Method for preparing crystalline insulin or insulin analog compositions

• US 10,124,040 B2
• Filed: 08/21/2015
• Issued: 11/13/2018
• Est. Priority Date: 08/26/2014
• Status: Active Grant

• Current Assignee: Merck Sharp & Dohme LLC (Merck & Co., Inc.)
• Original Assignee: Merck Sharp & Dohme Corporation (Merck & Co., Inc.)
• Inventors: Watson, Douglas S., Ortigosa, Allison C., Sleevi, Mark C., Story, Kathryn M.
• Primary Examiner(s): Packard, Benjamin J
• Application Number:
  US15/505,124
• Publication Number:
  US 20170209545A1
• Time in Patent Office: 1,180 Days
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61K 47/02 Inorganic compounds
• CPC Class Codes: A61K 47/10 Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• CPC Class Codes: A61K 9/143 with inorganic compounds
• CPC Class Codes: A61K 9/145 with organic compounds
• CPC Class Codes: C07K 14/62 Insulins

Abstract

A method for crystallizing insulin or insulin analogs under alkaline conditions in the presence of zinc, purifying the crystals by filtering through a filter, and drying the crystals captured on the filter to produce crystalline insulin or insulin analog compositions is described. Method has been exemplified with insulin lispro.

Claims

1. A method for preparing insulin lispro crystals comprising:

(a) providing a solution comprising the insulin lispro, a water miscible organic solvent, and a crystal stabilizing agent, wherein the solution has a pH that is about 8.0; and

(b) adding a zinc salt to the solution at a temperature of about 25°

C. to provide a crystalizing solution, cooling the solution to about 5°

C., and incubating the crystalizing solution for a time sufficient for the insulin lispro to produce the insulin lispro crystals.

2. The method in claim 1, wherein the solution comprises an organic acid or salt selected from the group consisting of acetic acid, acetate, citric acid, citrate, and glycine.

3. The method in claim 2, wherein the organic acid or salt comprises acetate.

4. The method in claim 1, wherein the solution comprises a base selected from the group consisting of potassium hydroxide, sodium hydroxide, and ammonium hydroxide.

5. The method in claim 4, wherein the base comprises ammonium hydroxide.

6. The method in claim 1, wherein the insulin lispro comprises a concentration in the range of 1.5 g/L of 2.5 g/L.

7. The method in claim 1, wherein the water miscible organic solvent comprises isopropanol.

8. The method in claim 1, wherein the crystal stabilizing agent comprises meta-cresol.

9. The method in claim 1, wherein the zinc salt comprises zinc chloride.

10. The method of claim 1, wherein the crystal stabilizing agent comprises meta-cresol and the solution further includes phenol at a concentration not more than 0.08 mM.

11. The method of claim 1, wherein the crystal stabilizing agent comprises meta-cresol and the solution does not include phenol.

12. The method in claim 1, wherein following the crystallization of the insulin lispro, a crystal slurry comprising the insulin lispro crystals is produced by (i) allowing the insulin lispro crystals in the crystalizing solution to settle in the crystalizing solution and the crystalizing solution decanted from the settled insulin lispro crystals or (ii) by centrifuging the crystallizing solution to remove the crystallizing solution from the insulin lispro crystals.

13. The method of claim 12, wherein the crystal slurry is applied to a filter apparatus to remove remaining crystalizing solution from the crystal slurry to produce a crystal cake bed and drying the crystal cake bed to provide the crystal insulin lispro.

14. The method of claim 12, wherein the crystal slurry is applied to a filter apparatus to remove remaining crystalizing solution from the crystal slurry to produce a crystal cake bed, washing the crystal cake bed with a cake wash solution comprising a water miscible organic solvent, a crystal stabilizing agent, and a zinc salt wherein the solution has a pH that of about 5.5, and drying the crystal cake bed to provide the crystal insulin lispro.

15. The method of claim 12, wherein the crystal slurry is applied to a centrifuge to remove remaining crystalizing solution from the crystal slurry, washing the crystals with a cake wash solution comprising a water miscible organic solvent, a crystal stabilizing agent, and a zinc salt wherein the solution has a pH that comprises about 4.5 to 8.5, and drying the crystals to provide the crystal insulin lispro.

16. A method for preparing insulin lispro crystals with 0.30% to 0.60% zinc content (dried basis), or about two to three zinc atoms per hexamer of insulin or insulin analog, comprising:

(a) providing a solution comprising the insulin lispro, a water miscible organic solvent, and a crystal stabilizing agent, wherein the solution has a pH that is about 8.0;

(b) adding zinc chloride to the solution at about 25°

C. to provide a crystalizing solution, cooling the crystalizing solution to about 5°

C., and incubating the crystalizing solution for a time sufficient produce insulin lispro crystals;

(c) removing the crystalizing solution from the insulin lispro crystals to produce a crystal slurry;

(d) applying the crystal slurry to a filter apparatus or centrifuge to remove remaining crystalizing solution from the decanted crystal slurry to produce a crystal cake bed;

(e) adding a cake wash solution comprises a water miscible organic solvent, a crystal stabilizing agent, and zinc salt, wherein the solution has a pH that is about 5.0 to the decanted crystal slurry to provide a mixture and incubating the mixture for a time sufficient to remove unbound zinc from the insulin lispro, and

(f) removing the cake wash solution from the mixture to provide the insulin or insulin analog crystals with 0.30% to 0.60% weight percent zinc content (dried basis), or approximately two to three zinc atoms per hexamer of insulin lispro.

17. A crystalline composition comprising insulin lispro, 0.30% to 0.60% zinc content (dried basis), or about two to three atoms of zinc per six molecules of the insulin or insulin analog, and meta-cresol wherein the insulin lispro crystals are cubic and have a 20-30 μ

m average crystal size or rhombohedral and have an average crystal size of greater than 40 μ

m.