REFOLDING PROTEINS USING A CHEMICALLY CONTROLLED REDOX STATE
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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.
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54 Claims
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1-24. -24. (canceled)
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25. A method of refolding proteins expressed in a non-mammalian expression system, the method comprising:
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(a) contacting the proteins with a preparation that supports the renaturation of at least one of the proteins to a biologically active form, to form a refold mixture, the preparation comprising; (i) a solution that comprises; (A) a thiol-pair ratio in the range of 0.001-100; and (B) a thiol-pair buffer strength, wherein the thiol-pair ratio and the thiol-pair buffer strength yield at least about 25% properly refolded protein; and (ii) at least one ingredient selected from the group consisting of a denaturant, an aggregation suppressor and a protein stabilizer; and (b) incubating the refold mixture. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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36. A method of refolding proteins expressed in a non-mammalian expression system, the method comprising:
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(a) generating a solution that comprises; (i) a thiol-pair ratio in the range of 0.001-100; and (ii) a thiol-pair buffer strength, wherein the thiol-pair ratio and the thiol-pair buffer strength yield at least about 25% properly refolded protein; (b) selecting at least one ingredient selected from the group consisting of a denaturant, an aggregation suppressor and a protein stabilizer; (c) combining the at least one ingredient with the solution of (a) to form a preparation that supports the refolding of the proteins; and (d) contacting the proteins with the preparation to achieve a chemically-controlled refold mixture; (e) incubating the refold mixture. - View Dependent Claims (37, 38, 39, 40, 41, 42, 43, 44, 45, 46)
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47. A method of refolding proteins expressed in a non-mammalian expression system, the method comprising:
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(a) contacting the proteins with a preparation that supports the renaturation of at least one of the proteins to a biologically active form, to form a refold mixture, the preparation comprising; (i) a solution that comprises; (A) a thiol-pair ratio in the range of 0.001-100; and (B) a thiol-pair buffer strength, wherein the thiol-pair ratio and the thiol-pair buffer strength yield about 30-80% properly refolded protein; and (ii) at least one ingredient selected from the group consisting of a denaturant, an aggregation suppressor and a protein stabilizer; and (b) incubating the refold mixture. - View Dependent Claims (48, 49, 50)
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51. A method of refolding proteins expressed in a non-mammalian expression system, the method comprising:
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(a) generating a solution that comprises; (i) a thiol-pair ratio in the range of 0.001-100; and (ii) a thiol-pair buffer strength, wherein the thiol-pair ratio and the thiol-pair buffer strength yield about 30-80% properly refolded protein; (b) selecting at least one ingredient selected from the group consisting of a denaturant, an aggregation suppressor and a protein stabilizer; (c) combining the at least one ingredient with the solution of (a) to form a preparation that supports the refolding of the proteins; (d) contacting the proteins with the preparation to achieve a chemically-controlled refold mixture; and (e) incubating the refold mixture. - View Dependent Claims (52, 53, 54)
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Specification