Purification device, and purification method
View Patent ↗Provided is a purification device for purifying a useful substance (i.e., target substance) without using an acidic eluent. The purification device purifying the target substance comprises a column and a filler packed in the column. The filler contains a carrier, a temperature responsive polyamino acid bonded to the carrier, and a binding substance bonded to the temperature responsive polyamino acid and specifically bonding to the target substance.
1. A purification device for purifying a target substance, comprising a column and a filler packed in the column, wherein
the filler comprises a carrier, a temperature responsive polyamino acid bonded to the carrier, and a binding substance bonded to the temperature responsive polyamino acid, which binding substance has been adapted for specifically bonding to the target substance to be purified,
wherein the binding substance is a protein having a cysteine (Cys) on the N-terminal thereof that binds to a thiol group on the C-terminal of the polyamino acid,
and the polyamino acid includes in side chains at least one of an amphipathic group, a hydrophobic group and a hydrophilic group.
2. The purification device as described in claim 1 , wherein
the temperature responsive polyamino acid changes a structure between a conformation in which the target substance is bounded to the binding substance and a conformation in which the target substance thus bonded to the binding substance is separated therefrom, and
the structural change occurs in the range from 0° C. to 60° C.
3. The purification device as described in claim 1 , wherein
the temperature responsive polyamino acid is at least a member selected from a group of polylysine, polyglutamic acid and polyasparagine.
4. The purification device as described in claim 1 , wherein the binding substance is Protein A and the target substance is an antibody.
5. The purification device as described in claim 1 , further comprising a temperature controller which is configured to change a temperature of the temperature responsive polyamino acid in the range from 0° C. to 60° C.
6. The purification device as described in claim 1 , wherein the temperature responsive polyamino acid includes a carboxyl butylamino group or a carboxyl propylamino group in the side chains.
7. The purification device as described in claim 1 , wherein the temperature responsive polyamino acid includes both a dodecylamino group and a hydroxy alkylamino group in the side chains.
8. The purification device as described in claim 7 , wherein the temperature responsive polyamino acid is a polyasparagine.
9. The purification device as described in claim 1 , wherein the temperature responsive polyamino acid includes a hydroxy alkylamino group in the side chains.
10. The purification device as described in claim 9 , wherein the temperature responsive polyamino acid is a polyglutamic acid.
11. A purification method for purifying a target substance, the method comprising:
a binding step of passing a solution containing a target substance and impurities through a column packed with a filler comprising a carrier, a temperature responsive polyamino acid bonded to the carrier and a binding substance bonded to the temperature responsive polyamino acid, which binding substance has been adapted for specifically bonding to the target substance to be purified, thereby making the target substance bond to the binding substance;
a cleaning step of cleaning an inside of the column via washing out the impurities without separating the target substance thus bonded to the binding substance;
a separation step of separating the target substance bonded to the binding substance via changing a temperature of the temperature responsive polyamino acid after the inside of the column is cleaned; and
an eluting step of eluting the separated target substance from the column,
wherein the binding substance is a protein having a cysteine (Cys) on the N-terminal thereof that binds to a thiol group on the C-terminal of the polyamino acid,
and the polyamino acid includes in side chains at least one of an amphipathic group, a hydrophobic group and a hydrophilic group.
12. The purification method as described in claim 11 , wherein
the temperature responsive polyamino acid changes a structure between a conformation in which the target substance is bonded to the binding substance and a conformation in which the target substance thus bonded to the binding substance is separated therefrom; and
the structural change occurs in the range from 0° C. to 60° C.
13. The purification method as described in claim 11 , wherein the temperature responsive polyamino acid is at least a member selected from a group of polylysine, polyglutamic acid and polyasparagine.
14. The purification method as described in claim 11 , wherein the binding substance is Protein A and the target substance is an antibody.
15. The purification method as described in claim 11 , wherein the separation step is performed in the range from 0° C. to 60° C.
16. The purification method as described in claim 11 , wherein the eluting step is performed at pH values from 5 to 9.
17. The purification method as described in claim 11 , wherein the temperature responsive polyamino acid includes a carboxyl butylamino group or a carboxyl propylamino group in the side chains.
18. The purification method as described in claim 11 , wherein the temperature responsive polyamino acid includes both a dodecylamino group and a hydroxy alkylamino group in the side chains.
19. The purification method as described in claim 18 , wherein the temperature responsive polyamino acid is a polyasparagine.
20. The purification method as described in claim 11 , wherein the temperature responsive polyamino acid includes a hydroxy alkylamino group in the side chains.
21. The purification method as described in claim 11 , wherein the temperature responsive polyamino acid is a polyglutamic acid.