Strong cation exchange chromatographic matrix and method for using same

Patent metadata

Field	Value
Publication number	US-11801505-B2
Application number	US-202217570891-A
Country	US
Kind code	B2
Filing date	Jan 7, 2022
Priority date	Sep 9, 2016
Publication date	Oct 31, 2023
Grant date	Oct 31, 2023

Abstract

A cation exchange chromatographic matrix comprising a base material, and a copolymer with one monomer unit having at least a sulfonic acid group, the copolymer being immobilized on the base material, wherein: the copolymer forms substantially no cross-linked structure, and the copolymer comprises neither acrylamide nor an acrylamide derivative as a monomer unit, or comprises acrylamide or an acrylamide derivative as a monomer unit in a range which has no substantial influence; the ratio of the mass of the copolymer to the mass of the base material is 5% or more and 200% or less; and the density of the sulfonic acid group is higher than 30 mmol/L and 200 mmol/L or lower.

First claim

The invention claimed is:

1. A purification method for purifying a physiologically active substance from a mixed solution containing impurities and the physiologically active substance, comprising
contacting the mixed solution with a cation exchange chromatographic matrix for biomolecule purification, wherein the cation exchange chromatographic matrix comprises:
a base material, and
a copolymer with one monomer unit having at least a sulfonic acid group, the copolymer being on a graft chain immobilized on the base material wherein:
the copolymer forms substantially no cross-linked structure, and the copolymer comprises neither acrylamide nor an acrylamide derivative as a monomer unit, or comprises acrylamide or an acrylamide derivative as a monomer unit at a mass percent of 10% or less based on all monomer units of the copolymer;
the ratio of the mass of the copolymer to the mass of the base material is 5% or more and 200% or less;
the density of the sulfonic acid group is higher than 30 mmol/L and 200 mmol/L or lower;
the mixed solution is contacted with the matrix in a flow-through mode; and
a mass of graft chain per the cation exchange chromatographic matrix volume is 0.03 g/mL or larger and 0.25 g/mL or smaller, wherein the mass of graft chain per the cation exchange chromatographic matrix volume is represented by the following expression:
sg (g/mL)=( w 2 −w 0 )/ v 1

wherein w 2 is the mass of the cation exchange chromatographic matrix obtained as a final product, v 1 is the volume of the cation exchange chromatographic matrix obtained as the final product, and w 0 is the mass of the base material before introduction of the graft chain.

2. The purification method according to claim 1 , wherein the pH of the mixed solution is 4.0 or higher and 10.0 or lower.

3. The purification method according to claim 1 , wherein the physiologically active substance is a monomer of an antibody protein.

4. The purification method according to claim 1 , wherein the impurities include dimeric and higher aggregates of the antibody protein.

5. The purification method according to claim 1 , wherein the recovery rate of the physiologically active substance is 80% or more.

6. The purification method according to claim 1 , wherein 100 mg or more of the antibody protein including the monomer and the aggregates is purified per mL of the cation exchange chromatographic matrix for biomolecule purification.

7. The purification method according to claim 1 , wherein the ratio of the aggregates is reduced by 50% or more when a solution of the antibody protein including the monomer and the aggregates is purified.

8. The purification method according to claim 1 , further comprising performing purification using an anion exchange chromatographic matrix before or after the purification using the cation exchange chromatographic matrix.

9. The purification method according to claim 8 , wherein the anion exchange chromatographic matrix is in a membrane form.

10. The purification method according to claim 8 , wherein the purification using an anion exchange chromatographic matrix is a flow-through mode.

11. The purification method according to claim 8 , further comprising performing affinity chromatography before the purification using the cation exchange chromatographic matrix and the purification using the anion exchange chromatographic matrix.

12. The purification method according to claim 8 , wherein buffer replacement is not performed in a series of purifications.

13. The purification method according to claim 11 , wherein the affinity chromatography is carried out in a bind and elute mode, and the physiologically active substance is eluted with a buffer consisting of a monovalent acid in the elution.

14. The purification method according to claim 13 , wherein the electrical conductivity of the buffer consisting of a monovalent acid is 10.0 mS/cm or lower.

15. The purification method according to claim 11 , further comprising adjusting the pH of the mixed solution to 4.0 or lower after performing the affinity chromatography.

16. The purification method according to claim 8 , wherein the electrical conductivity of the mixed solution containing the physiologically active substance is 10 mS/cm or lower in a series of purifications.

17. The purification method according to claim 1 , wherein the molar percent of the monomer unit having the sulfonic acid group in the copolymer is smaller than the molar percent of a neutral monomer unit having no charge.

18. The purification method according to claim 1 , wherein the mass percent of the monomer unit having the sulfonic acid group in the copolymer is smaller than the mass percent of a neutral monomer unit having no charge.

19. The purification method according to claim 1 , wherein the monomer unit having the sulfonic acid group is a glycidyl methacrylate derivative.

20. The purification method according to claim 17 , wherein the neutral monomer unit comprises at least a hydrophilic monomer unit derived from (meth)acrylate compounds selected from the group consisting of 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, 2-hydroxypropyl acrylate, 2-hydroxypropyl methacrylate, 3-hydroxypropyl acrylate, 3-hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, 4-hydroxybutyl methacrylate, 2-(dimethylamino)ethyl acrylate, and 2-(dimethylamino)ethyl methacrylate, and mixtures thereof, and the molar ratio of the hydrophilic monomer unit to the neutral monomer unit in the copolymer is 50% or more.

21. The purification method according to claim 18 , wherein the neutral monomer unit comprises at least a hydrophilic monomer unit derived from (meth)acrylate compounds selected from the group consisting of 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, 2-hydroxypropyl acrylate, 2-hydroxypropyl methacrylate, 3-hydroxypropyl acrylate, 3-hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, 4-hydroxybutyl methacrylate, 2-(dimethylamino)ethyl acrylate, and 2-(dimethylamino)ethyl methacrylate, and mixtures thereof, and the ratio of the mass of the hydrophilic monomer unit to the total mass of the neutral monomer unit in the copolymer is 50% or more.

22. The purification method according to claim 1 , wherein the cation exchange chromatographic matrix contains no carboxyl group, or has a density of a carboxyl group lower than the density of the sulfonic acid group.

23. The purification method according to claim 1 , wherein the base material is in a membrane form.

24. The purification method according to claim 1 , wherein the cation exchange chromatographic matrix for biomolecule purification is structured and configured for antibody protein purification.

25. The purification method according to claim 2 , wherein the pH of the mixed solution is 5.0 or higher and 7.0 or lower.

26. The purification method according to claim 6 , wherein 500 mg or more of the antibody protein including the monomer and the aggregates is purified per mL of the cation exchange chromatographic matrix for biomolecule purification.

Assignees

• Asahi Kasei Medical Co Ltd

Inventors

• Taniguchi Hiroki

Classifications

• B01J39/26 Primary

  Cation exchangers for chromatographic processes · CPC title

• B01D15/1871

  placed in series · CPC title

• B01D15/36

  involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion · CPC title

• B01D15/362

  Cation-exchange · CPC title

• B01D15/363

  Anion-exchange · CPC title