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US 12,269,843 B2Granted
Refolding proteins using a chemically controlled redox state
AssigneeAmgen Inc.
InventorsJoseph Edward Shultz, Roger Hart, Ronald Nixon Keener, III
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Abstract
A method of refolding proteins expressed in non-mammalian cells present in concentrations of 2.0 g/L or higher is disclosed. The method comprises identifying the thiol pair ratio and the redox buffer strength to achieve conditions under which efficient folding at concentrations of 2.0 g/L or higher is achieved and can be employed over a range of volumes, including commercial scale.
Claims9
A method for reproducibly obtaining proper refolding of proteins expressed in a non-mammalian expression system, the method comprising:
- (a) selecting a preparation that includes:
- at least one ingredient selected from the group consisting of a denaturant, an aggregation suppressor and a protein stabilizer;
- a concentration of oxidant;
- and
- a concentration of reductant;
- (b) selecting a desired yield of properly refolded proteins from a first concentration of proteins that is 1.0 g/l or greater;
- (c) determining a thiol-pair ratio of the oxidant and the reductant in the preparation based on the desired yield of properly refolded proteins;
- (d) determining a thiol-pair buffer strength for the preparation based on the determined thiol-pair ratio of (c), wherein the thiol-pair ratio and thiol-pair buffer strength are coordinated to achieve the desired yield of properly refolded proteins;
- (e) incubating the refold mixture to obtain the desired yield of properly refolded proteins;
- (f) selecting a second concentration of proteins;
- (g) adjusting, if necessary, the thiol-pair ratio for the second concentration of proteins, based on the desired yield of properly refolded proteins;
- and
- (h) adjusting the thiol-pair buffer strength for the second concentration of proteins until a final thiol-pair ratio is achieved, based on the adjusted thiol-pair ratio to achieve the same desired yield of properly refolded proteins for the second concentration.
10 more elements
Dependencies are inferred from the claim text.