CRYSTALLIZATION OF ANTI-CD20 ANTIBODIES

• US 20110020322A1
• Filed: 06/17/2010
• Published: 01/27/2011
• Est. Priority Date: 12/21/2007
• Status: Abandoned Application

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Lobo, Brian, Oshodi, Shadia Abike, Breece, Timothy, Wilkins, James A.
• Application Number:
  US12/818,017
• Publication Number:
  US 20110020322A1
• Time in Patent Office: Days
• US Class Current: 424/130.1
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/065 Purification, fragmentation
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C07K 2299/00 Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Abstract

The present invention relates generally to crystalline forms of anti-CD20 antibodies and purification of anti-CD20 antibodies involving crystallization.

Claims

1. A method of purifying a CD20 antibody from a mixture, comprising crystallizing the CD20 antibody and recovering the CD20 antibody from said mixture.

2. The method of claim 1 wherein said mixture has not been subjected to prior lyophilization.

3. The method of claim 1 wherein said mixture is concentrated Harvested Cell Culture Fluid (HCCF) of a recombinant cell culture producing the CD20 antibody.

4. The method of claim 3 wherein the cell culture is a mammalian cell culture.

5. The method of claim 4 wherein the mammalian cells are Chinese Hamster Overy (CHO) cells.

6. The method of claim 4 wherein the purification is performed in the absence of a Protein A purification step.

7. The method of claim 6 wherein the purification is performed in the absence of a cation exchange chromatography step.

8. The method of claim 4 wherein the purification additionally comprises a viral filtration step and an anion exchange chromatography step.

9. The method of claim 8 comprising the steps of (a) crystallizing the CD20 antibody, (b) dissolving the CD20 antibody crystals in a buffer, (c) subjecting the solution obtained from step (b) to anion exchange chromatography, and (d) concentrating the eluate obtained from the anion exchange chromatography.

28. A method for treating a CD20-associated condition or disease comprising administering to a mammalian subject an effective amount of a CD20 antibody purified by the method of claim 1 or claim 21.

10. A method of purifying a CD20 antibody from concentrated Harvested Cell Culture Fluid (HCCF) of mammalian cells, comprising the steps of (a) concentrating the HCCF, (b) crystallizing the CD20 antibody, (c) dissolving the CD20 crystals to obtain a CD20 solution, (d) subjecting the CD20 solution to purification on an anion exchange column, and (c) isolating the CD20 antibody.

11. The method of claim 10 wherein the CD20 antibody is a 2H7 antibody.

12. The method of claim 11 wherein the CD20 antibody is selected from the group consisting of 2H7 CD20 antibody variants A-I listed in Table 1.

13. The method of claim 12 wherein the CD20 antibody is selected from the group consisting of 2H7 CD20 antibody variants A, C and H listed in Table 1, having VL and VI-1 pairs of SEQ ID NOs:

1 and 2;

SEQ ID NOs;

3 and 4; and

SEQ ID NOs;

3 and 5, respectively.

14. The method of claim 11 wherein the HCCF is concentrated to a CD20 antibody concentration at about or greater than 1.5 mg/ml.

15. The method of claim 10 wherein crystallization is performed at a pH of about 6.0 to about 8.0.

16. The method of claim 15 wherein crystallization is performed at a pH of 7.8+/−

0.2.

17. The method of claim 10 wherein crystallization is performed at a temperature of about 4°

C. to about 40°

C.

18. The method of claim 17 wherein crystallization is performed at a temperature of about 37°

C.

19. The method of claim 10 wherein crystallization is induced by one or more precipitant selected from the group consisting of PBS, NaCl, Na₂SO₄, KCl, K₂SO₄, Na₂HPO₄, and KH₂PO₄.

20. The method of claim 19 wherein the precipitant is KH₂PO₄.

21. A method of purifying a CD20 antibody from concentrated Harvested Cell Culture Fluid (HCCF) of mammalian cells, comprising the steps of (a) concentrating the HCCF, (b) diafiltering the HCCF with a high salt concentration at a pH that inhibits crystallization, (c) crystallizing the CD20 antibody by raising the pH, (d) dissolving the CD20 antibody crystals to obtain a CD20 antibody solution, (e) subjecting the CD20 antibody solution to purification on an anion exchange column, and (f) isolating the resultant purified CD20 antibody.

22. The method of claim 21 wherein the CD20 antibody is selected from the group consisting of 2H7 CD20 antibody variants A-I listed in Table 1.

23. The method of claim 22 wherein the CD20 antibody is selected from the group consisting of 2H7 CD20 antibody variants A, C and H listed in Table 1, having VL and VH pairs of SEQ ID NOs:

1 and 2;

SEQ ID NOs;

3 and 4; and

SEQ ID NOs;

3 and 5, respectively.

24. A crystal of a CD20 antibody.

25. The crystal of claim 24 which has a microneedle, needle, globular or globular peanut morphology.

26. A composition comprising a crystal of claim 24.

27. The composition of claim 26 which is a pharmaceutical composition, comprising one or more pharmaceutically acceptable excipients.