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.
Claims17
(canceled)
A method for optimizing the yield of a properly refolded protein form by generating a preparation that supports the renaturation of a protein to the properly refolded protein form, wherein the protein is expressed in a non-mammalian expression system, the method comprising:
- (a) selecting a desired yield of the properly refolded protein form based on a prior yield of the refolded protein form;
- (b) selecting a thiol-pair ratio of an oxidant and a reductant in the preparation used to obtain the desired yield of the properly refolded protein form;
- (c) identifying a thiol-pair buffer strength of the oxidant and the reductant for the preparation based on the solubility of the preparation in combination with the desired yield of the properly refolded protein form;
- and
- (d) generating the preparation.
A method for identifying conditions that promote proper refolding of proteins expressed in a non-mammalian expression system, and generating a preparation therefor, 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;
- an oxidant;
- and
- a reductant;
- (b) selecting a desired minimum yield of properly refolded proteins from a first concentration of proteins;
- (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 solubility of the preparation and the desired yield of properly refolded proteins;
- and
- (e) preparing the preparation.
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 minimum yield of properly refolded proteins from a first concentration of proteins;
- (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 solubility of the preparation and the desired yield of properly refolded proteins;
- and
- (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;
- (h) adjusting, if necessary, the thiol-pair buffer strength for the second concentration of proteins, based on the solubility of the preparation and the desired yield of properly refolded proteins for the second concentration.
Dependencies are inferred from the claim text.