Methods for reducing chromatin content in protein preparations by treatment with alkyl cations

Patent metadata

Field	Value
Publication number	US-10023609-B2
Application number	US-201415121686-A
Country	US
Kind code	B2
Filing date	Feb 27, 2014
Priority date	Feb 27, 2014
Publication date	Jul 17, 2018
Grant date	Jul 17, 2018

Abstract

A method of reducing aggregate content in a preparation having a target protein includes contacting the preparation with an alkyl cation to form a mixture, and contacting the mixture with at least one functionalized solid to remove excess alkyl cation.

First claim

The invention claimed is:

1. A method of reducing aggregate content in a preparation comprising an antibody, the method comprising: (a) contacting the preparation with an alkyl cation to form a mixture, wherein the alkyl cation is selected from a bromide or a chloride salt of a quaternary ammonium cation selected from the group consisting of cetrimonium (N,N,N-Trimethyl-1-hexadecanammonium), N,N,N-Trimethyl-1-heptadecanammonium, N,N,N-Trimethyl-1-octadecanammonium, N,N,N-Trimethyl-1-pentadecanammonium, and N,N,N-Trimethyl-1-tetradecanammonium; and (b) contacting the mixture with at least one functionalized solid to remove excess alkyl cation, wherein the functionalized solid comprises tris(2-ethylaminoethyl)amine (TREN), thereby providing a solution comprising the antibody and having a reduced aggregate content compared to the preparation prior to (a).

2. The method of claim 1 , further comprising contacting the mixture with an aryl cation.

3. The method of claim 2 , wherein the aryl cation is ethacridine or methylene blue.

4. The method of claim 1 , wherein the alkyl cation comprises a bromide or a chloride salt of cetrimonium (N,N,N-Trimethyl-1-hexadecanammonium).

5. The method of claim 1 , wherein the alkyl cation is present in a concentration range selected from the group consisting of: (a) from about 0.001% to about 1%; (b) from about 0.01 to about 1%, and (c) from about 0.02 to about 0.03% (weight/volume).

6. The method of claim 1 , wherein an operating conductivity of the mixture is within a range selected from the group consisting of: (a) from about 0.1 to about 50 mS/cm; (b) from about 1 to about 30 mS/cm; and (c) from about 5 to about 15 mS/cm.

7. The method of claim 1 , wherein an operating pH of the mixture is in a range selected from the group consisting of: (a) from about 4 to about 10; (b) from about 5 to about 9; and from about 6 to about 8.

8. The method of claim 1 , wherein the mixture further comprises an antiviral agent that is not the alkyl cation, the antiviral agent being selected from the group consisting of chlorhexidine, benzalkonium chloride, methylene blue, ethacridine, and tri(n-butyl)phosphate.

9. The method of claim 1 , wherein a surface of the at least one functionalized solid promotes a chemical interaction selected from the group consisting of: electrostatic interactions, hydrophobic interactions, hydrogen bonding, and metal affinity.

10. The method of claim 1 , wherein the at least one functionalized solid is particulate.

11. The method of claim 1 , wherein the antibody is a recombinant protein.

12. The method of claim 1 , wherein the protein preparation is one selected from the group consisting of a cell-containing cell culture harvest, a substantially cell-free cell culture harvest, and a partially purified protein.

13. A method of reducing the aggregate content in a preparation comprising a soluble antibody, the method comprising:
(a) contacting the preparation with an alkyl cation and allantoin to form a mixture, wherein the alkyl cation is present at a concentration from about 0.001% to about 0.1% (weight/volume), wherein the alkyl cation is selected from a bromide or a chloride salt of a quaternary ammonium cation selected from the group consisting of cetrimonium (N,N,N-Trimethyl-1-hexadecanammonium), N,N,N-Trimethyl-1-heptadecanammonium, N,N,N-Trimethyl-1-octadecanammonium, N,N,N-Trimethyl-1-pentadecanammonium, and N,N,N-Trimethyl-1-tetradecanammonium; and
(b) removing solids from the mixture, thereby providing a soluble antibody having a reduced aggregate content compared to the preparation prior to (a).

14. The method of claim 13 , wherein the antibody is an antibody of a class selected from IgG, IgA, IgE, IgD and IgM.

15. The method of claim 13 , further comprising contacting the mixture with a functionalized solid comprising tris(2-ethylaminoethyl)amine (TREN).

16. The method of claim 13 , wherein the allantoin is at a concentration between about 0.6% and 2% (weight/volume).

17. The method of claim 13 , wherein the alkyl cation is a bromide or a chloride salt of cetrimonium (N,N,N-Trimethyl-1-hexadecanammonium).

Assignees

• Agency Science Tech & Res
• Agency For Science Research And Tech

Inventors

• Gagnon Peter Stanley

Classifications

• C07K1/145 Primary

  by extraction or solubilisation · CPC title

• B01D15/361

  Ion-exchange · CPC title

• B01D15/327

  with hydrophobic interaction · CPC title

• C07K1/32 Primary

  as complexes · CPC title

• C07K16/00

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