Matrix for separation of polyethers and method of separation
View Patent ↗The present invention relates to a separation matrix comprised of a support to the surfaces of which polymer chains have been coupled, wherein each polymer chain presents recurring proton-donating groups and at least the surface of the support is substantially hydrophilic. In the most advantageous embodiment, the support is porous cross-linked agarose, the polymers are poly(acrylic acid) and the proton-donating groups are carboxyl groups. The matrix is useful e.g. to remove PEG from pegylated and/or native compounds in a liquid. Accordingly, the invention also encompasses a method, such as a chromatographic method, wherein the separation matrix according to the invention is used, for example as a pre-treatment of a reaction mixture that comprises unreacted PEG, pegylated proteins and native proteins.
1. A method of separating at least one polyether target substance from other components of a liquid, comprising the steps:
(a) providing a separation matrix comprised of a support having surfaces of which polymer chains have been coupled, wherein each polymer chain presents recurring proton-donating groups and at least the surface of the support is substantially hydrophilic;
(b) contacting, at a pH below about 6, the liquid with said separation matrix to allow hydrogen bonding between the proton-donating groups of the separation matrix and proton-accepting ether oxygens of the polyether target substance(s); and
(c) recovering one or more target substances from the separation matrix by contacting said matrix with an eluent, which eliminates hydrogen bonding or competes with the target for hydrogen bonding to the support.
2. The method of claim 1 , wherein the proton-donating groups of the separation matrix are predominantly carboxyl groups.
3. The method of claim 1 , wherein the polymer chains of the separation matrix are predominantly poly(acrylic acid) chains.
4. The method of claim 1 , wherein in step (b), the pH is below about 4, and preferably below about 3.
5. The method of claim 1 , wherein the eluent of step (c) comprises an increasing pH gradient.
6. The method of claim 1 , wherein the polyether target substance is PEG or a pegylated compound, such as a protein.
7. The process of purifying a pegylated compound from a reaction mixture, which comprises a pre-treatment step to remove PEG and a subsequent step to isolate one or more pegylated compounds, wherein the pre-treatment is the method of claim 1 .
8. The process of claim 7 , wherein the compounds are proteins.
9. The process of claim 7 , wherein the subsequent step to isolate one or more pegylated compounds is a liquid chromatography step.