METHOD FOR THE PURIFICATION OF RECOMBINANT BLOOD COAGULATION FACTOR IX ENRICHED IN SULFATED AND/OR PHOSPHORYLATED MOLECULES
The present invention relates to a method for the purification of rFIX using anion exchange chromatography in the pseudo-affinity mode, wherein said method comprises a wash step with a wash buffer having a salt concentration of more than 200 mM. The purification according to the invention provides a method to enrich rFIX molecules which have been posttranslationally modified by sulfation and/or phosphorylation. The present invention further relates to purified rFIX compositions enriched in monosulfated and/or monophosphorylated rFIX molecules.
1 - 7 . (canceled)
8 . A purified composition comprising rFIX obtained by a method, the method for obtaining the composition comprising the steps:
a) loading a starting composition comprising rFIX onto an anion exchange column.,
b) washing the anion exchange material using a wash buffer which has a salt concentration of more than 200 mM (High Salt Wash Buffer), and
c) eluting the rFIX from the anion exchange material using an elution buffer comprising divalent cations, and
d) collecting the eluate.
9 . The purified composition according to claim 8 , wherein said purified composition comprises an increased relative amount of monosulfated and/or monophosphorylated rFIX molecules compared to the relative amount of monosulfated and/or monophosphorylated rFIX present in the High Salt Wash Buffer fraction.
10 . The purified composition according to claim 8 , wherein said purified composition comprises a relative amount of monosulfated and monophosphorylated rFIX molecules which is increased at least 3 fold compared to the relative amount of monosulfated and monophosphorylated rFIX present in the High Salt Wash Buffer fraction.
11 . The purified composition according to claim 8 , wherein said purified composition comprises a relative amount of at least 30% of monosulfated and/or monophosphorylated rFIX molecules.
12 . The purified composition according to claim 8 , wherein said purified composition comprises a relative amount of at least 50% of monosulfated and monophosphorylated rFIX molecules.
13 . The purified composition according to claim 8 , wherein the rFIX is human rFIX.
14 . The purified composition according to claim 8 , wherein the wash buffer has a conductivity of at least 21 mS/cm at 25° C.
15 . The purified composition according to claim 8 , wherein the wash buffer wherein the wash buffer has a conductivity of 21±1 mS/cm at 25° C.
16 . The purified composition according to claim 8 , wherein the elution buffer has a higher conductivity than the starting composition comprising rFIX.
17 . The purified composition according to claim 8 , wherein the elution buffer has a lower conductivity than the wash buffer which has a salt concentration of more than 200 mM, and which has a higher conductivity than the composition comprising rFIX.
18 . The purified composition according to claim 8 , wherein the elution buffer has a conductivity of at least 10 mS/cm at 25° C.
19 . The purified composition according to claim 8 , wherein the elution is conducted with a gradient of elution buffers having different salt concentrations.
20 . The purified composition according to claim 8 , wherein eluting the rFIX from the anion exchange material includes using a gradient of anions in an elution buffer comprising divalent cations.
21 . The purified composition according to claim 8 , wherein the concentration of divalent cations in the elution buffer is from 0.5 mM to 10 mM.
22 . The purified composition according to claim 8 , wherein the concentration of divalent cation in the elution buffer is from 2 mM to 5 mM.
23 . The purified composition according to claim 8 , wherein the concentration of divalent cation in the elution buffer is 2 mM.
24 . The purified composition according to claim 8 , wherein the divalent cation in the elution buffer is Ca 2+ .
25 . The purified composition according to claim 8 , wherein the elution buffer has a pH of from 6 to 9.
26 . The purified composition according to claim 8 , wherein the elution buffer has a pH of from 7 to 8.
27 . The purified composition according to claim 8 , wherein the elution buffer has a pH of 7.4±0.2.