Method for producing recombinant adenovirus

• US 6,485,958 B2
• Filed: 12/16/1998
• Issued: 11/26/2002
• Est. Priority Date: 07/01/1996
• Status: Expired due to Fees

• Current Assignee: Centelion
• Original Assignee: Gencell SA (France) (Sanofi )
• Inventors: Blanche, Francis, Guillaume, Jean-Marc
• Primary Examiner(s): MOSHER, MARY
• Application Number:
  US09/202,545
• Publication Number:
  US 20020028497A1
• Time in Patent Office: 1,441 Days
• Field of Search: 435/235.1, 435/239, 435/320.1, 424/199.1, 424/233.1, 424/93.2
• US Class Current: 435/239
• CPC Class Codes: B01D 15/363 Anion-exchange
• CPC Class Codes: B01D 61/145 Ultrafiltration
• CPC Class Codes: B01J 41/20 Anion exchangers for chromatographic processes
• 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
• CPC Class Codes: G01N 30/02 Column chromatography

Abstract

The invention concerns a method for producing recombinant adenovirus by which viral DNA is introduced in a packaging cell culture and the viruses produced are harvested after liberation in the supernatant. The invention also concerns the viruses produced and their use.

Claims

1. A method for producing recombinant adenoviruses, the method comprising introducing adenoviral DNA into a culture of encapsidation cells, and harvesting adenoviruses following their release into culture supernatant without lysis of the encapsidation cells by an external factor.

2. The method according to claim 1, wherein the harvesting is performed when at least 50% of the adenoviruses have been released into the supernatant.

3. The method according to claim 1, wherein the harvesting is performed when at least 70% of the adenoviruses have been released into the supernatant.

4. The method according to claim 1, wherein the harvesting is performed when at least 90% of the adenoviruses have been released into the supernatant.

5. The method according to claim 1, wherein the harvesting is by ultrafiltration.

6. The method according to claim 5, wherein the ultrafiltration is tangential ultrafiltration.

7. The method according to claim 5, wherein the ultrafiltration is performed on a membrane having a cut-off of less than 1000 kDa.

8. The method according to claim 1, wherein the harvesting is by anion-exchange chromatography.

9. The method according to claim 8, wherein the anion-exchange chromatography is performed on a strong anion-exchange resin.

10. The method according to claim 9, wherein the strong anion-exchange resin is selected from the group consisting of Source Q, Mono Q, Q Sepharose, Poros HQ, Poros QE, a Fractogel TMAE and Toyopearl Super Q.

11. The method according to claim 1, wherein the harvesting is by gel permeation chromatography.

12. The method according to claim 11, wherein th gel permeation chromatography is performed on a support selected from the group consisting of Sephacryl S-500 HR, Sephacryl S-1000 SF, Sephacryl S-1000 HR, Sephacryl S-2000, Superdex 200 HR, Sepharose 2B, 4B or 6B and TSK G6000 PW.

13. The method according to claim 1, wherein the harvesting is by ultrafiltration followed by anion-exchange chromatography.

14. The method according to claim 13, wherein the harvesting is by ultrafiltration followed by anion-exchange chromatography and then gel permeation chromatography.

15. The method according to claim 1, wherein the encapsidation cell transcomplements adenovirus E1 functions.

16. The method according to claim 15, wherein the encapsidation cell transcomplements adenovirus E1 and E4 functions.

17. The method according to claim 15, wherein the encapsidation cell transcomplements adenovirus E1 and E2a functions.

18. The method according to claim 15, wherein the cell is an embryonic human kidney cell, a human retinoblast or a cell from a human carcinoma.

19. The method according to claim 10, wherein the strong anion exchange resin is Source 15 Q.