Method for Activating Prethrombin-1
AssigneeZymoGenetics, Inc.
InventorsRobert W. Mallet, Christopher J. Stenland, Jonathon C. Boone, John W. Forstrom, Karen S. De Jongh
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Abstract
Methods for converting prethrombin-1 to thrombin are disclosed. An aqueous solution of prethrombin-1 is applied to oscutarin-C immobilized on a solid support so as to provide from 500 mg to 4000 mg of prethrombin-1 per mL of the solid support and a contact time between the prethrombin-1 and the oscutarin-C of from 1.8 to 3.5 minutes. The resulting active thrombin may be captured on an ion exchange chromatography medium or an affinity chromatography medium.
Claims19
A method for converting prethrombin-1 to thrombin comprising:
- providing prethrombin-1 at a concentration of 0.1 mg/mL to 10 mg/mL in an aqueous solution of 30 mM to 110 mM NaCl at pH 6.4-8.0;
- providing oscutarin-C immobilized on a solid support;
- and
- applying the aqueous solution to the immobilized oscutarin-C so as to provide from 500 mg to 4000 mg of prethrombin-1 per mL of the solid support and a contact time between the prethrombin-1 and the oscutarin-C of from 1.8 to 3.5 minutes inclusive, whereby the prethrombin-1 is cleaved to produce thrombin, and a thrombin-containing solution is obtained.
A method for converting prethrombin-1 to thrombin comprising:
- providing prethrombin-1 at a concentration of 1.0 mg/mL to 10 mg/mL in an aqueous solution of 30 mM to 110 mM NaCl at pH=6.8-8.0,
- providing oscutarin-C immobilized on a solid support at a concentration of from from 0.1 mg to 20 mg of oscutarin-C per mL of support;
- applying the prethrombin-1 to the immobilized oscutarin-C so as to provide a ratio of prethrombin-1:oscutarin-C of from 500 mg to 4000 mg of prethrombin-1 per mL of the solid support and a contact time between the prethrombin-1 and the oscutarin-C of from 1.8 to 3.5 minutes at a temperature of from 20° C.-30° C., whereby the prethrombin-1 is cleaved to produce thrombin, and a thrombin-containing solution is obtained;
- applying the thrombin-containing solution to immobilized PABA whereby the thrombin is bound to the PABA;
- washing the bound thrombin to remove impurities;
- and
- recovering the bound thrombin from the immobilized PABA.
Dependencies are inferred from the claim text.