CORRECTLY FOLDED ETANERCEPT IN HIGH PURITY AND EXCELLENT YIELD
First Claim
1. A method of reducing formation of etanercept aggregates and fragments in an aqueous injectable pharmaceutical formulation containing about 25 to about 75 mg/ml etanercept, comprising:
- a. removing incorrectly folded and aggregated etanercept from an etanercept-containing protein mixture to form an etanercept composition containing at least 90 wt. % correctly folded etanercept;
b. combining the etanercept composition with about 0.1 to 2 weight percent of sucrose and an amino acid that is not arginine and is not cysteine, one or more tonicity modifiers selected from the group consisting of sodium chloride, potassium chloride, sodium sulfate, and sodium citrate, and an aqueous buffer selected from the group consisting of phosphate, histidine, citrate, maleate, tartrate, succinate, 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, andwherein 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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Accused Products
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.
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Citations
20 Claims
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1. A method of reducing formation of etanercept aggregates and fragments in an aqueous injectable pharmaceutical formulation containing about 25 to about 75 mg/ml etanercept, comprising:
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a. removing incorrectly folded and aggregated etanercept from an etanercept-containing protein mixture to form an etanercept composition containing at least 90 wt. % correctly folded etanercept; b. combining the etanercept composition with about 0.1 to 2 weight percent of sucrose and an amino acid that is not arginine and is not cysteine, one or more tonicity modifiers selected from the group consisting of sodium chloride, potassium chloride, sodium sulfate, and sodium citrate, and an aqueous buffer selected from the group consisting of phosphate, histidine, citrate, maleate, tartrate, succinate, 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 wherein 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)
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- 16. A method of treating a subject in need of treatment for rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Wegener'"'"'s disease (granulomatosis), Crohn'"'"'s disease (or inflammatory bowel disease), chronic obstructive pulmonary disease (COPD), Hepatitis C, endometriosis, asthma, cachexia, psoriasis, or atopic dermatitis comprising administering to the subject an aqueous pharmaceutical composition comprising about 25 to about 75 mg/ml of an etanercept-containing protein mixture containing at least 90 wt. % correctly folded etanercept, about 0.1 to 2 weight percent of sucrose and an amino acid that is not arginine and is not cysteine, one or more tonicity modifiers selected from the group consisting of sodium chloride, potassium chloride, sodium sulfate, and sodium citrate, and an aqueous buffer selected from the group consisting of phosphate, histidine, citrate, maleate, tartrate, succinate, acetate, tris-(hydroxymethyl)-aminomethane (tris), and bicarbonate, the aqueous pharmaceutical composition having a pH of about 6.2 to 7.4, wherein 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.
- 19. A vial, syringe, or injector pen containing a stable aqueous etanercept composition comprising about 25 to about 75 mg/ml of an etanercept-containing protein mixture containing at least 90 wt. % correctly folded etanercept, about 0.1 to 2 weight percent of sucrose and an amino acid that is not arginine and is not cysteine, one or more tonicity modifiers selected from the group consisting of sodium chloride, potassium chloride, sodium sulfate, and sodium citrate, and an aqueous buffer selected from the group consisting of phosphate, histidine, citrate, maleate, tartrate, succinate, acetate, tris-(hydroxymethyl)-aminomethane (tris), wherein the stable aqueous etanercept composition is less immunogenic than a commercially available etanercept-containing composition and contains less incorrectly folded etanercept than a commercially available etanercept-containing composition.
Specification