Method of purification of viral vectors

• US 5,837,520 A
• Filed: 03/07/1995
• Issued: 11/17/1998
• Est. Priority Date: 03/07/1995
• Status: Expired due to Term

• Current Assignee: Canji Inc. (Merck & Co., Inc.)
• Original Assignee: Canji Inc. (Merck & Co., Inc.)
• Inventors: Liu, Xiaodong, Huyghe, Bernard G., Shabram, Paul W., Shepard, H. Michael
• Primary Examiner(s): Chambers, Jasemine C.
• Assistant Examiner(s): HAUDA, KAREN MARIE
• Application Number:
  US08/400,793
• Time in Patent Office: 1,351 Days
• Field of Search: 435/235.1, 435/239, 435/320.1, 435/803, 435/69.1, 424/93.2, 530/412
• US Class Current: 435/239
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• CPC Class Codes: C08F 265/02 on to polymers of acids, salts or anhydrides
• CPC Class Codes: C09D 11/106 containing macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds
• 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: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding

Abstract

The invention provides a method for purifying viral vectors containing therapeutic genes for use in gene therapy. The invention comprises a method of purification from a cell lysate of a recombinant viral vector containing a therapeutic gene, which comprises: a) treating said lysate with an enzymatic agent that selectively degrades both unencapsulated DNA and RNA; b) chromatographing the treated lysate from step a) on a first resin; and c) chromatographing the eluant from step b) on a second resin; wherein one resin is an anion exchange resin and the other is an immobilized metal ion chromatography (IMAC) resin.

Claims

1. A method for purification of an intact viral particle from a cell lysate, the method comprising:

a) treating said cell lysate which contains said intact viral particle with an enzymatic agent that selectively degrades both unencapsulated DNA and RNA;

b) chromatographing the treated lysate from step a) on a first resin; and

c) chromatographing the eluant from step b) on a second resin;

wherein one resin is an anion exchange resin and the other is an immobilized metal ion affinity resin.

2. The method of claim 1, wherein the first resin is an anion exchange resin and the second resin is an immobilized metal affinity resin.

3. The method of claim 1, which comprises the additional step of filtering the treated lysate from step (a).

4. The method of claim 1, which comprises the additional step of buffering the pH of the cell lysate between about 5.0 and about 9.0 before applying it to the first resin.

5. The method of claim 1, wherein the viral particle is a retrovirus.

6. The method of claim 1, wherein the viral particle is an adenovirus.

7. The method of claim 6, wherein the adenoviral particle is a recombinant viral particle which comprises a tumor suppressor gene.

8. The method of claim 1, wherein the adenoviral particle is a type 2 or type 5 adenovirus.

9. The method of claim 8, wherein the adenoviral particle is a type 5 adenovirus.

10. The method of claim 7, wherein the tumor suppressor gene is a wild-type p53 gene.

11. The method of claim 1, wherein the anion exchange resin is chosen from the group consisting of DEAE, TMAE, DMAE, QAE and PEI.

12. The method of claim 11, wherein the anion exchange resin is DEAE resin.

13. The method of claim 1, wherein the immobilized metal affinity resin is charged with a divalent cation of a metal chosen from the group consisting of cobalt, nickel, copper, and zinc.

14. The method of claim 13, wherein the immobilized metal affinity resin is a TED or an IDA resin.

15. The method of claim 14, wherein the immobilized metal affinity resin is an IDA-(cross-linked agarose) resin.

16. The method of claim 13, wherein the divalent cation is zinc.

17. The method of claim 1, wherein the enzymatic agent that selectively degrades both unencapsulated DNA and RNA is one or more enzymes.

18. The method of claim 17, wherein the one or more enzymes are endonucleases.

19. The method of claim 18, wherein the enzyme is a mixture of RNase and DNase.

20. A method for purification of intact viral particles from a cell lysate, the method comprising the steps of:

a) treating said cell lysate which contains said intact viral particle with an enzymatic agent that selectively degrades both unencapsulated DNA and RNA;

b) chromatographing the treated lysate from step a) on a first resin, and

c) chromatographing the eluant from step b) on a second resin;

wherein;

one resin is an anion exchange resin and the other is a hydrophobic interaction chromatography resin;

or

one resin is a cation exchange resin and the other is either a hydrophobic interaction chromatography resin or an immobilized metal ion affinity resin.

21. The method of claim 20, wherein one resin is an anion exchange resin and the other is a hydrophobic interaction chromatography resin.

22. The method of claim 21, wherein the first resin is an anion exchange resin and the second is a hydrophobic interaction chromatography resin.

23. The method of claim 20, wherein one resin is a cation exchange resin and the other is an immobilized metal ion affinity resin.

24. The method of claim 20, wherein one resin is a cation exchange resin and the other is a hydrophobic interaction chromatography resin.

25. A method of determining the number of intact viral particles in a sample, the method comprising:

a) chromatographing the sample containing the intact viral particles on an anion exchange resin;

b) monitoring the absorbance of the eluate from the chromatography of step (a) at a selected wavelength; and

c) determining the total number of intact viral particles in the sample by comparing the absorbance value obtained in step (b) to a standard curve which relates absorbance to number of viral particles.

26. The method of claim 25, wherein the anion exchange resin has QAE groups covalently attached to a polystyrene/divinyl benzene copolymer support.

27. The method of claim 25, wherein the method further comprises chromatographing the eluate from the chromatography of step (a) on a second resin, wherein the second resin is an immobilized metal ion affinity resin.