Methods of adenovirus purification

• US 7,326,555 B2
• Filed: 05/13/2003
• Issued: 02/05/2008
• Est. Priority Date: 05/14/2002
• Status: Active Grant

• Current Assignee: Merck Sharp & Dohme LLC (Merck & Co., Inc.)
• Original Assignee: Merck & Co., Inc.
• Inventors: To, Chi Shung Brian, Goerke, Aaron R, Lee, Ann L., Konz, John O. Jr.
• Primary Examiner(s): Chen; Stacy B.
• Application Number:
  US10/512,779
• Publication Number:
  US 20050153420A1
• Time in Patent Office: 1,729 Days
• Field of Search: None
• US Class Current: 435/239
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/10343 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2710/10351 Methods of production or purification of viral material
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

A process for purifying virus particles, especially recombinant adenovirus vector particles, is presented. The process relies on various combinations of cell lysis, detergent-based precipitation of host cell contaminants away from the virus, depth filtration or centrifugation, ultrafiltration, nuclease digestion and chromatography to robustly and economically produce highly purified product. This process results in contaminating DNA levels which are consistently below detectable levels.

Claims

1. A method of purifying adenovirus particles from a cell culture medium comprising adenovirus particles which comprises:

a) lysing host cells within the cell culture medium;

b) selectively precipitating impurity DNA away from the adenovirus particles by adding a selective precipitation agent to the cell culture medium, wherein the selective precipitation agent is selected from the group consisting of domiphen bromide (DB), cetylpyridinium chloride (CPC), cetyltrimethylammonium bromide (CTAB), benzethonium chloride (BTC), tetradecyltrimethylammonium bromide or chloride (TTA), and polyethylene imine (PEI);

c) clarifying the cell culture medium; and

d) recovering purified adenovirus particles from the cell culture medium;

wherein at least 80% of impurity DNA is precipitated away from the cell culture medium.

2. The method of claim 1 which further comprises:

e) conducting ultrafiltration on the adenovirus-containing medium recovered from step d); and

f) recovering concentrated, purified adenovirus particles.

3. The method of claim 1 wherein step d) is performed by a method selected from the group consisting of depth filtration, dead end filtration, microfiltration, centrifugation or a combination thereof.

4. The method of claim 1 wherein step d) is performed by depth filtration.

5. The method of claim 1 wherein step d) is performed by the combination of centrifugation and depth filtration.

6. The method of claim 2 wherein the purified adenovirus particles recovered from step f) are subjected to cation exchange chromatography.

7. The method of claim 1 wherein the clarified medium recovered from step d) is treated with a nuclease.

8. The method of claim 1 which further comprises:

e) subjecting the adenovirus-containing medium recovered from step d) to anion exchange chromatography;

f) diafiltering the adenovirus-containing eluate recovered from step e); and

g) recovering concentrated, purified adenovirus.

9. The method of claim 8 wherein step d) is performed by a method selected from the group consisting of depth filtration, dead end filtration, microfiltration, centrifugation or a combination thereof.

10. The method of claim 1 which further comprises:

e) conducting a first ultrafiltration process on the adenovirus-containing medium recovered from step d);

f) subjecting the adenovirus-containing medium recovered from step e) to anion exchange chromatography;

g) conducting a second ultrafiltration process on the adenovirus-containing medium recovered from step f);

h) optionally subjecting the adenovirus particles recovered from step g) to cation exchange chromatography; and

i) recovering concentrated, purified adenovirus.

11. The method of claim 10 wherein step d) is performed by a method selected from the group consisting of depth filtration, dead end filtration, microfiltration, centrifugation or a combination thereof.

12. The method of claim 10 wherein step d) is performed by depth filtration.

13. The method of claim 10 wherein step d) is performed by the combination of centrifugation and depth filtration.

14. The method of claim 1 which further comprises subjecting medium comprising the purified adenovirus particles to anion exchange chromatography wherein said anion exchange chromatography employs a buffer comprising a detergent.

15. The method of claim 14 wherein the detergent is a non-ionic surfactant.

16. The method of claim 15 wherein the detergent is polysorbate-80 (PS-80).

17. The method of claim 16 wherein the concentration of PS-80 is 0.1%.

18. The method of claim 1 which further comprises a diafiltration step into a buffer with a pH between 6.5 and 8.0.

19. The method of claim 18 wherein diafiltration is into a buffer comprising 50 mM HEPES, 2 mM MgCl2, 1 M NaCl, pH7.5.

20. The method of claim 18 wherein the buffer further comprises polysorbate-80 (PS-80).