IP Library › Granted Patent US 12,570,691
Granted Patent B2
US 12,570,691 · App. 17/155,935 · Granted Mar 10, 2026

Purification of chimeric FVIII molecules

Inventors: Lily Zhu (Belmont, MA); Anxhela Kole (Quincy, MA); John Kulman (Belmont, MA); Marisol Acosta (Worcester, MA)
Assignee: BIOVERATIV THERAPEUTICS INC.
C07K1/36C07K14/755C07K2319/31
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Quick Facts
Patent No.
US 12,570,691
App. No.
17/155,935
Granted
Mar 10, 2026
Kind
B2
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.

Claims (56)

1 . 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, and

(iv) eluting the chimeric protein with an AEX chromatography elution buffer, the AEX chromatography elution buffer comprising:

about 1 mM to about 1M N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES),

at least 400 mM sodium chloride, and

wherein the AEX chromatography elution buffer has a pH from about 6.0 to about 8.0, thereby providing the purified chimeric protein.

2 . The method of claim 1 , wherein the FVIII-specific affinity chromatography elution buffer has a pH of about 7.4.

3 . The method of claim 1 , wherein the AEX chromatography elution buffer comprises about 10 mM HEPES.

4 . The method of claim 1 , wherein the AEX chromatography elution buffer has a pH of about 7.8.

5 . The method of claim 1 , wherein the FVIII-specific affinity chromatography elution buffer comprises histidine, calcium chloride, arginine, and propylene glycol.

6 . The method of claim 5 , wherein the FVIII-specific affinity chromatography elution buffer comprises from about 30 mM to about 100 mM histidine.

7 . The method of claim 6 , wherein the FVIII-specific affinity chromatography elution buffer comprises about 50 mM histidine.

8 . The method of claim 5 , wherein the FVIII-specific affinity chromatography elution buffer comprises about 50 mM calcium chloride.

9 . The method of claim 5 , wherein the FVIII-specific affinity chromatography elution buffer comprises about 900 mM arginine.

10 . The method of claim 5 , wherein the FVIII-specific affinity chromatography elution buffer comprises from about 30% to about 60% propylene glycol.

11 . The method of claim 10 , wherein the FVIII-specific affinity chromatography elution buffer comprises about 45% propylene glycol.

12 . The method of claim 1 , wherein the FVIII-specific affinity chromatography elution buffer has a pH from about 6.0 to about 8.0.

13 . The method of claim 1 , wherein the method further comprises subjecting the chimeric protein to one or more additional purification and/or separation steps.

14 . The method of claim 1 , further comprising subjecting the chimeric protein to a tangential flow filtration step prior to step (i).

15 . The method of claim 1 , wherein the AEX chromatography elution buffer does not comprise calcium chloride.

16 . The method of claim 1 , wherein the method further comprises subjecting the eluted chimeric protein resulting from step (iv) to one or more additional purification and/or separation steps.

17 . The method of claim 16 , wherein the one or more additional purification and/or separation steps are selected from cation exchange chromatography, hydrophobic interaction chromatography, size-exclusion chromatography, multimodal chromatography, reversed phase chromatography, chromatofocusing, filtration, viral inactivation, and precipitation.

18 . The method of claim 1 , wherein the chimeric protein comprises the FVIII protein and a VWF fragment.

19 . The method of claim 18 , wherein the FVIII protein comprises a full or partial deletion of the B domain.

20 . The method of claim 19 , wherein the VWF fragment prevents or inhibits binding of endogenous VWF to the FVIII protein.

21 . The method of claim 19 , 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.

22 . The method of claim 21 , 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).

23 . The method of claim 22 , wherein the XTEN sequence is inserted into the FVIII protein.

24 . The method of claim 23 , wherein the XTEN sequence is inserted into the FVIII protein immediately downstream of residue 745 of the FVIII polypeptide.

25 . A method of purifying a chimeric protein comprising a factor VIII (FVIII) protein and a von Willebrand Factor (VWF) protein or a VWF fragment, 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, the FVIII-specific affinity chromatography elution buffer comprising:

50 mM histidine,

50 mM calcium chloride,

900 mM arginine-HCl,

45% propylene glycol, and

wherein the FVIII-specific affinity chromatography elution buffer has a pH of about 7.4;

(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;

(iv) eluting the chimeric protein with an AEX chromatography elution buffer, the AEX chromatography elution buffer comprising:

10 mM HEPES, and

at least 400 mM sodium chloride, and

wherein the AEX chromatography elution buffer has a pH of about 7.8 thereby providing the purified chimeric protein.

26 . The method of claim 25 , further comprising subjecting the chimeric protein to a tangential flow filtration step prior to step (i).

27 . The method of claim 25 , wherein the factor VIII-specific affinity chromatography elution buffer comprises a detergent, wherein the detergent is selected from the group consisting of polysorbate 20, polysorbate 80, polyethylene glycol, p-(1,1,3,3-tetramethylbutyl)-phenyl ether, octylphenoxypolyethoxyethanol, 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS), and 3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonate (CHAPSO).

28 . The method of claim 25 , further comprising washing chimeric protein bound to the affinity chromatography column with a FVIII-specific affinity chromatography wash buffer.

29 . The method of claim 25 , wherein the AEX chromatography elution buffer further comprises tris-(hydroxymethyl)aminoethane.

30 . The method of claim 25 , wherein the AEX chromatography elution buffer comprises a detergent, wherein the detergent is selected from the group consisting of polysorbate 20, polysorbate 80, polyethylene glycol, p-(1,1,3,3-tetramethylbutyl)-phenyl ether, octylphenoxypolyethoxyethanol, 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS), and 3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonate (CHAPSO).

31 . The method of claim 25 , further comprising: subjecting the eluted chimeric protein to one or more additional purification and/or separation steps, wherein the one or more additional purification and/or separation are selected from cation exchange chromatography, hydrophobic interaction chromatography, size-exclusion chromatography, multimodal chromatography, reversed phase chromatography, chromatofocusing, filtration, viral inactivation, and precipitation.

32 . The method of claim 25 , wherein the AEX chromatography elution buffer does not comprise calcium chloride.

33 . The method of claim 25 , wherein the anion exchange resin is strong quaternary ammonium (Q) anion exchanger.

34 . The method of claim 25 , wherein the chimeric protein comprises the FVIII protein and a VWF fragment.

35 . The method of claim 34 , 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.

36 . The method of claim 35 , wherein the VWF fragment prevents or inhibits binding of endogenous VWF to the FVIII protein.

37 . The method of claim 35 , 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).

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jan 16, 2026
From: ZHU, LILY; KOLE, ANXHELA; KULMAN, JOHN; ACOSTA, MARISOL
To: BIOGEN MA INC.
Reel/Frame 073493/0802 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jan 16, 2026
From: BIOGEN MA INC.
To: BIOVERATIV THERAPEUTICS INC.
Reel/Frame 074394/0401 →
Continuity (3)
Continuation 14910555
Provisional Application 61863810 · Aug 8, 2013
Related Publication 20210261607A1 · Aug 26, 2021
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