Chromatographic purification of virus preparations with negatively charged particles

Patent metadata

Field	Value
Publication number	US-9902942-B2
Application number	US-201314768213-A
Country	US
Kind code	B2
Filing date	Feb 22, 2013
Priority date	Feb 22, 2013
Publication date	Feb 27, 2018
Grant date	Feb 27, 2018

Abstract

A method of purifying a sample that includes a desired virus includes the steps of (i) providing a packed chromatographic column having negatively charged porous particles, (ii) equilibrating the column to the conditions to which the desired virus in the sample is to elute, (iii) contacting the sample with the packed chromatographic column such that the sample volume applied to the packed chromatographic column is less than or equal to the interparticle space of the negatively charged porous particles within the packed chromatographic column, (iv) eluting the desired virus from the packed chromatographic column, where the desired virus is in a purer state and in the conditions to which the packed chromatographic column was equilibrated.

First claim

The invention claimed is:

1. A method of purifying a sample comprising a bacteriophage the method comprising the steps of:
(i) providing a packed chromatographic column comprising negatively charged porous particles wherein the particles have pores smaller in size than the bacteriophage;
(ii) equilibrating the packed chromatographic column with an equilibration buffer having a sufficient amount of salt, citrate or an acetate that yields a column conductivity in a range from 0.1 mS/cm to 30 mS/cm at a pH in a range from 5 to 7, wherein the bacteriophage does not substantially bind to the equilibrated packed chromatography column;
(iii) contacting the sample to the packed chromatographic column of (ii), wherein a sample volume applied consists of a volume that is less than or equal to a void volume of the negatively charged porous particles within the equilibrated packed chromatographic column; and
(iv) collecting a column effluent from the packed chromatographic column, wherein the column effluent comprises the bacteriophage in a more pure state compared to the sample applied in (iii) and, wherein the method does not use a column washing step or elution step.

2. The method of claim 1 , wherein the sample is unpurified, at an intermediate level of purity, highly purified, or concentrated.

3. The method of claim 1 , wherein the bacteriophage has been concentrated prior to its application to the column by ultrafiltration.

4. The method of claim 1 , wherein the sample has been previously exposed to a negatively charged surface or surfaces under conditions that provide a substantial absence of binding of the virus to the surface or surfaces prior to its application to the packed chromatographic column, and wherein the negatively charged surface or surfaces comprise a membrane, a monolith, a fiber or plurality of fibers, a particle or plurality of particles, or a compound construction thereof.

5. The method of claim 4 , wherein the negatively charged surface or surfaces further comprise secondary reactivities comprising the ability to participate in hydrophobic interactions, metal affinity interaction, or combinations thereof.

6. The method of claim 1 , wherein the packed chromatographic column is packed solely with the negatively charged porous particles and the sample volume is less than 40% of a volume of the packed chromatographic column.

7. The method of claim 1 , wherein the equilibration buffer has a pH in a range of from about 4 to about 7, or from about 4.5 to about 6.0.

8. The method of claim 1 , wherein the equilibration buffer comprises a conductivity value in a range of from about 0.1 mS/cm to about 15 mS/cm.

9. The method of claim 1 , wherein the sample comprises a sample application condition for contacting the sample with the packed chromatographic column, the sample application condition comprising a pH in a range from about 2 to about 10, or a range of conductivity from a non-zero conductivity up to a conductivity corresponding to a saturated solution of a particular salt or combination of salts.

10. The method of claim 1 , wherein the negatively charged porous particles comprise cation exchange particles, wherein electronegativity is optionally conferred to the negatively charged porous particles partly by a moiety selected from the group consisting of a carboxyl, a sulfa, or a phosphor moiety.

11. The methods of claim 1 , wherein the negatively charged porous particles comprise one or more secondary chemical functionalities selected from the group consisting of anion exchange, hydrophobic interactions, hydrogen bonding, pi-pi interactions, and metal chelation.

12. The method of claim 1 , comprising the additional step of contacting the sample with one or more contaminant-dissociating agents prior to the step of contacting the sample with the packed chromatographic column.

13. The method of claim 12 , wherein the one or more contaminant-dissociating agents are selected from the group consisting of salts, nonionic organic polymers, organic solvents, surfactants, and chaotropes.

14. The method of claim 13 , wherein the one or more contaminant-dissociating agents comprise a nonionic organic polymer selected from the group consisting of polyethylene glycol, polypropylene glycol and polybutylene glycol.

15. The method of claim 14 , wherein the nonionic organic polymer has an average molecular weight in a range from about 130 D to about 1000 D.

16. The method of claim 13 , wherein the one or more contaminant-dissociating agents comprise an organic solvent selected from the group consisting of ethylene glycol, propylene glycol, butylene glycol, dimethylsulfoxide, ethanol, and phenoxyethanol.

17. The method of claim 12 , wherein the one or more contaminant-dissociating agents are provided at a concentration range from about 0.01% to about 25% weight/volume.

18. The method of claim 13 , wherein the one or more contaminant-dissociating agents comprise a surfactant selected from the group consisting of Tween, triton, CHAPS, CHAPSO and octyl glucoside, wherein the surfactant is provided at a concentration range from about 0.001% to about 1% weight/volume or from about 0.001% to about 0.1% weight/volume.

Assignees

• Agency Science Tech & Res

Inventors

• Gagnon Peter Stanley

Classifications

• C12N2710/00051

  Methods of production or purification of viral material · CPC title

• B01D15/363

  Anion-exchange · CPC title

• B01D61/145

  Ultrafiltration · CPC title

• C12N7/00 Primary

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• B01D15/424

  Elution mode · CPC title