PURIFICATION OF CHIMERIC FVIII MOLECULES
The invention is directed to methods of purifying a chimeric protein comprising subjecting the chimeric protein to a factor VIII-specific affinity chromatography, and subjecting the chimeric protein to an AEX chromatography; wherein the chimeric protein comprises a factor VIII protein or a fragment thereof. The chimeric protein purified by the present methods shows improved factor VIII activity.
1 - 89 . (canceled)
90 . A method of purifying a chimeric protein comprising a factor VIII (FVIII) protein and a von Willebrand Factor (VWF) protein or a VWF fragment, the method comprising:
(i) subjecting the chimeric protein to a FVIII-specific affinity chromatography column,
(ii) eluting the chimeric protein with a FVIII-specific affinity chromatography elution buffer,
(iii) subjecting the chimeric protein to an anion exchange (AEX) chromatography column, wherein the AEX chromatography column comprises an anion exchange resin comprising one or more quaternary amino groups.
91 . The method of claim 90 , wherein the FVIII protein comprises a full or partial deletion of the B domain.
92 . The method of claim 91 , wherein the FVIII protein comprises a partial deletion of the B domain.
93 . The method of claim 91 , wherein the FVIII protein comprises a full deletion of the B domain.
94 . The method of claim 90 , wherein the chimeric protein comprises the FVIII protein and a VWF fragment.
95 . The method of claim 94 , wherein the FVIII protein is linked to a first Fc region, the VWF fragment comprises a D′ domain and a D3 domain of VWF and is linked to a second Fc region, and the first Fc region and the second Fc region form a disulfide bond.
96 . The method of claim 95 , wherein the chimeric protein further comprises an XTEN sequence that consists of 4 to 6 types of amino acids selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P).
97 . The method of claim 96 , wherein the XTEN sequence is inserted into the FVIII protein.
98 . The method of claim 97 , wherein the XTEN sequence is inserted into the FVIII protein immediately downstream of residue 745 of the FVIII polypeptide.
99 . The method of claim 90 , wherein the FVIII-specific affinity chromatography elution buffer comprises histidine, calcium chloride, arginine, and propylene glycol.
100 . The method of claim 90 , wherein the FVIII-specific affinity chromatography elution buffer has a pH from about 6.0 to about 8.0.
101 . The method of claim 90 , wherein the method further comprises subjecting the chimeric protein to one or more additional purification and/or separation steps.
102 . The method of claim 90 , further comprising subjecting the chimeric protein to a tangential flow filtration step prior to step (i).
103 . The method of claim 99 , wherein the FVIII-specific affinity chromatography elution buffer comprises from about 30 mM to about 100 mM histidine.
104 . The method of claim 103 , wherein the FVIII-specific affinity chromatography elution buffer comprises about 50 mM histidine.
105 . The method of claim 99 , wherein the FVIII-specific affinity chromatography elution buffer comprises about 50 mM calcium chloride.
106 . The method of claim 99 , wherein the FVIII-specific affinity chromatography elution buffer comprises about 900 mM arginine.
107 . The method of claim 99 , wherein the FVIII-specific affinity chromatography elution buffer comprises from about 30% to about 60% propylene glycol.
107 . The method of claim 107 , wherein the FVIII-specific affinity chromatography elution buffer comprises about 45% propylene glycol.