Liquid purification or separation – Processes – Making an insoluble substance or accreting suspended...
Reexamination Certificate
2001-10-03
2002-09-24
Drodge, Joseph W. (Department: 1723)
Liquid purification or separation
Processes
Making an insoluble substance or accreting suspended...
C210S638000, C210S639000, C210S774000, C210S805000, C210S806000, C530S422000, C530S427000
Reexamination Certificate
active
06454950
ABSTRACT:
TECHNICAL FIELD
The present invention concerns separations in aqueous phase systems containing at least one aqueous phase containing a thermoseparating polymer. The compounds to be separated may be of biological nature (biomolecule) having for instance lipid, peptide and/or carbohydrate structure.
TECHNICAL BACKGROUND
The traditional aqueous two-phase systems have been the PEG/dextran and PEG/salt systems (PEG=poly(ethylene glycol). The former system derives from polymer-polymer incompatibility and the latter 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 (Albertsson, Partitian of cell particles and macromolecules, 3
rd
ed., Wiley, New York; and Walter et al. (Ed.), Aqueous two-phase systems, meth. Enzym. 228 (1994 (Academic Press, London)). The PEG/salt systems are mainly used in large scale enzyme extractions (Cordes et al., Meth. Enzymol. 228 (1994) 640-608 (Academic Press, London).
More novel two-phase systems have utilized polymers that have a solubility in water that decreases upon increasing the temperature (thermoseparating polymers). In these cases 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, i.e. the temperature at which the phases start to separate out. The cloud point for an aqueous solution of a given polymer depends on polymer concentration and amount and type of other components added (for instance cloud point-decreasing agents). The lowest cloud point is called the lowest critical solution temperature (LCST) (Saeki et al., Polymer 17 (1976) 685-688). This phenomenon also occurs in water solution of non-ionic surfactants e.g. Triton X-114, C12E05. Applications of thermoseparations in biotechnology have been reviewed (Galaev et al, Enzyme Microb. Technol. 15(1993) 354-366). If not otherwise specified, LCST will refer to the lowest cloud point for a system consisting of a pure polymer and pure water.
Many thermoseparating polymers contain ethylene oxide groups. PEG as such is one example but its cloud point is too high (above 100° C.) for use in a thermoseparating process for the separation of biomaterials Ethylene oxide (EO)-propylene oxide (PO) random copolymers have lower cloud points. Ucon 50 HB-5100 and Breox 50A 1000 are random copolymers composed of 50% EO and 50% PO groups (EOPO-polymers). Both copolymers have a LCST at 50° C.
Phase systems of polymers having various EO/PO ratios have been studied in the separation of proteins (Harris et al., Biosep. 2 (1991) 237-246; Alred et al., J. Chromatogr. 659 (1994) 289-298; Persson et al., Chromatogr. 711 (1998) 97-109; Berggren et al., J. Chromatogr. A 718 (1995) 67-79; Johanssson et al., Biochim. Biophys. Acta 1290 (1996) 289-298). Thermoseparating polymers consisting of blocks of EO and PO units (Pluronics) and their use for separation of an enzyme have been described (Tani et al., Analytical Science 13 (1997) 925-929). The polymers used by Tani et al have a hydrophilic EO block in the middle and less hydrophilic PO blocks as terminal groups or vice versa (EOPO block copolymer).
Two of the inventors (Folke Tjerneld and Josefine Persson) are also an inventor in WO 9811127 and WO 981140 both of which describe purification of apolipoprotein in aqueous two phase systems containing a thermoseparating polymer.
Partition of amino acids and oligopeptides having various hydrophobicity and net charge has been studied in the water/Ucon system (Johansson et al., Biosep. 5 (1995) 269-279; (Johansson et al., Biochim. Biphys Acta 1335 (1996) 315-325).
Disadvantages with thermoseparating polymers used so far and objectives with the invention.
In phase systems containing thermoseparating polymers the polymer phase so far always has contained relatively large concentrations of polymer (above 40%) with most proteins exclusively distributing to the water phase. It would be beneficial a) to have systems in which there are less polymer in the polymer phase, b) to be able to direct the compound to be separated back and forth between the phases and c) to have separation processes in which recirculation of the thermoseparating polymer is facilitated.
DEFINITIONS
Polymers:
When the term polymer is used in the present invention it refers to water-soluble/water-miscible polymers if not otherwise emphasised. By a polymer is further meant that it contains ≧20 monomeric units that may be different or equal.
Thermoseparating polymers are polymers having water-solubilities that decrease upon increasing the temperature.
Cloud point-decreasing agents are compounds that when added to a polymer-water solution decrease the cloud point of the solution.
Cloud point-increasing agents are compounds that, when added to a solution of a thermoseparating polymer in water increase the cloud point of the solution.
Incompatible polymers. In the context of the present invention a polymer is incompatible with another polymer if two aqueous phases can be formed when both polymers are mixed with water. The definition also encompasses that the first polymer occurs predominantly in one phase and the second polymer predominantly in the other phase.
Micell-forming and non-micell-forming polymers:
Micell-forming properties of a thermoseparating polymer are reflected in cloud point variation when the polymer concentration is varied in a solution consisting of pure water and the polymer. See
FIG. 1
where the slightly concave curvature of Breox PAG 50A 1000 (International Speciality Chemicals Lt, Southampton, U.K.) and the strong concave curvature of the hydrophobically modified ethylene oxide propylene oxide random copolymer (HM-EOPO polymer) used in the experimental part reflect that Breox is non-micell forming and the HM-EOPO polymer micell-forming. For the sake of simplicity and in the context of the preferred modes of the invention, a micell-forming thermoseparating polymer is a polymer that, when mixed with water to the concentration of its LCST and raising the temperature from below to above this temperature, will be able to give two phases, both of which have a water concentration of at least 70% such as 80%. Many of these polymers exhibit a pronounced minimum at their LCST. Compare the curves for HM-EOPO polymer (micell-forming) with Breox (non-micell-forming) in
FIG. 1. A
non micell-forming polymer is a water-soluble/water-miscible polymer that does not comply with the definition of a micell-forming polymer. The percentage values above and below are based on w/w if not otherwise specified.
A non-micell-forming polymer is normally incompatible with a micell-forming polymer meaning that a non-micell-forming polymer often will act as cloud point-decreasing agents in mixtures of water and micell-forming polymers and vice versa for micell-forming polymers when introduced into mixtures of water and non-micell forming polymers.
SUMMARY OF THE INVENTION
It has now been discovered that the objectives discussed above will be met in case one selects a thermoseparating polymer that is micell-forming as defined above. Accordingly the present invention concerns a method for the separation of a predetermined compound comprising
i) partitioning the compound in a two-phase system (system A) in which at least one of the phases (phase
1
) and the other is poor (phase
2
) in a thermoseparating polymer (I),
ii) collecting one phase (phase
1
or phase
2
) containing said compound, and
iii) if desired, further working up said compound from the phase collected in step ii.
The characteristic feature of the method is that
a) polymer (I) is a micell-forming thermoseparating polymer, that when mixed with water to the concentration of its lowest critical solution temperature (LCST and raising the temperature from below to above this temperature is capable to give two phases, both of which have a water concentration of at least 70%, and
b) phase
2
possibly contains at least one agent that is cloud point-decreasing for the th
Johansson Hans Olof
Persson Josefine
Tjerneld Folke
Amersham Pharmacia Biotech AB
Drodge Joseph W.
Ronning, Jr. Royal N.
Ryan Stephen G.
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