Novel antibody purification method, antibody obtained therefrom, novel antibody purification method using cation exchanger, and antibody obtained therefrom

Patent metadata

Field	Value
Publication number	US-2016237113-A1
Application number	US-201415022890-A
Country	US
Kind code	A1
Filing date	Sep 16, 2014
Priority date	Sep 17, 2013
Publication date	Aug 18, 2016
Grant date	—

Abstract

The first embodiment of the present invention is a method for purifying an antibody or a substance derive from an antibody, wherein a carrier 1 having an affinity ligand with affinity for the antibody or the substance derived from the antibody and a carrier 2 having a cation exchange group are used to prepare an integrated column 1 connecting a column containing the carrier 1 and a column containing the carrier 2 or a column 2 having the mixture of the carrier 1 and carrier 2, the antibody or the substance derived from the antibody is applied to the column 1 or the column 2, and then the adsorbed antibody or substance derived from the antibody is eluted from the column 1 or the column 2. The second embodiment of the present invention is a method for using a carrier having a cation exchange group, wherein a solution containing an antibody or a substance derived from an antibody is applied to a carrier having a cation exchange group having a carboxyl group-containing ligand and pKa of 4.0 or more, and the antibody or the substance derived from the antibody is eluted by an acidic buffer having pH of 4.0 or less.

First claim

1 : A method for purifying an antibody or a substance derived from an antibody, wherein
a carrier 1 having an affinity ligand with affinity for the antibody or the substance derived from the antibody and a carrier 2 having a cation exchange group are used to prepare an integrated column 1 connecting a column containing the carrier 1 and a column containing the carrier 2 or a column 2 having a mixture of the carrier 1 and the carrier 2, the antibody or the substance derived from the antibody is adsorbed on the column 1 or the column 2, and then the adsorbed antibody or substance derived from the antibody is eluted from the column 1 or the column 2.

2 : A method for purifying an antibody or a substance derived from an antibody with a carrier 1 having an affinity ligand with affinity for the antibody or the substance derived from the antibody and a carrier 2 having a cation exchange group, wherein
a solution containing the antibody or the substance derived from the antibody is applied to an integrated column 1 connecting a column containing the carrier 1 and a column containing the carrier 2 in series or a column 2 having a mixture of the carrier 1 and the carrier 2, and an eluate is passed through the integrated column or the mixed column to elute the adsorbed antibody or the substance derived from the antibody.

3 : The method according to claim 1 , wherein the antibody or the substance derived from the antibody is eluted by the eluate having an acidic pH with an ionic strength linear gradient.

4 : The method according to claim 1 , wherein the antibody or the substance derived from the antibody is eluted by the eluate having an acidic pH with an ionic strength step gradient.

5 : The method according to claim 2 , wherein
the integrated column or the mixed column is equilibrated with an equilibration buffer, the solution containing the antibody or the substance derived from the antibody is applied to the integrated column or the mixed column, and the integrated column or the mixed column is washed with a wash buffer having higher pH than that of the eluate and lower ionic strength than those of the equilibration buffer and the solution containing the antibody or the substance derived from the antibody before elution.

6 : The method according to claim 2 , wherein
the integrated column or the mixed column is equilibrated with an equilibration buffer, the solution containing the antibody or the substance derived from the antibody is applied to the integrated column or the mixed column, the integrated column or the mixed column is washed with a washing buffer 1 having higher pH than that of the eluate and ionic strength greater than or equal to those of the equilibration buffer and the solution containing the antibody or the substance derived from the antibody, and is washed with a washing buffer 2 having higher pH than that of the eluate and lower ionic strength than those of the equilibration buffer and the solution containing the antibody or the substance derived from the antibody before elution.

7 : The method according to claim 1 , wherein the carrier 1 has protein A, protein G, protein L or an analog thereof as a ligand.

8 : The method according to claim 1 , wherein the carrier 1 has protein A or an analog thereof as a ligand.

9 : The method according to claim 1 , wherein the antibody or the substance derived from the antibody is immunoglobulin G, immunoglobulin G derivative, or Fc-containing molecule.

