Methods and compositions with reduced opalescence

• US 7,744,890 B2
• Filed: 10/12/2007
• Issued: 06/29/2010
• Est. Priority Date: 10/12/2006
• Status: Expired due to Fees

• Current Assignee: Wyeth (Pfizer Inc.)
• Original Assignee: Wyeth LLC (Pfizer Inc.)
• Inventors: Luisi, Donna L., Nichols, Pilarin Elizabeth
• Primary Examiner(s): Saunders; David A
• Application Number:
  US11/974,312
• Publication Number:
  US 20080154023A1
• Time in Patent Office: 991 Days
• Field of Search: None
• US Class Current: 424/177.1
• CPC Class Codes: A61K 39/39591 Stabilisation, fragmentation

Abstract

Methods for reducing the opalescent appearance of a protein preparation by modifying the ionic strength of the preparation, as well as compositions, e.g., pharmaceutical compositions, of concentrated protein with decreased opalescence are disclosed. Purification methods which monitor and/or reduce the salt concentrations at selected steps are also disclosed.

Claims

1. A method for decreasing the opalescence of an antibody preparation, the method comprising:

(a) determining a second virial coefficient of an antibody in the antibody preparation; and

(b) if the second virial coefficient of the antibody is negative, reducing the ionic strength of the antibody preparation such that the opalescence of the antibody preparation is reduced.

2. The method of claim 1, wherein the antibody comprises a human, humanized, chimeric, CDR-grafted, or an in vitro generated antibody, or an antigen-binding fragment thereof.

3. The method of claim 1, wherein the antibody comprises an antibody having IgG1 or an IgG4 heavy chain constant region.

4. The method of claim 1, wherein the second virial coefficient of the antibody is determined at a salt concentration ranging from about 50 to 200 mM.

5. The method of claim 4, wherein the second virial coefficient of the antibody is determined to be about −

1 to −

10×

10<sup>−

3</sup>, or about −

1 to −

10×

10<sup>−

4</sup> mol-mg/g² at 100 mM sodium chloride.

6. The method of claim 1, wherein the antibody is present at a concentration of about 5 to 300 mg/ml.

7. The method of claim 1, wherein the ionic strength of the antibody preparation is reduced by decreasing the concentration of a salt present in the preparation.

8. The method of claim 7, wherein the salt present in the preparation is selected from the group consisting of sodium chloride, calcium chloride, magnesium chloride and sodium phosphate.

9. The method of claim 7, wherein the concentration of the salt is reduced to at least about two-, three-, five-, ten- or one hundred-fold lower than the initial concentration in the antibody preparation.

10. The method of claim 7, wherein the ionic strength of the antibody preparation is reduced by replacing a first salt with a second salt, wherein the first salt is NaCl, and the second salt is selected from the group consisting of Na₂HPO₄, MgCl₂, and CaCl₂.

11. The method of claim 7, wherein the ionic strength of the antibody preparation is reduced by replacing a first salt with a second salt, wherein the first salt is Na₂HPO₄, and the second salt is selected from the group consisting of MgCl₂ and CaCl₂.

12. The method of claim 1, wherein the ionic strength of the antibody preparation is reduced by ultra-filtration or dialysis.

13. The method of claim 1, wherein the ionic strength of the antibody preparation is reduced in a process of purifying the antibody, wherein the process of purifying the antibody comprises one or more of centrifugation, filtration, chromatography, lyophilization or reconstitution.

14. The method of claim 1, further comprising evaluating the opalescence of the antibody preparation.

15. The method of claim 14, wherein the opalescence of the antibody preparation is evaluated by determining the turbidity of the solution, a change in the second virial coefficient of the antibody, or a change in the formation of high molecular weight species.

16. The method of claim 15, wherein the opalescence is reduced to about 2 or 1 European Pharmacopoeia standard or less.

17. The method of claim 15, wherein the second virial coefficient changes from about −

1 to −

10×

10<sup>−

3</sup> to about −

1 to −

10×

10<sup>−

4</sup>.

18. The method of claim 15, wherein the high molecular weight species is reduced by an amount of about two-, three-, four-, five-, six-, seven-, eight-, nine-, ten-, fifty- or one hundred-fold.

