Process for refolding recombinant chymotrypsin

Patent metadata

Field	Value
Publication number	US-10301611-B2
Application number	US-201515526779-A
Country	US
Kind code	B2
Filing date	Nov 13, 2015
Priority date	Nov 18, 2014
Publication date	May 28, 2019
Grant date	May 28, 2019

Abstract

A process for refolding recombinant chymotrypsin produced from prokaryote host cells is described. In particular, the present invention provides a process for refolding recombinant chymotrypsin produced from E. coli is described.

First claim

What is claimed:

1. A process for refolding recombinant chymotrypsinogen produced from a prokaryote host cell comprising:
(a) providing the recombinant chymotrypsinogen at a concentration of about 5 to 15 g/L in a solubilization solution comprising a chaotropic agent, a buffer agent, and a low molecular weight reducing agent in the absence of a low molecular weight oxidizing agent, wherein the solubilization solution does not include a low molecular weight oxidizing agent to provide a redox pair comprising the low molecular weight reducing agent and a low molecular weight oxidizing agent; and
(b) infusing the solubilization solution comprising the recombinant chymotrypsin over time into a diluent to provide a refold solution comprising the chaotropic agent at about 25% to 30% of its concentration in the solubilization solution, a buffer agent, and the low molecular weight reducing agent in the absence of a low molecular weight oxidizing agent to provide a refold solution comprising the recombinant chymotrypsinogen at a concentration between 1 and 5 g/L and incubating the refold solution for a time sufficient for the recombinant chymotrypsinogen to refold into a conformation characteristic of native chymotrypsinogen and form the disulfide bonds characteristic of native chymotrypsinogen, wherein the refold solution does not include a low molecular weight oxidizing agent to provide a redox pair comprising the low molecular weight reducing agent and a low molecular weight oxidizing agent.

2. The process of claim 1 , wherein the chaotropic agent is guanidinium chloride.

3. The process of claim 1 , wherein the chaotropic agent in the refold solution is about 1.4 to 1.8 M.

4. The process of claim 1 , wherein the reducing agent is cysteine or cysteine hydrochloride.

5. The process of claim 1 , wherein the low molecular weight reducing agent in the solubilization solution comprising the recombinant chymotrypsinogen is at a concentration sufficient to provide about 1 to 15 SH residues of the low molecular weight reducing agent per cysteine residue of the recombinant chymotrypsinogen.

6. The process of claim 1 , wherein the low molecular weight reducing agent in the refold solution comprising the recombinant chymotrypsinogen is at a concentration sufficient to provide about 1 to 30 SH residues of the low molecular weight reducing agent per cysteine residue of the recombinant chymotrypsinogen.

7. The process of claim 1 , wherein the recombinant chymotrypsinogen comprises the amino acid sequence for porcine or bovine chymotrypsin.

8. A process for preparing recombinant chymotrypsin comprising:
(a) providing solubilized recombinant chymotrypsinogen at a concentration of about 5 to 15 g/L in a solubilization solution comprising a chaotropic agent, a buffer agent, and a first low molecular weight reducing agent in the absence of a low molecular weight oxidizing agent at a pH of about the pI of chymotrypsinogen or greater, wherein the solubilization solution does not include a low molecular weight oxidizing agent to provide a redox pair comprising the low molecular weight reducing agent and a low molecular weight oxidizing agent;
(b) infusing the solubilization solution comprising the recombinant chymotrypsinogen into a diluent to provide a refold solution comprising the chaotropic agent at about 25% to 30% of its concentration in the solubilization solution, a buffer agent, and the first low molecular weight reducing agent in the absence of a low molecular weight oxidizing agent or a second low molecular weight reducing agent in the absence of a low molecular weight oxidizing agent at a pH of about the pI of chymotrypsinogen to provide a refold solution comprising the recombinant chymotrypsinogen at a concentration greater than 1 g/L and less than 12.5 g/L, or of about 1.5 g/L, wherein the refold solution does not include a low molecular weight oxidizing agent to provide a redox pair comprising the first or second low molecular weight reducing agent and a low molecular weight oxidizing agent;
(c) incubating the refold solution comprising the recombinant chymotrypsinogen for a time sufficient for the recombinant chymotrypsinogen to refold into a conformation characteristic of native chymotrypsinogen and form the disulfide bonds characteristic of native chymotrypsinogen; and
(d) diluting the refold solution and incubating the diluted refold solution for a time sufficient for the recombinant chymotrypsinogen therein to auto-catalyze to provide the recombinant chymotrypsin.

9. The process of claim 8 , wherein the recombinant chymotrypsin is subjected to a chromatography step to remove peptides from the auto-catalysis of the recombinant chymotrypsinogen.

10. The process of claim 9 , wherein the recombinant chymotrypsin obtained from the chromatography step is concentrated to provide a composition comprising the recombinant chymotrypsin at a concentration of about 20 to 80 mg/mL.

11. The process of claim 9 , wherein the chromatography is affinity chromatography.

12. The process of claim 11 , wherein the affinity chromatography is performed on a matrix comprising benzamidine.

13. The process of claim 8 , wherein the recombinant chymotrypsinogen comprises the amino acid sequence for porcine or bovine chymotrypsin.

14. The process of claim 8 , wherein the solubilized recombinant chymotrypsinogen is obtained from inclusion bodies isolated from prokaryote host cells transformed with an expression vector comprising a nucleic acid molecule encoding the recombinant chymotrypsinogen and fermented under conditions for producing the recombinant chymotrypsinogen.

15. The process of claim 14 , wherein the prokaryote host cell is E. coli.

16. The process of claim 1 , wherein the buffer agent is ethanolamine.

17. The process of claim 1 , wherein the reducing agent is cysteine or cysteine hydrochloride.

18. The process of claim 8 , wherein the buffer agent is ethanolamine.

19. The process of claim 8 , wherein the reducing agent is cysteine or cysteine hydrochloride.

Assignees

• Merck Sharp & Dohme

Inventors

• Sleevi Mark C
• Lile Jack
• Grimison Bryn

Classifications

• C12Y304/21001

  Chymotrypsin (3.4.21.1) · CPC title

• C12N9/6427 Primary

  Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4) · CPC title