Purifying insulin using cation exchange and reverse phase chromatography in the presence of an organic modifier and elevated temperature

Patent metadata

Field	Value
Publication number	US-11198718-B2
Application number	US-201916551974-A
Country	US
Kind code	B2
Filing date	Aug 27, 2019
Priority date	Mar 14, 2014
Publication date	Dec 14, 2021
Grant date	Dec 14, 2021

Abstract

A process is described for purifying insulin and insulin analogs that comprises high-pressure liquid chromatography with an acidic cation exchange medium performed in the presence of a water miscible organic modifier and at an elevated temperature followed by reverse phase chromatography performed in the presence of a water miscible organic modifier and at an elevated temperature.

First claim

What is claimed is:

1. A process for isolating properly folded insulin or insulin analog from an aqueous mixture comprising the insulin or insulin analog and related impurities, wherein the process comprises:
(a) performing an acid-stable cation exchange chromatography with the aqueous mixture at a differential pressure of about 0.276 MPa in the presence of hexylene glycol at a concentration of about 30% by volume and at an outlet temperature between 41° C. to 45° C. under isocratic conditions to yield a first insulin or insulin analog mixture; and
(b) performing a reverse phase high performance liquid chromatography with a silica-based reverse phase resin on the first insulin or insulin analog mixture in the presence of isopropanol and at an outlet temperature between 40° C. to 46° C. to provide a second mixture comprising the isolated properly folded insulin or insulin analog.

2. The process of claim 1 , wherein the acid-stable cation exchange chromatography has an outlet temperature of about 42° C.

3. The process of claim 1 , wherein the reverse phase high performance liquid chromatography has an outlet temperature of about 43° C.

4. The process of claim 1 , wherein the insulin analog comprises an acid-stable insulin analog, which is stable and soluble in acidic or weakly acidic solutions and insoluble or partially insoluble at physiological pH.

5. The process of claim 4 , wherein the acid-stable insulin analog comprises insulin glargine.

6. The process of claim 1 , wherein the insulin analog comprises a pI-shifted insulin analog in which the pI of the insulin analog is less than or greater than the pI of native human insulin.

7. The process of claim 6 , wherein the pI of the pI shifted insulin analog comprises a pI greater than 5.6 or less than 5.4.

8. The process of claim 6 , wherein the pI-shifted insulin analog comprises a pI from between 5.8 to 8.0.

9. The process of claim 1 , wherein the insulin analog comprises an insulin analog selected from the group consisting of insulin glargine, insulin aspart, insulin glulisine, and insulin lispro.

10. A process for purifying a properly folded insulin or insulin analog from a crude mixture comprising the insulin or insulin analog and related impurities, the process comprising:
(a) providing a crude mixture of insulin or insulin analog in 50 mM acetic acid, pH 3.5 and diluting the crude mixture with hexylene glycol to about 0.3 to 0.4 g/L and then adjusting the pH to about 4.2 to produce a diluted crude mixture, wherein the mass of hexylene glycol to insulin or insulin analog in the diluted crude mixture is about 0.395 kg hexylene glycol/kg insulin or insulin analog;
(b) applying the diluted crude mixture to a temperature-stable cation exchange chromatography matrix;
(c) washing the matrix with a first wash solution comprising a concentration of mineral salt of about 10 to 25 mM and hexylene glycol at a concentration of about 30% by volume and then washing the matrix with a second wash solution comprising a concentration of mineral salt greater than the concentration of mineral salt in the first wash solution and less than the concentration of mineral salt capable of eluting the insulin or insulin analog from the matrix wherein the second wash solution comprises hexylene glycol at a concentration of about 30% by volume;
(d) eluting the insulin or insulin analog from the matrix with an eluting solution comprising the concentration of mineral salt capable of eluting the insulin or insulin analog from the matrix and hexylene glycol at a concentration of about 30% by volume to provide a second mixture; the temperature-stable cation exchange chromatography being performed under a differential pressure of about 0.276 MPa and at an outlet temperature of about 42° C.; and
(e) diluting the second mixture with water to provide a diluted second mixture comprising the insulin or insulin analog at a concentration of less than 24 g/L and applying the diluted second mixture to a reverse phase high performance liquid chromatography matrix comprising an acetate buffer at a pH of about 3.0 and isopropanol at a concentration of about 5% by volume; and
eluting the insulin or insulin analog with a linear gradient of isopropanol in an acetate buffer at a pH of about 3.0 increasing concentration from about 13 to 15 percent by volume to about 25 to 27 percent by volume to provide a mixture of the properly folded insulin or insulin analog; the reverse phase high performance liquid chromatography being performed at an outlet temperature of about 43° C.

11. The process of claim 10 , wherein step (e) further comprises washing the matrix with a first solution comprising about 4 to 7 percent by volume of isopropanol in an acetate buffer at a pH of about 3.0; and then washing the matrix with a linear gradient of the isopropanol in an acetate buffer at a pH of about 3.0 of increasing concentration from about 4 to 7 percent by volume to about 13 to 15 percent by volume.

12. The process of claim 10 , wherein the insulin analog comprises an acid-stable insulin analog, which is stable and soluble in acidic or weakly acidic solutions and insoluble or partially insoluble at physiological pH.

13. The process of claim 12 , wherein the acid-stable insulin analog comprises insulin glargine.

14. The process of claim 10 , wherein the insulin analog comprises a pI-shifted insulin analog in which the pI of the insulin analog is less than or greater than the pI of native human insulin.

15. The process of claim 14 , wherein the pI of the pI shifted insulin analog comprises a pI greater than 5.6 or less than 5.4.

16. The process of claim 14 , wherein the pI-shifted insulin analog comprises a pI from between 5.8 to 8.0.

17. The process of claim 10 , wherein the insulin analog comprises an insulin analog selected from the group consisting of insulin glargine, insulin aspart, insulin glulisine, and insulin lispro.

Assignees

• Merck Sharp & Dohme

Inventors

• Ortigosa Allison D
• Coleman Michael P
• George Shibu T
• Rauscher Michael A
• Sleevi Mark C
• Chow Kartoa

Classifications

• C07K1/20

  Partition-, reverse-phase or hydrophobic interaction chromatography · CPC title

• C07K14/62 Primary

  Insulins · CPC title

• C07K1/18

  Ion-exchange chromatography · CPC title