19. The method of claim 1, wherein the second virial coefficient of the antibody is determined by a static light scattering and Zimm analysis.

20. A method for reducing the formation of high molecular weight species in an antibody preparation, the method comprising:

(a) providing an antibody preparation comprising an antibody determined to have a negative second virial coefficient;

(b) reducing the ionic strength of the antibody preparation such that the high molecular weight species in the antibody preparation are reduced.

21. The method of claim 20, wherein the antibody comprises a human, humanized, chimeric, CDR-grafted, or an in vitro generated antibody, or an antigen-binding fragment thereof.

22. The method of claim 20, wherein the antibody comprises an IgG1 or an IgG4 heavy chain constant region.

23. The method of claim 20, wherein the antibody of the antibody preparation has a negative second virial coefficient in a salt concentration ranging from about 50 to 200 mM.

24. The method of claim 23, wherein the second virial coefficient is about −

1 to −

10×

10<sup>−

3</sup>, or about −

1 to −

10×

10<sup>−

4</sup> mol-mg/g² in an antibody preparation containing 100 mM sodium chloride.

25. The method of claim 20, wherein the antibody is present at a concentration of about 5 to 300 mg/ml.

26. The method of claim 20, wherein the ionic strength of the antibody preparation is reduced by decreasing the concentration of a salt present in the preparation.

27. The method of claim 26, wherein the salt present in the preparation is selected from the group consisting of sodium chloride, calcium chloride, magnesium chloride and sodium phosphate.

28. The method of claim 26, wherein the concentration of the salt is reduced to at least about two-, three-, five-, ten- or one hundred-fold lower than the initial concentration in the antibody preparation.

29. The method of claim 26, wherein the ionic strength of the antibody preparation is reduced by replacing a first salt with a second salt, wherein the first salt is NaCl, and the second salt is selected from the group consisting of Na₂HPO₄, MgCl₂, and CaCl₂.

30. The method of claim 26, wherein the ionic strength of the antibody preparation is reduced by replacing a first salt with a second salt, wherein the first salt is Na₂HPO₄, and the second salt is selected from the group consisting of MgCl₂ and CaCl₂.

31. The method of claim 20, wherein the ionic strength of the antibody preparation is reduced by ultra-filtration or dialysis.

32. The method of claim 20, wherein the ionic strength of the antibody preparation is reduced in a process of purifying the antibody, wherein the process of purifying the antibody comprises one or more of centrifugation, filtration, chromatography, lyophilization or reconstitution.

33. The method of claim 20, further comprising evaluating the opalescence of the antibody preparation.

34. The method of claim 33, wherein the opalescence of the antibody preparation is evaluated by determining the turbidity of the solution, a change in the second virial coefficient of the antibody, or a change in the formation of high molecular weight species.

35. The method of claim 34, wherein the opalescence is reduced to about 2 or 1 European Pharmacopoeia standard or less.

36. The method of claim 34, wherein the second virial coefficient changes from about −

1 to −

10×

10<sup>−

3</sup> to about −

1 to −

10×

10<sup>−

4</sup>.

37. The method of claim 20, wherein the high molecular weight species is reduced by an amount of about two-, three-, four-, five-, six-, seven-, eight-, nine-, ten-, fifty- or one hundred-fold.

38. A method of decreasing the formation of high molecular weight species in an antibody preparation, the method comprising:

(a) providing an antibody preparation comprising an antibody determined to have a negative second virial coefficient in 100 mM sodium chloride solution;

(b) reducing the ionic strength in the antibody preparation such that the formation of high molecular weight species in the antibody preparation is reduced.

39. A method of improving the efficiency of a purification process of an antibody in an antibody preparation, the method comprising:

evaluating whether the antibody in the antibody preparation has a negative second virial coefficient at one or more salt concentrations;

reducing the ionic strength in the antibody preparation, such that the high molecular weight species in the antibody preparation is reduced, thereby improving the efficiency of a purification process of an antibody preparation.

40. The method of claim 39, wherein the ionic strength of the antibody preparation is reduced by one or more steps selected from filtration, replacing a salt used in the antibody preparation with a lower opalescent inducer, or reducing the concentration of a salt present in the purification process.