Refolding of recombinant proteins

Patent metadata

Field	Value
Publication number	US-2016137690-A1
Application number	US-201514922802-A
Country	US
Kind code	A1
Filing date	Oct 26, 2015
Priority date	Jul 14, 2006
Publication date	May 19, 2016
Grant date	—

Abstract

Processes are provided for recovering and purifying refolded recombinant proteins produced in heterologous host cells, which includes the step of refolding the protein in a high pH buffer.

First claim

1 . A process for recovering a refolded recombinant protein from a prokaryotic cell culture, the process comprising the steps of:
(a) isolating a recombinant protein from the prokaryotic cell culture; (b) solubilizing said protein in a first buffered solution, pH greater than 9, comprising a first chaotropic agent; (c) refolding said solubilized protein in a second buffered solution, pH>9 but ≦11, comprising a second chaotropic agent and two or more reducing agents, and addition of air or oxygen for such a time and under such conditions that refolding of the recombinant protein occurs; and (d) recovering said refolded recombinant protein.

2 . The process of claim 1 , wherein the recombinant protein is a growth factor.

3 . The process of claim 2 , wherein the growth factor is vascular endothelial growth factor (VEGF).

4 . The process of claim 3 , wherein the VEGF is VEGF 165 .

5 . The process of claim 1 , wherein the first and second chaotropic agents are urea.

6 . The process of claim 1 , wherein the first buffered solution or the second buffered solution further comprises arginine.

7 . (canceled)

8 . The process of claim 1 , wherein the first buffered solution comprises 1 M Urea, 300 mM arginine, 10 mM CHES or TRIS, 5 mM EDTA, pH 11, final concentration.

9 . (canceled)

10 . The process of claim 1 , wherein the second buffered solution comprises 1 M Urea, 15 mM cysteine, 2 mM DTT, 100 mM arginine, 10 mM CHES or TRIS, 5 mM EDTA, pH 10, final concentration.

11 . (canceled)

12 . The process of claim 1 , wherein the recombinant protein is incubated in the first buffered solution for at least 1 hour.

13 . The process of claim 12 , wherein the incubation is performed at 2-40° C.

14 . The process of claim 1 , wherein the solubilized protein is incubated in the second buffered solution for about 3 to 24 hours.

15 . The process of claim 14 , wherein the incubation is performed at 2-40° C.

16 . The process of claim 1 , wherein the two or more reducing agents comprise cysteine and DTT.

17 . The process of claim 1 , wherein the addition of air or oxygen is provided at aka =0.001 to 0.1 min −1 .

18 . The process of claim 1 , further comprising stabilizing the refolded recombinant protein by adding nitrogen.

19 . The process of claim 1 , wherein said recovery step (d) comprises clarifying the second buffered solution with the recombinant protein and sequentially contacting said refolded recombinant protein to a mixed mode chromatographic support, a cationic chromatographic support, and a first hydrophobic chromatographic support, and selectively eluting the refolded recombinant protein from each support.

20 . The process of claim 19 , wherein the clarifying step comprises: adding detergent to a final concentration of 1%, adjusting pH to about 8.5-9.5, incubating solution for 1 to 10 hours at 25-30° C., centrifuging the solution; and filtering liquid recovered from the centrifugation step.

21 . The process of claim 19 , further comprising contacting said refolded recombinant protein to a second hydrophobic chromatographic support or an ion exchange support and selectively eluting the refolded recombinant protein from the support.

22 . The process of claim 19 or 21 , wherein said first hydrophobic chromatographic support is selected from the group consisting of butyl-, propyl-, octyl-, phenyl-, and aryl-agarose resins.

23 . A process for recovering a refolded recombinant protein from a prokaryotic cell culture, the method comprising the steps of:
(a) isolating a recombinant protein from the prokaryotic cell culture; (b) solubilizing and refolding said protein in a combo buffered solution, pH>9 but ≦11, with addition of air or oxygen; and, (c) recovering said refolded recombinant protein.

24 . The process of claim 23 , wherein the recovery step comprises clarifying the combo solution with the recombinant protein and sequentially contacting said refolded recombinant protein to a mixed mode chromatographic support, a cationic chromatographic support, and a first hydrophobic chromatographic support, and selectively eluting the refolded recombinant protein from each support.

25 . The process of claim 24 , wherein the clarifying step comprises: adding detergent to a final concentration of 1%, adjusting pH to about 8.5-9.5, incubating solution for 1 to 10 hours at 25-30° C., centrifuging the solution; and filtering liquid recovered from the centrifugation step.

26 . The process of claim 23 , wherein the combo buffered solution comprises 1 M Urea, 15 mM cysteine, 2 mM DTT, 100 mM arginine, 10 mM CHES or TRIS, 5 mM EDTA, pH 10, final concentration.

27 . (canceled)

28 . The process of claim 23 , wherein the recombinant protein is incubated in the combo buffered solution for about 3 to 24 hours.

29 . The process of claim 28 , wherein the incubation is performed at 2-40° C.

30 . The process of claim 24 , further comprising contacting said refolded recombinant protein to a second hydrophobic chromatographic support or an ion exchange support and selectively eluting the refolded recombinant protein from the support.

31 . A method for purifying a recombinant protein, the method comprising the steps of:
sequentially contacting a recombinant protein with a mixed mode support, a cationic chromatographic support, a first hydrophobic interaction chromatographic support, and selectively eluting the recombinant protein from each support.

32 . The method of claim 30 , further comprising contacting the recombinant protein with a second hydrophobic chromatographic support or an ion exchange support, and selectively eluting the recombinant protein from the support.

33 . The process of claim 21 , wherein said second hydrophobic chromatographic support is selected from the group consisting of butyl-, propyl-, octyl-, phenyl-, and aryl-agarose resins.

Assignees

• Genentech Inc

Inventors

• Pizarro Shelly
• Sanchez Ailen
• Schmelzer Charles H

Classifications

• C07K1/14 Primary

  Extraction; Separation; Purification · CPC title

• C07K1/36 Primary

  by a combination of two or more processes of different types · CPC title

• C07K14/475

  Growth factors; Growth regulators · CPC title

• C07K1/1133

  by redox-reactions involving cystein/cystin side chains · CPC title

• C07K1/20

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