Methods for cultivating cells and propagating viruses

• US 6,146,891 A
• Filed: 01/28/1998
• Issued: 11/14/2000
• Est. Priority Date: 01/31/1997
• Status: Expired due to Fees

• Current Assignee: Schering Corporation (Merck & Co., Inc.)
• Original Assignee: Schering Corporation (Merck & Co., Inc.)
• Inventors: Condon, Russell G.G., Connelly, Nancy V., Yabannavar, Vijay, Frei, Andreas, Glowacki, Edward, Batandolo, Serge
• Primary Examiner(s): Scheiner, Laurie
• Application Number:
  US09/014,444
• Time in Patent Office: 1,021 Days
• Field of Search: 435/41, 435/69.1, 435/235.1, 435/239, 435/243, 435/289.1, 435/325, 435/378, 435/380, 435/395, 435/403
• US Class Current: 435/378
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2531/00 Microcarriers
• CPC Class Codes: C12N 2710/10321 Viruses as such, e.g. new isolates, mutants or their genomic sequences
• 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 5/0075 using microcarriers
• 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

This invention relates to methods for the cultivating cells, and in particular to methods for propagating viruses.

Claims

1. A method for cultivating cells, comprising:

(a) cultivating the cells on a first batch of microcarriers until the cells are substantially confluent;

(b) detaching the cells from the microcarriers without removing the microcarriers from suspension and without allowing the microcarriers to settle;

(c) after step (b), adding a second batch of microcarriers; and

(d) cultivating the cells further.

2. The method of claim 1 wherein the cells are detached from the first batch of microcarriers by adding trypsin.

3. The method of claim 1 wherein the step of detaching the cells from the first batch of microcarriers comprises the following steps:

(a) washing the microcarriers and attached cells to remove soluble materials;

(b) contacting the microcarriers and washed cells with a chelating agent;

(c) removing the chelating agent;

(d) trypsinizing the cells for a short period to detach the cells from the microcarriers; and

(e) neutralizing the trypsin by adding protein, wherein (a)-(e) are conducted in a single cultivation vessel.

4. The method of claim 3 wherein the protein added in (e) is serum.

5. The method of claim 3, wherein the cultivation vessel is a bioreactor.

6. The method of claim 3 wherein the chelating agent is EDTA.

7. The method of claim 3 wherein the trypsin is used in (d) at a concentration of from about 0.05% to about 0.1% for 5 to 10 minutes.

8. The method of claim 3 wherein the trypsin is used in step (d) at a concentration of from about 0.065% to about 0.075% for about 8 minutes.

9. The method of claim 1, wherein a virus capable of infecting the cells is added during (d).

10. The method of claim 9 wherein the virus is added when the cells have become substantially confluent on the microcarriers.

11. The method of claim 9 wherein the virus comprises an adenoviral vector.

12. The method of claim 9 wherein the virus carries a recombinant gene.

13. The method of claim 12 wherein the virus carries a tumor suppressor gene.

14. The method of claim 13 wherein the virus is ACN53.

15. The method of claim 14 wherein the cells are 293 cells.

16. The method of claim 15 wherein the cells are incubated for from 3 to 4 days.

17. The method of claim 1 wherein (c) is carried out in a larger bioreactor than used in (b).

18. The method of claim 1 wherein the microcarriers have a particle size in the range of 100 to 250 microns.

19. The method of claim 1, wherein steps (a) and (b) are carried out in the same vessel.

20. The method of claim 1, wherein steps (a) through (d) are carried out without removing the microcarriers from suspension and without allowing the microcarriers to settle.

21. The method of claim 1, wherein existing culture media is removed during step (b) and new culture media is added.

22. The method of claim 21, wherein the existing culture media is removed through a rotating spinfilter.

23. The method of claim 1, wherein the microcarriers are treated to enhance cell adhesion.

24. The method of claim 22, wherein the microcarriers are coated with collagen.