to be integrated in one step. (Ferreira, J. Chromatography A, 1195 (2008) 94-100).

ATPS formed by mixing two water soluble incompatible polymers have been used for separation of sensitive biological materials. The most well known are the PEG/dextran system (PEG=poly(ethyleneglycol) and PEG/phosphate system). It is the possibility to form two phase systems with high water concentration in both phasses that makes the PEG/dextran and PEG/phosphate useful for sensitive biological materials. (Hans-Olof Johansson, Bioseparation 7: 259-267, 1999).

The traditional aqueous two phase systems have been the PEG/dextran which dervies from polymer-polymer incompatability and PEG/salt systems which derives from salting out of the polymer with a salt. The PEG/dextran systems are used for small scale separations of macromolecules, membranes, cell particles and cells. The PEG/salt systems are mainly used in large scale enzyme extractions. (Tjerneld (US 6,454,950). 

More novel two-phase systems have utlized polymers that have a solubility in water that decreases upon increasing the temperature (thermoseparating polymers). In these case two macroscopic phases (one polymer enriched bottom phase and one water rich top phase) can be obtained upon heating a solution of the polymer a few degrees above the cloud point (the temperature at which the phases start to searate out). The cloud point for an aqueous solution of a given polymer depends on polymer concentration and amount and type of other components added. The lowest cloud point is called the lowest critical solution temperature (LCST). (Tjerneld, US 6,454,950). 

Aqueous polymer two phase-systems: 

Polymers used in two phase systems are aqueous in the sense that they form aqueous phases when combined with water. Aqueous polymer two phase-systems are more useful for biologicals. One reason for this is the high water concentration in both phases. They are formed by mixing certain hydrophilic and typically neutral polyers in aqueous solution. These include dextran (polyclucose) and poly(ethylene glycol) (PEG) as well as polysucrose (such as Ficoll™) and PEG or linear polyacrylamid and PEG. Typically concentrations of each polymer are 50-10% w/w. At such concentrations, entropic and other forces tend to drive the formation of two phases both of which are typically greater than 90% (ww) water. The phases are typically enriched in one polymer and have low interfacial tension. Phase density differences drive the phases to separate by gravity or centrifugation. One advantage of the PEG and Dextran type of two-phase syste is that target proteins may partition in favour of the PEG enriched, less dense, upper phase while cell debris may partition (or sediment) to the interface or complementary lower phase. Independent of the challenge of adding and then removing two polymers from the bioprocess stream is the cost of polymers such as dextran and PEG (WO2010/080062).

In the biotechnical field, aqueous polymer two phase systems formed with two polymers or with one polymer in presence of significant added salt are of general interest. Many undesired components such as cell debris will tend to appreciably partition to the lower (dextran-rich or salt richs, respectively) phase in a PEG and dextran, or a PEG and salt two phase system. Drawbacks include phase component cost, phase component removal and effect of phase components on other downstream operations.

The application of an ATPS at process-scale has been hampered by the complexity of the system combined with the fact that partition mechanisms are poorly understood and method development is fairly empirical. The partitioning of biological compounds in an ATPS depends not only on the physico-chemical properties of the biomolecule, such as surface hydrophobicity, charge and size, but also on the system composition.  (Azevedo, “Chromatography-free recovery of biopharmaceutical through aqueous two-phase processing” Trends in Biotechnology, (2009), 27(4):240-247.

Conditions: PEG characteristics, including weight, size and concentration are very important factors in the properties of the phase forming system. Higher molecular weight of PEG has less coefficient factor and then lower polymer concentraiotn needed for high separation. Increases in salt concentraiton also result in an increase in partition cefficients of bioproducts in upper phase or interface due to salting out. Partitioning of proteins and enzymes to the phases in the ATPE system also depends on their isoelectric points. The pH of the system, however, affects the cahrge of target protein and ion composition as well as introduces differential partitioning into the tow phases. A pH value above 7 is suitable fot eh PEG/phosphate system and a pH below 6.5 is compatible with the PEG/sulphate sustem. Most fo the biomolecuels, especially proteins and enzymes, are stable at neutral pH that is favorable condition to conduct ATP partitioning. The selection of salts for ATPS depends on their ability to promote hydrophobic interactions between biomolecuels. The PEG/phosphate system is widely used for recovery of bioproducts. Other salts having similar properties to phosphate, such as sulphate and citrate, have also been used. (Goja J. Bioproces Biotechniq 2013, 4:1)

PEG/Salt Systems

The impact of salt concentration has been widely studies. Increases in salt concentraiton result in an increase in partition coefficients of bioproducts in upper pahse or interface due to salting out. In general, proteins with the negative charge tend to partition to the top phase in PEG/Salt systems while those with the positive charge usually go to the bottom phase. (Goja J. Bioproces Biotechniq 2013, 4:1) 

PEG/dextran system: The PEG/dextran system is a particulalry mild system and is thus used for more sensitive biological materiasl such as cells and cell organells. (Hans-Olof Johansson, Bioseparation 7: 259-267, 1999).

