Methods and compositions with reduced opalescence

• US 20080154023A1
• Filed: 10/12/2007
• Published: 06/26/2008
• Est. Priority Date: 10/12/2006
• Status: Active Grant

• Current Assignee: Wyeth (Pfizer Inc.)
• Original Assignee: Wyeth (Pfizer Inc.)
• Inventors: Luisi, Donna L., Nichols, Pilarin Elizabeth
• Granted Patent: US 7,744,890 B2
• Time in Patent Office: Days
• US Class Current: 530/387.3
• 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:

modifying the ratio of the ionic strength to the antibody concentration of the antibody preparation such that the opalescent appearance of the antibody preparation is reduced, wherein the antibody preparation, prior to modifying the ionic strength, shows an opalescence greater than about 2 European Pharmacopoeia standard at a pharmaceutically effective concentration of about 100 mg/ml.

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

4. The method of claim 1, wherein the antibody preparation comprises an anti-GDF8 antibody.

5. The method of claim 1, wherein the antibody of the antibody preparation has a negative second virial coefficient in a salt concentration ranging from about 50 to 200 mM.

6. The method of claim 5, wherein the second virial coefficient of the antibody is about −

1 to −

10×

10<sup>−

3</sup>, or about −

1 to −

10×

10<sup>−

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

7. The method of claim 1, wherein the antibody of the antibody preparation has a positive virial coefficient in a salt concentration ranging from about 50 to 200 mM.

8. The method of claim 7, wherein the second virial coefficient of the antibody is about 1 to 10×

10<sup>−

4</sup> or about 1 to 10×

10<sup>−

3</sup> mol-mg/g² in a preparation containing 100 mM sodium chloride.

9. The method of claim 1, wherein the antibody is present in the antibody preparation at a concentration of about 5 to 300 mg/ml, prior to or after performing the method.

10. 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.

11. The method of claim 10, wherein the ionic strength of the antibody preparation is reduced by decreasing the concentration of a salt present in the preparation, wherein the salt present in the preparation is selected from the group consisting of sodium chloride, calcium chloride, magnesium chloride and sodium phosphate.

12. The method of claim 10, wherein the ionic strength of the antibody preparation is reduced by decreasing the concentration of a salt present in the preparation, wherein the concentration of the salt is reduced to at least about two-, three-, five-, ten- or one hundred-fold lower than the concentration in the opalescent preparation.

13. The method of claim 10, wherein the ionic strength of the antibody preparation is modified 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 NaHPO4, MgCl₂, and CaCl₂.

14. The method of claim 10, wherein the ionic strength of the antibody preparation is reduced by replacing a first salt with a second salt, wherein the first salt is NaHPO4, and the second salt is selected from the group consisting of MgCl₂ and CaCl₂.

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

16. The method of claim 1, further comprising:

evaluating the opalescence of the antibody preparation;

or

determining the salt concentration in the antibody preparation;

wherein the ionic strength of the antibody preparation is modified by a process comprising one of more of;

applying one or more filtration methods or reducing the salt concentration of one or more steps in the process of purifying the antibody, wherein the process of purifying the antibody comprises one or more of centrifugation, filtration, chromatography, lyophilization or reconstitution.

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

18. The method of claim 17, wherein the opalescence of the antibody preparation is detected by evaluating the turbidity of the solution, evaluating a change in the second virial coefficient, or evaluating a change in the formation of high molecular weight species.

19. The method of claim 18, wherein the opalescence is reduced to about 2 or 1 European Pharmacopoeia standard or less.

20. The method of claim 18, wherein the second virial coefficient changes from about −

1 to −

10×

10<sup>−

3</sup> to about −

1 to −

10×

10<sup>−

4</sup>.

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

52. An antibody preparation produced by the method of claim 1.

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

modifying the ratio of the ionic strength to the antibody concentration of the antibody preparation such that the high molecular weight species in the protein preparation are reduced.

23. The method of claim 22, wherein the antibody preparation comprises a human, humanized, chimeric, CDR-grafted, or an in vitro generated antibody, or an antigen-binding fragment thereof.

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

25. The method of claim 22, wherein the antibody preparation comprises an anti-GDF8 antibody.

26. The method of claim 22, wherein the antibody of the antibody preparation has a negative second virial coefficient in a salt concentration ranging from about 50 to 200 mM.

27. The method of claim 26, 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.

28. The method of claim 22, wherein the antibody of the antibody preparation has a positive virial coefficient in a salt concentration ranging from about 50 to 200 mM.

29. The method of claim 28, wherein the second virial coefficient of the antibody is about 1 to 10×

10<sup>−

4</sup> or about 1 to 10×

10<sup>−

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

30. The method of claim 22, wherein the antibody is present at a concentration of about 5 to 300 mg/ml, prior to or after practicing the method.

31. The method of claim 22, wherein the ionic strength of the antibody preparation is reduced by decreasing the concentration of a salt present in the preparation.

