PURIFICATION OF A BIVALENTLY ACTIVE ANTIBODY USING A NON-CHROMATOGRAPHIC METHOD

• US 20090240040A1
• Filed: 06/01/2007
• Published: 09/24/2009
• Est. Priority Date: 06/01/2006
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Semetey, Vincent, Krishnamurthy, Vijay M., Kaufman, George K., Bilgicer, Zihni Basar, Estroff, Lara A., Whitesides, George M., Thomas, Samuel W.
• Granted Patent: US 8,124,743 B2
• Time in Patent Office: Days
• US Class Current: 530/419
• CPC Class Codes: C07K 16/065 Purification, fragmentation
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/54 F(ab')2
• CPC Class Codes: Y10S 436/822 Identified hapten

Abstract

The present invention discloses a method of purifying bivalent antibodies or antibody fragments that are active at both Fab sites from a source of antibodies or antibody fragments using a non-chromatographic method that includes inducing the formation of cyclic immunoglobulin aggregates by addition of multivalent hapten to a salt solution of soluble antibodies or antibody fragments, wherein the multivalent hapten possesses a linker between the two haptens effective to prevent the binding of both haptens of the ligand to the same antibody or antibody fragment.

Claims

1. A method of purifying bivalent antibodies or antibody fragments that are active at both Fab sites from a source of antibodies or antibody fragments using a non-chromatographic method comprising:

(a) inducing the formation of cyclic immunoglobulin aggregates by addition of multivalent hapten to a salt solution of soluble antibodies or antibody fragments, wherein said multivalent hapten possesses a linker between the multiple haptens effective to prevent the binding of the multiple haptens of the ligand to the same antibody or antibody fragment.

2. The method of claim 1, further comprising

removing high molecular weight proteins by salt precipitation and recovery of antibody or antibody fragment that remains soluble prior to step (a).

3. The method of claim 1 or 2, further comprising

dissolving cyclic immunoglobulin aggregates recovered from (a) and dissociating them from multivalent hapten by addition of excess monovalent hapten.

4. The method of claim 3, further comprising

dialyzing the antibodies or antibody fragments to remove said monovalent haptens.

5. The method of claim 1, wherein said antibodies are monoclonal antibodies.

6. The method of claim 5 wherein said monoclonal antibodies are IgGs.

7. The method of claim 5 wherein said monoclonal antibodies are IgEs.

8. The method of claim 1, wherein said antibodies are polyclonal antibodies.

9. The method of claim 1, wherein said source is ascites.

10. The method of claim 1, wherein said salt is ammonium sulfate.

11. The method of claim 1, wherein the K_d^affinity≦

10 nM.

12. The method of claim 1, wherein the inducing step (a) is carried out in about a 35% ammonium sulfate solution.

13. The method of claim 1, wherein the multiple haptens of the multivalent hapten are the same.

14. The method of claim 1, wherein the multiple haptens of the multivalent hapten are different.

15. The method of claim 3, wherein the hapten of the monovalent hapten is the same as the hapten(s) of the multivalent hapten.

16. The method of claim 3, wherein the hapten of the monovalent hapten is different from the hapten(s) of the multivalent hapten.

17. The method of claim 1, wherein at least one hapten of the multivalent hapten is conjugated to a cross-linker by an amine-reactive chemical reaction.

18. The method of claim 1, wherein at least one hapten of the multivalent hapten is conjugated to a cross-linker by a thiol-reactive chemical reaction.

19. The method of claim 1, wherein at least one hapten of the multivalent hapten is conjugated to a cross-linker by a carboxylate-reactive chemical reaction.

20. The method of claim 1, wherein at least one hapten of the multivalent hapten is conjugated to a cross-linker by a hydroxyl-reactive chemical reaction.

21. The method of claim 1, wherein at least one hapten of the multivalent hapten is conjugated to a cross-linker by an aldehyde- or ketone-reactive chemical reaction.

22. The method of claim 1, wherein at least one hapten of the multivalent hapten is conjugated to a cross-linker by an active hydrogen-reactive chemical reaction.

23. The method of claim 1, wherein at least one hapten of the multivalent hapten is conjugated to a cross-linker by a photoreactive chemical reaction.

24. The method of claim 1, wherein the purity of the purified antibody or antibody fragment is in the range of about 30 to % pure.

25. The method of claim 1, wherein said multivalent hapten is bivalent.

26. The method of claim 25, wherein the linker length separating the hapten molecules is from 0.6 nm to 10 nm.