Process for the purification of factor VIII and factor VIII obtained by said process

Claim 2

Process for the purification of Factor VIII from human plasma comprising treating a solution containing Factor VIII with a ion exchange chromatographic column applying as equilibrating buffer and as eluent for the adsorbed Factor VIII high ionic strength saline solutions at acid pH and collecting the eluate in presence of stabilizers and optionally of an antiprotease.

Claim 3

Process according to claim 1, wherein the resin is an anion exchange resin.

Claim 4

Process according to claim 2, wherein the ion exchange resin comprises quaternary amino groups.

Claim 5

Process according to claims 1 to 3, wherein the equilibrating buffer consists of 10-30 mM Tris, 150-300 mM NaCI, 5-15 mM CaCl 2 and pH is comprised from 6.4 and 6.8.

Claim 6

Process according to claim 4, wherein the equilibrating buffer consists of 20 mM Tris, 250 mM NaCI, 10 mM CaCl 2 and pH 6.6.

Claim 7

Process according to claims 1 to 5, wherein the eluent for Factor VIII consists of 10-30 mM Tris, 450-650 mM NaCI, 5-15 mM CaCl 2 and pH is comprised from 6.4 and 6.8.

Claim 8

Process according to claim 6, wherein the eluent for Factor VIII is 20 mM Tris, 550 mM NaCI, 10 mM CaCl 2 and pH 6.6.

Claim 9

Process according to claims 1 to 7, wherein the eluted Factor VIII is stabilized with:

Claim 10

Process according to claim 8, wherein lysine or histidine are used as antiprotease.

Claim 11

Process according to claim 9, wherein the antiprotease is used in concentration 10 mM.

Claim 12

Process according to claims 1 to 10, wherein: A) Human fresh frozen plasma is thawed with stirring, collecting the cryoprecipitate by centrifugation. B) The cryoprecipitate is solubilized in distilled heparinized water, the solution is treated with an aluminium hydroxide suspension and then with PEG 4000, the pH is adjusted with acetic acid 1 N to a value 6.4-6.6 and the solution is thereafter cooled to 9°-11°C; the proteins adsorbed upon aluminium hydroxide and those precipitated are then removed by centrifugation. C) The supernatant obtained in the previous stage is treated on chromatographic column containing an anion exchange resin by using an equilibrating buffer 20 mM Tris, 250 mM NaCI, 10 mM CaCl 2 and pH 6.6; the resin is then washed with the aforementioned buffer in order to eliminate the undesired proteins and the factor von Willebrand and Factor VIII:C is then recovered by eluting with 20 mM Tris, 550 mM NaCI, 10 mM CaCl 2 and pH 6.6; the so obtained Factor VIII is stabilized with The obtained eluate is concentrated, diafiltrated and further concentrated (final volume = 1/4 starting volume). D) The Factor VIII solution is pasteurized, concentrated, dispensed into vials and lyophilized.

Claim 13

Process according to claim 11, wherein the equilibrating buffer consists of 20 mM Tris, 250 mM NaCI, 10 mM CaC1 2 and pH 6.6

Claim 14

Process according to claim 11, wherein the eluent buffer for Factor VIII is 20 mM Tris, 550 mM NaCI, 10 mM CaCl 2 and pH 6.6.

Claim 15

Factor VIII obtained according to the processes of claims 1 to 13.

Claim 16

Factor VIII according to claim 14, having specific activity higher than 300 U.I. / mg protein.

Claim 17

Use of Factor VIII according to claims 14 and 15 for the preparation of pharmaceutical compositions useful in the substitution therapy of haemophilia A.