Preparation of an acylated protein powder

• US 5,700,904 A
• Filed: 06/07/1995
• Issued: 12/23/1997
• Est. Priority Date: 06/07/1995
• Status: Expired due to Fees

• Current Assignee: Eli Lilly and Company
• Original Assignee: Eli Lilly and Company
• Inventors: Baker, Jeffrey Clayton, Moser, Brian A., Shrader, Warren E.
• Primary Examiner(s): Russel, Jeffrey E.
• Application Number:
  US08/484,220
• Time in Patent Office: 930 Days
• Field of Search: 530/303, 530/305, 530/344, 530/417, 530/410, 530/345, 530/419, 514/3, 514/4, 514/21
• US Class Current: 530/305
• CPC Class Codes: C07K 1/30 by precipitation
• CPC Class Codes: C07K 14/62 Insulins

Abstract

A method for recovering an acylated protein as a powder, especially in cases where said acylated protein is one that resists isolation by isoelectric precipitation from aqueous solutions of the protein, said method comprising in combination adjusting said aqueous solution to near the isoelectric pH of the protein and providing a suitable alcohol concentration to cause precipitation of the protein in the form of filterable particles at said pH.

Claims

1. A process for forming a fatty acid-acylated protein precipitate, which comprises, adjusting the pH of a solution comprising the fatty acid-acylated protein and adding alcohol, such that the fatty acid-acylated protein precipitates.

2. The process of claim 1, wherein adjusting the pH precedes adding alcohol.

3. The process of claim 1, wherein adding alcohol precedes adjusting the pH.

4. The process of claim 1, wherein the fatty acid-acylated protein is selected from the group consisting of a fatty acid-acylated insulin, a fatty acid-acylated insulin analog, and a fatty acid-acylated proinsulin.

5. The process of claim 4, wherein the pH is adjusted to a pH between about 4.0 and about 6.0.

6. The process of claim 5, wherein the fatty acid-acylated protein is B29-N.sup.ε

-fatty acid-acylated human insulin.

7. The process of claim 6, wherein the fatty acid-acylated protein is B29-N.sup.ε

-palmitoyl-acylated human insulin.

8. The process of claim 6, wherein the fatty acid-acylated protein is B29-N.sup.ε

-myristoyl-acylated human insulin.

9. The process of claim 5, wherein the fatty acid-acylated protein is B28-N.sup.ε

-fatty acid-acylated Lys^B28, Pro^B29 -human insulin.

10. The process of claim 9, wherein the fatty acid-acylated protein is B28-N.sup.ε

-palmitoyl-acylated Lys^B28, Pro^B29 -human insulin.

11. The process of claim 9, wherein the fatty acid-acylated protein is B28-N.sup.ε

-myristoyl-acylated Lys^B28, Pro^B29 -human insulin.

12. The process of claim 5, wherein the fatty acid-acylated protein is B1-N.sup.α

-fatty acid-acylated human insulin.

13. The process of claim 5, wherein the fatty acid-acylated protein is B1-N.sup.α

-fatty acid-acylated Lys^B28,Pro^B29 -human insulin.

14. The process of claim 5, wherein the alcohol is ethanol, and wherein ethanol is added in an amount to yield a concentration of ethanol in the solution of about 20% to about 35% by volume.

15. A process for producing a fatty acid-acylated protein powder, which comprises,

a) recovering the fatty acid-acylated protein precipitate produced by the process of claim 1;

and

b) drying the recovered precipitate of step a) to produce a powder comprising the fatty acid-acylated protein.