Method of isolation of polypeptide-aptamer-polymer conjugates

Patent metadata

Field	Value
Publication number	US-2018354987-A1
Application number	US-201715619148-A
Country	US
Kind code	A1
Filing date	Jun 9, 2017
Priority date	Jun 9, 2017
Publication date	Dec 13, 2018
Grant date	—

Abstract

A method for purification of a target in a solution includes providing a target in a solution, the solution comprising one or more contaminants, providing a polymer conjugate in the solution, wherein the polymer conjugate is configured to specifically bind the target, incubating the solution at a first temperature to facilitate binding of the polymer conjugate to the target, providing an environmentally-responsive block copolymer in the solution, wherein the environmentally-responsive block copolymer comprises one or more of an ethylene oxide (EO), a propylene oxide (PO), or an EO/PO block copolymer, and heating the solution comprising the environmentally-responsive block copolymer and the polymer conjugate bound to the target at a second temperature to initiate a liquid-liquid phase separation, wherein the liquid-liquid phase separation produces an aqueous phase and a liquid polymer phase, and wherein the liquid polymer phase comprises the polymer conjugate bound to the target.

First claim

1 . A method for purification of a target in a solution, comprising:
providing a target in a solution, the solution comprising one or more contaminants; providing a polymer conjugate in the solution, wherein the polymer conjugate is configured to specifically bind the target; incubating the solution at a first temperature to facilitate binding of the polymer conjugate to the target; providing an environmentally-responsive block copolymer in the solution, wherein the environmentally-responsive block copolymer comprises one or more of an ethylene oxide (EO), a propylene oxide (PO), or an EO/PO block copolymer; and heating the solution comprising the environmentally-responsive block copolymer and the polymer conjugate bound to the target at a second temperature to initiate a liquid-liquid phase separation, wherein the liquid-liquid phase separation produces an aqueous phase and a liquid polymer phase, and wherein the liquid polymer phase comprises the polymer conjugate bound to the target.

2 . The method of claim 1 , wherein the environmentally-responsive block copolymer is a thermoresponsive block copolymer.

3 . The method of claim 2 , wherein the thermoresponsive block copolymer has a cloud point between 21° C. and 45° C.

4 . The method of claim 1 , further comprising:
separating the aqueous phase from the liquid polymer phase; treating the liquid polymer phase such that the target dissociates from the polymer conjugate to generate unbound target; and eluting the unbound target, wherein the unbound target is substantially isolated from the liquid polymer phase after the eluting.

5 . The method of claim 1 , wherein the polymer conjugate comprises an aptamer.

6 . The method of claim 1 , wherein the polymer of the polymer conjugate is poly(N-isopropylacrylamide) (pNIPAM).

7 . The method of claim 1 , wherein the target is one or more of a contaminant or a biotherapeutic protein.

8 . The method of claim 1 , wherein the environmentally-responsive block copolymer comprises one or more of polypropylene oxide 1000 (PPG 1000), Pluronic L31, polypropylene oxide 725 (PPG 725), and Pluronic 17R4.

9 . The method of claim 1 , wherein the polymer of the polymer conjugate is a thermoresponsive polymer, wherein a cloud point of the polymer of the polymer conjugate is different than a cloud point of the environmentally-responsive block copolymer.

10 . The method of claim 1 , wherein the second temperature is higher than the first temperature, and wherein the second temperature is lower than 45° C.

11 . A method for selective purification of a target in a solution, comprising:
binding a target to an aptamer of a polymer-aptamer conjugate in a solution, wherein the aptamer of the polymer-aptamer conjugate is configured to specifically bind the target; separating the solution into two liquid phases using a thermoresponsive block copolymer, wherein the thermorepsonsive block copolymer is one of an ethylene oxide (EO), a propylene oxide (PO), or an EO/PO block copolymer, wherein the thermoresponsive block copolymer has a cloud point between 25° C. and 41° C., and wherein the two liquid phases comprise an aqueous phase and a liquid polymer phase, and wherein the liquid polymer phase comprises the target bound to the polymer-aptamer conjugate.

12 . The method of claim 11 , comprising incubating the solution containing the target and the polymer-aptamer conjugate at a first temperature to facilitate target binding.

13 . The method of claim 12 , comprising heating the solution containing the target bound to the polymer-aptamer conjugate and the thermoresponsive block copolymer to a second temperature to initiate the separating, wherein the second temperature is higher than the first temperature.

14 . The method of claim 11 , wherein the target is one or more of a contaminant, or a biotherapeutic protein.

15 . The method of claim 11 , further comprising:
extracting the aqueous phase from the liquid polymer phase; treating the liquid polymer phase such that the target dissociates from the polymer-aptamer conjugate to generate unbound target; and eluting the target.

16 . The method of claim 11 , wherein the polymer of the polymer-aptamer conjugate is a thermoresponsive polymer, wherein a cloud point of the polymer of the polymer-aptamer conjugate is different than the cloud point of the environmentally-responsive block copolymer.

17 . The method of claim 11 , wherein the target bound to the polymer-aptamer conjugate is not in the aqueous phase after the separating.

18 . A method for selective purification of a target in a solution, comprising:
adding a polymer-aptamer conjugate to a solution comprising a target, wherein the polymer-aptamer conjugate is configured to bind the target, wherein the polymer of the polymer-aptamer conjugate comprises a thermoresponsive polymer having a first cloud point, and wherein the aptamer of the polymer-aptamer conjugate comprises a DNA aptamer; incubating the solution at a first temperature to facilitate binding of the polymer-aptamer conjugate to the target; adding a thermoresponsive block copolymer to the solution, wherein the thermoresponsive block copolymer is one of an ethylene oxide (EO), a propylene oxide (PO), or an EO/PO block copolymer and has a second cloud point between 25° C. and 41° C.; and heating the solution at a second temperature to initiate a first liquid-liquid phase separation, wherein the second temperature is higher than the first temperature, wherein the first liquid-liquid phase separation produces a first aqueous phase and a first liquid polymer phase, wherein the first aqueous phase comprises unbound species, and wherein the first liquid polymer phase comprises the target bound to the polymer-aptamer conjugate.

19 . The method of claim 18 , further comprising:
extracting the aqueous phase from the liquid polymer phase; reducing heat of solution to a third temperature, wherein the third temperature is lower than the cloud point of the of the thermoresponsive block copolymer; adding a buffer to the solution, wherein the buffer is configured to unbind the polymer-aptamer conjugate from the target to produce an unbound target; heating the solution to a fourth temperature to initiate a second liquid-liquid phase separation, wherein the second liquid-liquid phase separation produces a second aqueous phase and a second liquid polymer phase, wherein the second aqueous phase comprises the unbound target, and wherein the second liquid polymer phase comprises the polymer-aptamer conjugate; removing the second liquid polymer phase; and eluting the unbound target from the second aqueous phase.

20 . The method of claim 18 , wherein the thermoresponsive block copolymer comprises one of polypropylene oxide 1000 (PPG 1000), Pluronic L31, polypropylene oxide 725 (PPG 725), and Pluronic 17R4.

Assignees

• Gen Electric

Inventors

• Kvam Erik Leeming
• Hay Bruce Allan
• Kovacs Ernest William

Classifications

• C08G2650/58

  Ethylene oxide or propylene oxide copolymers, e.g. pluronics · CPC title

• B01D11/0492

  Applications, solvents used · CPC title

• C07K16/00 Primary

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C12N15/115

  Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers} · CPC title

• C12N2310/16

  Aptamers · CPC title