32. The method of claim 31, wherein the ionic strength of the antibody preparation is reduced by decreasing the concentration of a salt present in the preparation, wherein the salt present in the preparation is selected from the group consisting of sodium chloride, calcium chloride, magnesium chloride and sodium phosphate.

33. The method of claim 31, wherein the ionic strength of the antibody preparation is reduced by decreasing the concentration of a salt present in the preparation, wherein the concentration of the salt is reduced to at least about two-, three-, five-, ten- or one hundred-fold lower than the concentration in the opalescent preparation.

34. The method of claim 31, wherein the ionic strength of the antibody preparation is modified 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 NaHPO₄, MgCl₂, and CaCl₂.

35. The method of claim 31, wherein the ionic strength of the antibody preparation is modified by replacing a first salt with a second salt, wherein the first salt is NaHPO₄, and the second salt is selected from the group consisting of MgCl₂ and CaCl₂.

36. The method of claim 22, wherein the ionic strength of the antibody preparation is modified by ultra-filtration or dialysis.

37. The method of claim 22, further comprising:

evaluating the opalescence of the antibody preparation;

or

determining the salt concentration in the antibody preparation;

wherein the ionic strength of the antibody preparation is modified by a process comprising one of more of;

applying one or more filtration methods or reducing the salt concentration of one or more steps in the process of purifying the antibody, wherein the process of purifying the antibody comprises one or more of centrifugation, filtration, chromatography, lyophilization or reconstitution.

38. The method of claim 22, further comprising evaluating the opalescence of the antibody preparation.

39. The method of claim 38, wherein the opalescence of the antibody preparation is detected by evaluating the turbidity of the solution, evaluating a change in the second virial coefficient, or evaluating a change in the formation of high molecular weight species.

40. The method of claim 39, wherein the opalescence is reduced to about 2 or 1 European Pharmacopoeia standard or less.

41. The method of claim 39, wherein the second virial coefficient changes from about −

1 to −

10×

10<sup>−

3</sup> to about −

1 to −

10×

10<sup>−

4</sup>.

42. The method of claim 39, wherein the amount of high molecular weight species is reduced by about two-, three-, four-, five-, six-, seven-, eight-, nine-, ten-, fifty- or one hundred-fold.

53. An antibody preparation produced by the method of claim 22.

43. A method of decreasing the formation of high molecular weight species in an antibody preparation, the method comprising modifying the ratio of the ionic strength to the antibody concentration in the antibody preparation such that the formation of high molecular weight species in the protein preparation is reduced, wherein the antibody in the antibody preparation has a negative virial coefficient in 100 mM sodium chloride solution.

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

(optionally) evaluating whether the antibody preparation forms an opalescent solution at one or more salt concentrations;

modifying the ratio of the ionic strength to the antibody concentration 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,

wherein the ionic strength of the solution is modified by one or more of;

applying one or more filtration methods, replacing the salt used in the protein preparation with a lower opalescent inducer, or reducing the ionic strength of one or more steps in the purification process.

45. A method of improving the production of an antibody, the method comprising:

recovering a first antibody solution; and

evaluating the level of opalescence or presence of high molecular weight species in the first antibody solution;

wherein (a) if the level of opalescence or high molecular weight species is equal to or less than a predetermined level, then selecting said first antibody solution for use in the antibody product;

or (b) if the level of opalescence or high molecular weight species is greater than a predetermined level, then selecting a second antibody solution having a second ionic strength.

46. The method of claim 45, further comprising repeating the evaluating step until the level of opalescence in the solution is equal to or less than the predetermined level.

47. The method of claim 45, wherein the antibody solution is recovered by a protein purification method chosen from one or more of:

antibody elution from a matrix, recovery of a dialyzed solution or other filtrate, solubilizing a dry preparation, or a chromatographic method.

48. The method of claim 47, wherein the antibody solution is recovered by a chromatographic method and the chromatographic method is selected from the group consisting of Protein A chromatography, affinity chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography, size exclusion chromatography, diafiltration, ultrafiltration, viral removal filtration, anion exchange chromatography, hydroxyapatite chromatography and cation exchange chromatography.

49. The method of claim 45, wherein the predetermined level of the antibody solution has a turbidity value of about 2.

50. A method of determining the propensity of an antibody preparation to have an opalescent appearance, the method comprising:

(optionally) identifying an antibody as having a tendency to self-aggregate;

providing one or more samples of the antibody preparation; and

detecting the formation of high molecular weight species upon increasing the salt concentration in the one or more samples, wherein an increase in the formation of high molecular weight species upon increasing salt concentration is indicative of an increased propensity of the antibody in the preparation to have an opalescent appearance.

51. A method of evaluating the opalescence of an antibody sample, the method comprising:

providing an antibody sample;

determining the turbidity or presence of high molecular weight species at one or more salt concentrations; and

providing a report on the level of opalescence in the antibody sample as a function of turbidity or presence of high molecular weight species at the one or more salt concentrations.

54. A pharmaceutically-acceptable antibody preparation comprising an antibody at a concentration of at least about 50 mg/ml in less than about 50 mM NaCl.