Method for purification of cleaved pro-insulin
View Patent ↗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%.
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).