Purification platform for bispecific antibodies

Patent metadata

Field	Value
Publication number	US-10626142-B2
Application number	US-201514808171-A
Country	US
Kind code	B2
Filing date	Jul 24, 2015
Priority date	Jul 26, 2014
Publication date	Apr 21, 2020
Grant date	Apr 21, 2020

Abstract

High resolution protein A chromatography employing a chaotropic agent and pH gradient or pH step elution buffer results in improved peak resolution between closely related molecular species. Bispecific antibodies containing a protein A-binding-ablating substitution CH3 domain paired with a protein A-binding CH3 domain are separated with high peak resolution from monospecific antibodies containing a protein A-binding-ablating substituted CH3 domain paired with the protein A-binding-ablating substituted CH3 domain and monospecific antibodies containing a protein A-binding CH3 domain paired with the protein A-binding CH3 domain. Useful chaotropic agents include magnesium chloride and calcium chloride.

First claim

The invention claimed is:

1. A method of making a protein comprising:
a. loading an affinity matrix with a mixture of multimeric proteins comprising (i) a first homodimer comprising two copies of a first polypeptide, and (ii) a heterodimer comprising the first polypeptide and a second polypeptide, wherein the first polypeptide has greater affinity for the affinity matrix than does the second polypeptide, wherein the affinity matrix comprises a Protein A ligand affixed to a substrate comprising a multiplicity of particles having a mean diameter of 45 μm and comprising pores having a mean diameter of 1100 Å; and
b. eluting and collecting the heterodimer from the affinity matrix in a buffer comprising CaCl 2 or MgCl 2 and having a first pH range,
wherein the first homodimer elutes from the affinity matrix in the buffer at a second pH range.

2. The method according to claim 1 , wherein the mixture of multimeric proteins comprises a second homodimer comprising two copies of the second polypeptide.

3. The method according to claim 2 , wherein the second homodimer elutes from the affinity matrix in the buffer at a third pH range, wherein the third pH range comprises a higher pH than the first pH range, which comprises a higher pH than the second pH range.

4. The method according to claim 1 , wherein the substrate comprises any one or more of agarose, poly(styrene divinylbenzene), polymethacrylate, cellulose, controlled pore glass, and spherical silica.

5. The method according to claim 1 , wherein 5 to 50 grams of protein is loaded per liter of affinity matrix.

6. The method according to claim 1 comprising the step of applying a pH gradient to the loaded affinity matrix of step (a).

7. The method according to claim 6 comprising the step of washing the loaded affinity matrix of step (a) with a solution at pH 6-8 prior applying the pH gradient.

8. The method according to claim 7 , wherein the pH gradient is run between pH 6 and pH 3.

9. The method according to claim 1 , wherein the first pH range is selected from a range within pH 5.5 and pH 3.6.

10. The method according to claim 9 , wherein the buffer comprises acetate.

11. The method according to claim 10 , wherein the buffer comprises 40 mM acetate.

12. The method according to claim 1 , wherein the buffer comprises CaCl 2 .

13. The method according to claim 12 , wherein the buffer comprises 250-500 mM CaCl 2 .

14. The method according to claim 1 , wherein the buffer comprises MgCl 2 .

15. The method according to claim 14 , wherein the buffer comprises 250-500 mM MgCl 2 .

16. The method according to claim 1 , wherein the heterodimer comprises a bispecific antibody.

17. The method according to claim 16 , wherein the first polypeptide comprises a CH3 domain that is capable of binding to Protein A and the second polypeptide comprises a CH3 domain that is not capable of binding to Protein A.

18. The method according to claim 17 , wherein the second polypeptide comprises a HY to RF substitution in its CH3 domain.

19. The method according to claim 1 , wherein the mixture of multimeric proteins is produced by a plurality of eukaryotic cells in a cell culture.

20. The method according to claim 19 , wherein the eukaryotic cells comprise Chinese hamster ovary (CHO) cells or derivatives thereof.

