Method for purification of cleaved pro-insulin

Patent metadata

Field	Value
Publication number	US-9409967-B2
Application number	US-201214348634-A
Country	US
Kind code	B2
Filing date	Sep 28, 2012
Priority date	Sep 30, 2011
Publication date	Aug 9, 2016
Grant date	Aug 9, 2016

Abstract

The present invention is within the field of biomolecule purification. More closely the invention relates to chromatographic purification of insulin using a specific kind of shell beads having an inner core and an outer functionalized layer. The method enables purification at high flow rates and high purity, over 90%.

First claim

The invention claimed is:

1. A method for purification of insulin from pro-insulin comprising the following steps:
loading a sample of cleaved pro-insulin onto a chromatography medium comprising porous shell beads having an inner core and an outer layer, wherein the inner core is non-functionalized while the outer layer is functionalized with ion exchange ligands;
adsorbing insulin on the ion exchange ligands; and
eluting insulin from the chromatography medium at a flow rate of 100-1000 cm/h, wherein the eluted insulin has a purity of more than 85%.

2. The method of claim 1 , wherein the porous shell beads are 20-100 μm in diameter.

3. The method of claim 1 , wherein the porous shell beads are 40-80 μm in diameter.

4. The method of claim 1 , wherein the functionalized layer of the porous shell beads comprises a 3-9 μm thick layer.

5. The method of claim 1 , wherein the functionalized layer of the porous shell beads comprises a 5-7 μm thick layer.

6. The method of claim 1 , wherein the ion exchange ligand is a strong cation exchange group selected from the group consisting of sulphonate (SO 3 − ), sulphate (—OSO 3 − ), phosphate (—OPO 3 2− ), and phosphonate (PO 3 2− ).

7. The method of claim 1 , wherein the inner core is filled with a polar polymer selected from the group consisting of agarose, dextran, cellulose, starch, and pullulan.

8. The method of claim 1 , wherein the pro-insulin is produced from bacteria.

9. The method of claim 1 , wherein the eluted insulin has a purity of more than 90%.

10. The method of claim 1 , wherein the flow rate is 300-600 cm/h.

11. The method of claim 1 , wherein the inner core is inactive.

12. The method of claim 1 , wherein the inner core is empty.

13. The method of claim 1 , wherein the porous shell beads are 60-80 μm in diameter.

14. The method of claim 1 , wherein the inner core is filled with a synthetic polymer selected from the group consisting of polyacrylic amide, polymethacrylic amide, and poly(hydroxyalkylacrylates).

Assignees

• Ge Healthcare Bio Sciences Ab
• Ge Healthcare Bioprocess R&D Ab

Inventors

• Brekkan Eggert
• Eriksson Kjell
• Johansson Bo-Lennart
• Shanagar Jamil

Classifications

• C07K14/62 Primary

  Insulins · CPC title

• B01D15/361

  Ion-exchange · CPC title

• B01J20/3212

  consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds · CPC title

• B01J39/26

  Cation exchangers for chromatographic processes · CPC title

• B01J20/288

  Polar phases · CPC title