PEG/phosphate system: is used for large scale purification of proteins. (Hans-Olof Johansson, Bioseparation 7: 259-267, 1999).

Fusion Proteins which direct Product Protein to Particular Phase

Hydrophobin-Protein fusions:

Hydrophobins are bipolar and small proteins, consisting of about 100-150 amino acids, of which 8 are cysteine residues. They are expressed by filamentous fungi to help the organisms adapt to the environment. Hydrophobins and hydrophobin fusion proteins can be purified and concentrated with a surfactant. (Joensuu (US 14/902851, published as US 2016-0159854)

Collen (Biochimica et Biophysica Acta (2002) 1569, pp. 139-150) discloses partitioning and purification of the fusion protein endoglucanase – hydrophobin I (EGI-HFBI) from a culture filtrate originating from Trichoderma reesei fermnetnation. The micelle extraction system was formed by mixxing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Rppal PES100. The detergent/polymer aqueous two-phase sytems resultsted in better separation compared to cloud point extraction in a Tris X-114/water system. After the primary recovery step, EGI-HFBI was back extracted into a water phase by adding (EOPO) copolymers to the micell rich pahse and a temerpature induced phase separation at 55C. 

Penttila (US7,335,492 and US 7,060,669) disclose the separation of molecules by fusing them with a targetting protein having the capability to carry the molecule to a desired phase in ATPS. Examples of molecuels suited as tareting proteins are hydrophobin like small proteins (hydrophobins). The hydrophoin like protein is bound to the product molecule to be separated. Phase forming materials and possibly also additional salts are added to a water solution containing the fusion moecule. The two phases are formed either by gravity settling or centrifugation. The targeting protein drives the product to for instance the detergent rich phase which can be the top or bottom phase. 

Insertion of Trp rich peptides into C terminus of Product protein:

Hassien (J Chromatography A, 668 (1994) 121-128) iclsoes insertion of AlaTrpTrpPro near the C temrinus of ZZT0. The Trip rich peptide strongly increasedthe partitioning of ZZTO into the PEG rich pahse in a PEG-potassium phosphate aqueous twp phase system. 

One polymer-Two Phase systems: Thermoseparating polymer/water two phase systems

Thermoresponsive hydrophilic polymers exhibit inverse thermal solubility such that as temperature is raised above a certain cloud temperature (Tc) which is related to the polymer’s lower critical solubility temperature (LCST), they self associate and start to form a unique polymer rich phase. Such polymers include copolymer or block copolymers frored with mixture of ethyelene oxide (EO) and propylene oxide (PO) monomeric groups, so called EOPO polymers, polysaccharides modified with EO, PO or similar groups (e.g., ethylhydroxyethycellulose or EHEC) or polyers formed using N-isopropylacrylamide (NIPAAM). 

The system is referred to as a “thermoseparated aqueous two phase system”. Two phases are formed by heating an aqueous solution of a thermoseparating polymer. Above a cricial solution teemperature, the cloud point, the solution will separate into two macroscopic phases. One of the phases (often the bottom phase) is enriched with polymer, the other is depleted. The lowest cloud point of the system (LCST) is the lower critical solution temperature. (Hans-Olof Johansson, Bioseparation 7: 259-267, 1999).

In general thermoseparating phases have normally been used together with dextran or similar polysaccharid in a two step process. Thus selectivity over target and contaminant protein occurs in the first partition step followed by use of temperature induced phase separation (of typically EOPO polymer rich phase) to isolate target and polymer into target containing aqueous phase floating on top of a self associated polymer rich denser phase. 

Modes of Operation

Continuous ATPS:

Palomares (MX2009013602) discloses a device for the continous recovery of bioparticles by means of aqueous two-phase systems which is perforemd in a continous mannter where the partition, separation and/or recvoery of proteins in continous systems. The singe device performs continous bioparticle priamry recvoery whithout requiring centrifugation, agitation and decantation, allowing biopartciles recovery more efficiently than recvoeyr processes sysing other systems and can be implemented onan industrial scale. The recovery of each phase is carried out in separate containers. 

Villegas (J. Sep. Wol. 2013, 36, 391-399) discloses the application of a continous aqueous two-phase system for the recovery of biomlecules. Compared with batch systems, continous operation increased partition coefficient with higher recovery efficiencies. Befiefly, an equilibrated 3 kg system phases were separated into two different containers and fed independently at 10 mL/min while spiked SPE sample was injected by a third inlet at 1 mL/min. Mixingagitation was accomplisehd with a static mixer. Phases were continously harvested at the end of the system, as well as the interphase by a coupled three way outlet connected.