Method for preparing a concentrate of factor XI
View Patent ↗The invention concerns a concentrate of human Factor XI having high specific activity prepared using a method comprising a filtration-adsorption step and a chromatography step on cation exchange resin. The concentrate obtained is fully adapted for therapeutic use as substitution therapy in cases of Factor XI deficiency.
1. A method for preparing a concentrate of Factor XI (FXI), comprising
a) filtration-adsorption of a supernatant of human plasma cryoprecipitate using a filter comprising cellulose and perlites and a charged resin, wherein the filter has a grade ranging from 0.1 to 0.4 μm, wherein the filtration-adsorption step comprises passing the supernatant of plasma cryoprecipitate through the filter to adsorb FXI on the filter, and desorbing or eluting the adsorbed FXI; and
b) chromatography of the product of step a) on a cation exchange resin to obtain a solution of Factor XI.
2. The method according to claim 1 , wherein the filter has a grade ranging from 0.2 to 0.4 μm.
3. The method according to claim 2 , wherein the filter has a grade ranging from 0.25 to 0.35 μm.
4. The method according to claim 3 , wherein the filter has a grade of 0.3 μm.
5. The method according to claim 1 , wherein the FXI adsorbed on the filter is desorbed or eluted using a solution adjusted to a pH of 5.5 to 6.5 comprising sodium citrate, disodium phosphate, potassium phosphate, disodium EDTA and sodium chloride, the concentration of said sodium chloride being higher than 0.5 M.
6. The method according to claim 5 , wherein the concentration of said sodium chloride in said solution is higher than or equal to 0.6 M.
7. The method according to claim 6 , wherein the concentration of said sodium chloride in said solution is higher than or equal to 0.75 M.
8. The method according to claim 7 , wherein the concentration of said sodium chloride in said solution is 1 M.
9. The method according to claim 1 , wherein step a) further comprises washing the filter prior to eluting the adsorbed FXI, using a buffer adjusted to a pH of 5.5 to 6.5 comprising sodium citrate, disodium phosphate, potassium phosphate and sodium chloride, the concentration of said sodium chloride being equal to or lower than 0.5 M.
10. The method according to claim 9 , wherein the concentration of said sodium chloride in said buffer is equal to or lower than 0.4 M.
11. The method according to claim 10 , wherein the concentration of said sodium chloride in said buffer is 0.3 M.
12. The method according to claim 1 , further comprising an additional step for viral inactivation between step a) and step b), and/or a virus removal step after step b).
13. The method according to claim 1 , wherein the cation exchange resin of step b) is equilibrated with a buffer adjusted to a pH of 5.5 to 6.5 comprising sodium citrate, sodium chloride, lysine and arginine.
14. The method according to claim 1 , wherein the cation exchange resin of step b) is loaded with the FXI resulting from step a), then washed with a buffer adjusted to a pH of 6.1 to 6.9 comprising sodium citrate, disodium phosphate, potassium phosphate, sodium chloride, lysine and arginine.
15. The method according to claim 1 , wherein the solution of Factor XI obtained at step b) is stabilised through the addition of 0.5 to 3 IU Antithrombin III, 0.5 to 4 IU of heparin and 0.5 to 2 IU of C1-inhibitor per 100 IU of Factor XI.
16. The method according to claim 1 , further comprising packaging the stabilised FXI solution as a pharmaceutical product.
17. The method according to claim 1 , further comprising lyophilisation of the Factor XI solution obtained from step b).