Purification of chimeric FVIII molecules
AssigneeBioverativ Therapeutics Inc.
InventorsLily Zhu, Anxhela Kole, John Kulman, Marisol Acosta
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Abstract
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.
Claims11
A method of purifying a chimeric protein comprising a factor VIII (“FVIII”) protein and a von Willebrand Factor (VWF) protein or fragment thereof,
- wherein the FVIII protein is linked to a first Fc region, the VWF protein 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,
- and
- wherein the method comprises the following steps performed in the following order:
- (i) subjecting the chimeric protein to a tangential flow filtration step,
- (ii) subjecting the chimeric protein to a FVIII-specific affinity chromatography, wherein the chimeric protein is eluted from a FVIII-specific affinity chromatography column using a FVIII-specific affinity chromatography elution buffer, and
- (iii) subjecting the chimeric protein to an anion exchange (AEX) chromatography, wherein the chimeric protein is eluted from an AEX chromatography column using an AEX chromatography elution buffer, wherein the AEX chromatography column comprises an anion exchange resin comprising one or more quaternary amino groups, and wherein the AEX chromatography elution buffer comprises:
- (a) about 1 mM to about 100 mM CaCl2,
- (b) from about 1 mM to about 100 mM HEPES, and
- (c) about 0.3 M to about 1.0 M NaCl, and
- (d) has a pH from about 5.0 to about 9.0,
- thereby providing the purified chimeric protein.
A method of purifying a chimeric protein comprising a factor VIII (“FVIII”) protein and a von Willebrand Factor (VWF) protein or fragment thereof,
- wherein the FVIII protein is linked to a first Fc region, the VWF protein 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, wherein the VWF protein prevents or inhibits binding of endogenous VWF to the FVIII protein,
- and
- wherein the method comprises the following steps performed in the following order:
- (i) subjecting the chimeric protein to a tangential flow filtration step,
- (ii) subjecting the chimeric protein to a FVIII-specific affinity chromatography, wherein the chimeric protein is eluted from a FVIII-specific affinity chromatography column using a FVIII-specific affinity chromatography elution buffer, and
- (iii) subjecting the chimeric protein to an anion exchange (AEX) chromatography, wherein the chimeric protein is eluted from an AEX chromatography column using an AEX chromatography elution buffer, wherein the AEX chromatography column comprises an anion exchange resin, and wherein the anion exchange resin is CAPTO-Q,
- thereby providing the purified chimeric protein,
- wherein the FVIII-specific affinity chromatography elution buffer comprises:
- (a) 50 mM histidine,
- (b) 0.9 M arginine-HCl,
- (c) 50 mM CaCl2),
- (d) 45% propylene glycol, and
- (e) about 0.05% TWEEN-20
- and has a pH of 7.2;
- and
- wherein the AEX chromatography elution buffer comprises:
- (a) 5 mM CaCl2,
- (b) 10 mM HEPES, and
- (c) about 0.35 M NaCl
- and has a pH of 7.2.
Dependencies are inferred from the claim text.