Correctly folded etanercept in high purity and excellent yield
First Claim
1. A method of reducing incorrectly folded etanercept in and stabilizing an aqueous injectable pharmaceutical formulation containing about 25 to about 75 mg/ml correctly-folded etanercept, comprising:
- a) removing incorrectly folded and aggregated etanercept from an etanercept-containing protein mixture using a mixed-mode chromatography procedure configured to produce an etanercept-containing composition such that the amount of said incorrectly folded etanercept is less than 5 wt % of said composition and wherein the amount of said correctly folded etanercept is greater than 95 wt %, wherein said incorrectly folded etanercept is an etanercept protein that has a conformation different from that of the correctly folded etanercept and said different conformation renders said incorrectly folded etanercept protein lacking in biological activity as a TNF inhibitor and said incorrectly folded etanercept is not an aggregate, wherein said mixed-mode chromatography procedure comprises binding correctly folded etanercept and incorrectly folded etanercept to a mixed-mode chromatography resin having both ion exchange and hydrophobic moieties and contacting the mixed-mode chromatography resin to elute with a salt solution at a pH between 4.5 and 8.5; and
b) combining the etanercept-containing composition formed in step (a) with a correctly folded etanercept-stabilizing formulation comprising about 0.1 to about 2 weight percent of a combination of sugar and an amino acid wherein the amino acid is not arginine and is not cysteine, 0 to about 25 mM NaCl, and an aqueous buffer selected from the group consisting of phosphate, histidine, citrate, maleate, tartrate, acetate, tris-(hydroxymethyl)-aminomethane (tris), and bicarbonate to form the aqueous injectable pharmaceutical formulation containing about 25 to about 75 mg/ml etanercept,wherein the aqueous injectable pharmaceutical formulation has a pH of about 6.2 to 7.4 and does not contain cysteine and does not contain arginine, andwherein the correctly folded etanercept-stabilizing formulation stabilizes said correctly-folded etanercept in said etanercept-containing composition such that the aqueous injectable pharmaceutical formulation is less immunogenic than a commercially available etanercept-containing composition and contains less incorrectly folded etanercept than a commercially available etanercept-containing composition.
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Abstract
A mixed mode chromatography method for separating correctly folded from incorrectly folded conformations of a given protein is provided. The method is highly effective in separating correctly folded etanercept from incorrectly folded etanercept and aggregates in commercially attractive yields capable of affording etanercept preparations having very high purity in terms of correctly folded etanercept versus incorrectly folded etanercept. The invention is further directed to protein preparations and formulations comprising correctly folded proteins obtained using the present methods, and methods of treatment using the high purity preparations obtained from the mixed mode method.
63 Citations
22 Claims
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1. A method of reducing incorrectly folded etanercept in and stabilizing an aqueous injectable pharmaceutical formulation containing about 25 to about 75 mg/ml correctly-folded etanercept, comprising:
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a) removing incorrectly folded and aggregated etanercept from an etanercept-containing protein mixture using a mixed-mode chromatography procedure configured to produce an etanercept-containing composition such that the amount of said incorrectly folded etanercept is less than 5 wt % of said composition and wherein the amount of said correctly folded etanercept is greater than 95 wt %, wherein said incorrectly folded etanercept is an etanercept protein that has a conformation different from that of the correctly folded etanercept and said different conformation renders said incorrectly folded etanercept protein lacking in biological activity as a TNF inhibitor and said incorrectly folded etanercept is not an aggregate, wherein said mixed-mode chromatography procedure comprises binding correctly folded etanercept and incorrectly folded etanercept to a mixed-mode chromatography resin having both ion exchange and hydrophobic moieties and contacting the mixed-mode chromatography resin to elute with a salt solution at a pH between 4.5 and 8.5; and b) combining the etanercept-containing composition formed in step (a) with a correctly folded etanercept-stabilizing formulation comprising about 0.1 to about 2 weight percent of a combination of sugar and an amino acid wherein the amino acid is not arginine and is not cysteine, 0 to about 25 mM NaCl, and an aqueous buffer selected from the group consisting of phosphate, histidine, citrate, maleate, tartrate, acetate, tris-(hydroxymethyl)-aminomethane (tris), and bicarbonate to form the aqueous injectable pharmaceutical formulation containing about 25 to about 75 mg/ml etanercept, wherein the aqueous injectable pharmaceutical formulation has a pH of about 6.2 to 7.4 and does not contain cysteine and does not contain arginine, and wherein the correctly folded etanercept-stabilizing formulation stabilizes said correctly-folded etanercept in said etanercept-containing composition such that the aqueous injectable pharmaceutical formulation is less immunogenic than a commercially available etanercept-containing composition and contains less incorrectly folded etanercept than a commercially available etanercept-containing composition. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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Specification