Method of storing a separation matrix

Patent metadata

Field	Value
Publication number	US-11708390-B2
Application number	US-201716095721-A
Country	US
Kind code	B2
Filing date	May 10, 2017
Priority date	May 11, 2016
Publication date	Jul 25, 2023
Grant date	Jul 25, 2023

Abstract

The present invention concerns a method of storing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support. The method comprises the steps of: a) providing a storage liquid comprising at least 50% by volume of an aqueous alkali metal hydroxide solution; b) permeating the separation matrix with the storage liquid; and c) storing the storage liquid-permeated separation matrix for a storage time of at least days. The alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO 51 or SEQ ID NO 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

First claim

The invention claimed is:

1. A method of storing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by SEQ ID NO 51 or SEQ ID NO 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine; and wherein the method comprises the steps of:
a) providing a storage liquid comprising at least 50% by volume of an aqueous alkali metal hydroxide solution;
b) permeating the separation matrix with the storage liquid; and
c) storing the storage liquid-permeated separation matrix for a storage time of at least 5 days.

2. The method of claim 1 , wherein the mutants comprise further mutations in one or more of positions 1, 2, 7, 10, 15, 20, 21, 24, 25, 28, 29, 32, 34, 35, 36, 39, 42 and 43 in SEQ ID NO 51 or 52.

3. The method according to claim 1 , wherein the glutamine residue at position 1 of SEQ ID NO 51 or 52 has been mutated to an alanine.

4. The method according to claim 1 , wherein the asparagine or glutamic acid residue at position 35 of SEQ ID NO 51 or 52 has been mutated to an alanine.

5. The method according to claim 1 , wherein the multimers of immunoglobulin-binding alkali-stabilized Protein A domains are homomultimers selected from the group consisting of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers or nonamers.

6. The method according to claim 1 , wherein the multimers of immunoglobulin-binding alkali-stabilized Protein A domains each comprise a C-terminal cysteine residue for covalent coupling to the porous support.

7. The method according to claim 1 , wherein the multimers of immunoglobulin-binding alkali-stabilized Protein A domains are coupled to the porous support via thioether links.

8. The method according to claim 1 , wherein the separation matrix comprises at least 11 mg/ml of the multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to the porous support.

9. The method according to claim 1 , wherein the porous support is highly cross-linked agarose beads.

10. The method according to claim 1 , wherein the aqueous alkali metal hydroxide solution is sodium hydroxide solution, potassium hydroxide solution or a mixture thereof.

11. The method according to claim 1 , wherein the aqueous alkali metal hydroxide solution has a molarity of from 10 mM to 100 mM.

12. The method according to claim 1 , wherein the storage liquid further comprises a C2-C7 alcohol.

13. The method according to claim 1 , wherein the storage liquid comprises at least 70% by volume aqueous alkali metal hydroxide solution.

14. The method according to claim 1 , wherein the storage time is at least 25 days.

15. The method according to claim 1 , wherein the separation matrix is cleaned and/or sanitized with a cleaning fluid prior to storing, wherein the cleaning fluid comprises at least 50% by volume of an aqueous alkali metal hydroxide solution, and wherein the aqueous alkali metal hydroxide solution has a molarity of from 500 mM to 5 M.

16. The method according to claim 1 , wherein the separation matrix retains at least 80% of its original dynamic binding capacity after step b).

17. The method of claim 1 , wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutated to a glutamic acid.

18. A separation matrix product comprising a storage receptacle, a separation matrix and a storage liquid; wherein the storage receptacle contains the separation matrix permeated with the storage liquid; wherein the separation matrix comprises multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by SEQ ID NO 51 or SEQ ID NO 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine; and wherein the storage liquid comprises at least 50% by volume of an aqueous alkali metal hydroxide solution.

19. The separation matrix product according to claim 18 , wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutated to a glutamic acid.

Assignees

• Cytiva Bioprocess R & D Ab

Inventors

• Forss Annika
• Rodrigo Gustav José
• Bjorkman Tomas
• Ander Mats
• Hansson Jesper Ulf

Classifications

• C07K1/22 Primary

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

• B01D15/00

  Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor · CPC title

• B01J20/286

  Phases chemically bonded to a substrate, e.g. to silica or to polymers · CPC title

• B01J20/3212

  consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds · CPC title

• B01J20/3274

  Proteins, nucleic acids, polysaccharides, antibodies or antigens · CPC title