21. The method according to claim 1 comprising the steps of:
c. applying the collected heterodimer of step (b) to a multimodal chromatography resin in a buffer having an acidic pH;
d. eluting the heterodimer from the multimodal chromatography resin in a buffer having a more alkaline pH; and
e. collecting the heterodimer.

22. A method of producing a protein comprising the steps of:
a. loading a first affinity matrix comprising a Protein A ligand affixed to a substrate with a mixture of multimeric proteins, the mixture comprising:
i. a first homodimer comprising two copies of a first polypeptide,
ii. a heterodimer comprising the first polypeptide and a second polypeptide, and
iii. a second homodimer comprising two copies of the second polypeptide;
b. washing the second homodimer from the first affinity matrix in a wash buffer;
c. eluting and collecting the first homodimer and the heterodimer from the first affinity matrix;
d. loading a second affinity matrix comprising a Protein A ligand affixed to a substrate with a mixture comprising the first homodimer and the heterodimer collected at step (c);
e. eluting and collecting the heterodimer from the affinity matrix in a buffer having a first pH range and comprising CaCl 2 or MgCl 2 ,
wherein, the first homodimer elutes from the affinity matrix in the buffer at a second pH range, and
wherein the first affinity matrix, the second affinity matrix, or both comprise a Protein A ligand affixed to the substrate, wherein the substrate is a particle and the first affinity matrix, the second affinity matrix, or both comprise a multiplicity of particles of a mean diameter of 45 μm and comprising pores having a mean diameter of 1100 Å.

23. The method according to claim 22 , wherein the substrate of the first affinity matrix, the second affinity matrix, or both comprise any one or more of agarose, poly(styrene divinylbenzene), polymethacrylate, controlled pore glass, and spherical silica agarose.

24. The method according to claim 22 , wherein 5 to 50 grams of protein in the mixture of multimeric proteins is loaded onto the first affinity matrix.

25. The method according to claim 24 , wherein 5 to 50 grams of protein in the mixture comprising the first homodimer and the heterodimer collected at step (c) is loaded per liter of second affinity matrix at step (d).

26. The method according to claim 22 comprising the step of applying a pH gradient to the loaded affinity matrix of step (d).

27. The method according to claim 26 , wherein the pH gradient is run between pH 6 and pH 3.

28. The method according to claim 22 , wherein the first pH range is between pH 5.5 and pH 3.6.

29. The method according to claim 22 , wherein the buffer of step (e) comprises acetate.

30. The method according to claim 29 , wherein the buffer comprises 40 mM acetate.

31. The method according to claim 22 , wherein the buffer of step (e) comprises CaCl 2 .

32. The method according to claim 31 , wherein the buffer comprises 250-500 mM CaCl 2 .

33. The method according to claim 22 , wherein the buffer of step (e) comprises MgCl 2 .

34. The method according to claim 33 , wherein the buffer comprises 250-500 mM MgCl 2 .

35. The method according to claim 22 , wherein the heterodimer comprises a bispecific antibody.

36. The method according to claim 35 , wherein the first polypeptide comprises a CH3 domain that is capable of binding to Protein A and the second polypeptide comprises a CH3 domain that is not capable of binding to Protein A.

37. The method according to claim 36 , wherein the second polypeptide comprises a HY to RF substitution in its CH3 domain.

38. The method according to claim 22 , wherein the mixture of multimeric proteins is produced by a plurality of eukaryotic cells in a cell culture.

39. The method according to claim 38 , wherein the eukaryotic cells comprise Chinese hamst

Assignees

• Regeneron Pharma

Inventors

• Tustian Andrew
• Endicott Christine
• Adams Benjamin
• Mattila John
• Bak Hanne

Classifications

• B01D15/3828

  Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography · CPC title

• C07K2317/14

  Specific host cells or culture conditions, e.g. components, pH or temperature · CPC title

• C07K2317/31

  multispecific · CPC title

• C07K1/22 Primary

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title