10 : The method according to claim 1 , wherein the antibody or the substance derived from the antibody is Fab, scFv, diabody, or a molecule containing a binding part to an antigen.

11 : The method according to claim 1 , wherein the carrier 2 has a carboxyl group as a ligand.

12 : The method according to claim 11 , wherein the carboxyl group is derived from an acidic amino acid.

13 : The method according to claim 1 , wherein the elution pH of the antibody or the substance derived from the antibody is less than 5.0.

14 : The method according to claim 1 , wherein 10% DBC of the carrier 1 to IgG at 6 minutes of contact time is 1 mg/mL or more and 100 mg/mL or less.

15 : The method according to claim 1 , wherein the carrier 2 has the ion exchange capacity of 0.001 mmol/mL or more and 0.5 mmol/mL or less.

16 : The method according to claim 1 , wherein the carrier 1 has the volume average particle diameter of 1 μm or more and 1000 μm or less, and the carrier 2 has the volume average particle diameter of 1 μm or more and 1000 μm or less.

17 : The method according to claim 1 , wherein
the solution containing the antibody or the substance derived from the antibody is applied to the carrier 2 having a cation exchange group having a carboxyl group-containing ligand and pKa of 4.0 or more, and the antibody or the substance derived from the antibody is eluted by an acidic buffer having pH of 4.0 or less.

18 : The method according to claim 1 , wherein 10% DBC of the carrier 2 to IgG at 6 minutes of contact time is 1 mg/mL or more and 200 mg/mL or less.

19 : The method according to claim 2 , wherein
the column packed with the carrier 1 having an affinity ligand is connected to the column packed with the carrier 2 having the cation exchange group such that the column packed with the carrier 2 is downstream to prepare the integrated column, the solution containing the antibody or the substance derived from the antibody is applied to the integrated column under condition of neutral pH, and the antibody or the substance derived from the antibody is eluted by an acidic buffer having pH of 4.0 or less.

20 : The method according to claim 2 , wherein the ratio of the carrier 1 to the carrier 2 of the integrated column is 1/20 or more and 20/1 or less on the basis of the volume.

21 : The method according to claim 2 , wherein
the carrier 1 having an affinity ligand is mixed with the carrier 2 having a cation exchange group to prepare the mixed column, the solution containing the antibody or the substance derived from the antibody is applied to the mixed column under condition of neutral pH, and the antibody or the substance derived from the antibody is eluted by an acidic buffer having pH of 4.0 or less.

22 : The method according to claim 2 , wherein the ratio of the carrier 1 to the carrier 2 of the mixed column is 1/20 or more and 20/1 or less on the basis of the volume.

23 : The method according to claim 1 , wherein the ratio of 10% DBC of the carrier 2 to IgG at 6 minutes of contact time relative to 10% DBC of the carrier 1 to IgG at 6 minutes of contact time under each adsorption condition is 1/10 times or more and 10 times or less.

24 : An antibody or a substance derived from an antibody purified with the method according to claim 1 .

25 : A method for using a carrier having a cation exchange group, wherein
a solution containing an antibody or a substance derived from an antibody is applied to a carrier 2 having a cation exchange group having a carboxyl group-containing ligand and pKa of 4.0 or more, and the antibody or the substance derived from the antibody is eluted by an acidic buffer having pH of 4.0 or less.

26 : The method according to claim 25 , wherein the carboxyl group-containing ligand is derived from an acidic amino acid.

27 : The method according to claim 25 , wherein 10% DBC of the carrier 2 to IgG at 6 minutes of contact t

Assignees

• Kaneka Corp

Inventors

• Minakuchi Kazunobu

Classifications

• C07K1/22 Primary

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• C07K16/1271

  Micrococcaceae (F); Staphylococcaceae (F), e.g. Staphylococcus (G) · CPC title

• C07K2317/622

  Single chain antibody (scFv) · CPC title

• C07K1/36

  by a combination of two or more processes of different types · CPC title

• C07K2317/626

  Diabody or triabody · CPC title