Factor X activation
View Patent ↗Methods and systems for activating Factor X are disclosed.
1. A method for activating Coagulation Factor X (FX) comprising:
a) providing an aqueous solution that comprises FX isolated from mammalian plasma or tissue culture cell supernatant;
b) contacting the aqueous solution with an anion-exchange material under binding conditions for a contact period of at least five hours; and
c) eluting the activated FX (FXa) from the anion-exchange material with an elution buffer.
2. The method of claim 1 , wherein the contact period is at least 10 hours.
3. The method of claim 1 , wherein the binding conditions comprise a conductivity of 8 mS/cm or less.
4. The method of claim 1 , wherein the binding conditions comprise a pH of between 8 and 10.
5. The method of claim 1 , wherein the FX is loaded at greater than 0.5 mg per mL of anion-exchange material.
6. The method of claim 1 , wherein the contact and eluting steps are performed at a temperature of greater than 8° C.
7. The method of claim 1 , wherein the elution buffer comprises at least one of a divalent cation and a counter-ion.
8. The method of claim 7 , wherein the divalent cation is Ca 2+ .
9. The method of claim 7 , wherein the counter-ion is at least one of chloride, acetate, or sulfate.
10. The method of claim 7 , wherein the concentration of the divalent cation or counter-anion or both in the elution buffer increases over time.
11. The method of claim 10 , wherein the concentration of the divalent cation increases from 1 to 100 mM.
12. The method of claim 10 , wherein the concentration of the divalent cation increases from 1 to 10 mM.
13. The method of claim 10 , wherein the concentration of the counter-anion increases from 1 to 1000 mM.
14. The method of claim 10 , wherein the concentration of the counter-anion increases from 9 to 200 mM.
15. The method of claim 1 , wherein the elution buffer has a conductivity between 1 and 100 mS/cm at 25° C.
16. The method of claim 1 , wherein the aqueous solution comprising FX is contacted with the anion-exchange material in the presence of ethylenediaminetetraacetic acid (EDTA).
17. The method of claim 1 , wherein the anion-exchange material comprises a ligand comprising at least one of quaternary ammonium [Q], diethylaminoethyl [DEAE], diethylaminopropyl [ANX], or primary amine.
18. The method claim 1 , wherein the anion-exchange material comprises a matrix derived from at least one of polystyrene, polymethylmetaacrylate, polyvinylbenzene, polyvinyl pyridine, cross-linked poly(styrene-divinylbenzene), sepharose, and cross linked agarose.
19. The method of claim 1 , further comprising, prior to step a), isolating FX from mammalian plasma to produce the aqueous solution.
20. The method of claim 1 , further comprising, prior to step a), isolating FX from tissue culture cell supernatant to produce aqueous solution.
21. The method of claim 1 , wherein the FX in the aqueous solution is isolated by precipitation, ultrafiltration, or chromatography.