Correctly folded etanercept in high purity and excellent yield
First Claim
1. An etanercept-containing protein mixture comprising correctly folded etanercept in an amount constituting greater than 95 wt % and less than 5 wt % incorrectly folded etanercept of the protein mixture, andwherein said etanercept-containing protein mixture contains 0 to 25 mM NaCl and does not contain arginine or cysteine, andwherein 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 protein mixture is obtained by using a mixed-mode chromatography method involving 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.
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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.
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28 Claims
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1. An etanercept-containing protein mixture comprising correctly folded etanercept in an amount constituting greater than 95 wt % and less than 5 wt % incorrectly folded etanercept of the protein mixture, and
wherein said etanercept-containing protein mixture contains 0 to 25 mM NaCl and does not contain arginine or cysteine, and 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 protein mixture is obtained by using a mixed-mode chromatography method involving 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.
Specification