IP Library › Granted Patent US 12,492,390
Granted Patent B2
US 12,492,390 · App. 18/298,742 · Granted Dec 9, 2025

Methods of purifying recombinant ADAMTS13 and other proteins and compositions thereof

Inventors: Meinhard Hasslacher (Vienna, AT); Christian Fiedler (Vienna, AT); Artur Mitterer (Orth/Donau, AT)
Assignee: TAKEDA PHARMACEUTICAL COMPANY LIMITED
C12N9/6489C12M47/12C12N11/14C12Y304/24087
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Quick Facts
Patent No.
US 12,492,390
App. No.
18/298,742
Granted
Dec 9, 2025
Kind
B2
Abstract

Provided herein are methods for purifying recombinant A Disintegrin-like and Metallopeptidase with Thrombospondin Type 1 Motif 13 (ADAMTS13) protein from a sample. The method comprises enriching for ADAMTS13 protein by chromatographically contacting the sample with hydroxyapatite under conditions that allow ADAMTS13 protein to appear in the eluate or supernatant from the hydroxylapatite. The methods may further comprise tandem chromatography with a mixed mode cation exchange/hydrophobic interaction resin that binds ADAMTS13 protein. Additional optional steps involve ultrafiltration/diafiltration, anion exchange chromatography, cation exchange chromatography, and viral inactivation. Also provided herein are methods for inactivating virus contaminants in protein samples, where the protein is immobilized on a support. Also provided herein are compositions of ADAMTS13 prepared according to said methods.

Claims (52)

1 . An in vitro method for purifying a therapeutic protein composition, said method comprising:

providing a solution comprising a therapeutic protein;

immobilizing said therapeutic protein on an anion exchange resin or cation exchange resin, wherein said immobilization step is performed in the absence of a solvent-detergent mixture; and

subsequently incubating said immobilized therapeutic protein, with a solvent-detergent mixture at concentrations and for a time period suitable to inactivate a lipid-enveloped virus, wherein said solvent-detergent mixture comprises a non-ionic detergent and an organic solvent and

wherein said method results in a reduction in formation of aggregates of said therapeutic protein as compared to a comparable method, wherein said comparable method comprises incubation with said solvent-detergent mixture while said therapeutic protein is not immobilized.

2 . The method according to claim 1 , wherein said therapeutic protein is at least one selected from ADAMTS13, Factor II, Factor VIII, Factor VIIa, Factor IX, von Willebrand factor, and anti-MIF antibody.

3 . The method according to claim 1 , wherein said therapeutic protein is ADAMTS13.

4 . The method according to claim 1 , wherein said solvent-detergent mixture comprises 0.3% Tri-N-butyl phosphate, 0.3% Polysorbate 80, and 1% of a detergent composition comprising octylphenol ethoxylates of general formula:

wherein the average value of N is approximately 9.5.

5 . The method according to claim 1 , wherein incubating with said solvent-detergent mixture is from about 30 minutes to about 1 hour.

6 . The method according to claim 1 , further comprising eluting said therapeutic protein from said anion exchange resin or cation exchange resin with a storage buffer.

7 . The method according to claim 6 , wherein said storage buffer has a pH of greater than 7.0 and comprises less than 10 mM calcium ions, a buffering compound, 0.05% non-ionic detergent, and a salt.

8 . The method according to claim 6 , wherein said therapeutic protein is immobilized on the cation exchange resin; and

wherein there is an ultrafiltration, diafiltration, or buffer exchange step subsequent to the eluting step from the cation exchange resin with the storage buffer.

9 . The method of claim 1 , wherein said therapeutic protein is immobilized on a cation exchange resin.

10 . The method of claim 1 , wherein said therapeutic protein is immobilized on an anion exchange resin.

11 . The method of claim 1 , wherein said solvent-detergent mixture comprises at least 0.1% solvent and at least 0.1% detergent.

12 . The method of claim 1 , wherein said method results in a reduction of at least about 50% in the formation of aggregates of said therapeutic protein as compared to solvent-detergent treatment while said therapeutic protein is not immobilized.

13 . The method of claim 1 , wherein said method results in a reduction of at least about 10% in the formation of aggregates of said therapeutic protein as compared to solvent-detergent treatment while said therapeutic protein is not immobilized.

14 . An in vitro method for purifying a composition comprising ADAMTS13, said method comprising:

providing a solution comprising ADAMTS13;

immobilizing said ADAMTS13 on a cation exchange resin, wherein said immobilization step is performed in the absence of a solvent-detergent mixture; and

subsequently incubating said immobilized ADAMTS13, with a solvent-detergent mixture at concentrations and for a time period suitable to inactivate a lipid-enveloped virus, wherein said solvent-detergent mixture comprises a non-ionic detergent and an organic solvent and

wherein said method results in a reduction in formation of aggregates of said ADAMTS13 as compared to a comparable method, wherein said comparable method comprises incubation with said solvent-detergent mixture while said ADAMTS13 is not immobilized.

15 . The method according to claim 14 , wherein said solvent-detergent mixture comprises 0.3% Tri-N-butyl phosphate, 0.3% Polysorbate 80, and 1% of a detergent composition comprising octylphenol ethoxylates of general formula:

wherein the average value of N is approximately 9.5.

16 . The method according to claim 14 , wherein incubating with said solvent-detergent mixture is from about 30 minutes to about 1 hour.

17 . The method according to claim 14 , further comprising eluting said ADAMTS13 from said cation exchange resin with a storage buffer.

18 . The method according to claim 17 , wherein said storage buffer has a pH of greater than 7.0 and comprises less than 10 mM calcium ions, a buffering compound, 0.05% non-ionic detergent, and a salt.

19 . The method according to claim 17 , wherein there is an ultrafiltration, diafiltration, or buffer exchange step subsequent to the eluting step from the cation exchange resin with the storage buffer.

20 . The method of claim 14 , wherein said solvent-detergent mixture comprises at least 0.1% solvent and at least 0.1% detergent.

21 . The method of claim 14 , wherein said method results in a reduction of at least about 50% in the formation of aggregates of said ADAMTS13 as compared to solvent-detergent treatment while said ADAMTS13 is not immobilized.

22 . The method of claim 14 , wherein said method results in a reduction of at least about 10% in the formation of aggregates of said ADAMTS13 as compared to solvent-detergent treatment while said ADAMTS13 is not immobilized.

23 . An in vitro method for inactivating a lipid-enveloped virus contaminant in a protein solution during the purification of a therapeutic protein composition, said method comprising:

providing the solution comprising the therapeutic protein;

immobilizing said therapeutic protein on an anion exchange resin or cation exchange resin, wherein said immobilization step is performed in the absence of a solvent-detergent mixture; and

subsequently incubating said immobilized therapeutic protein, with a solvent-detergent mixture at concentrations and for a time period suitable to inactivate the lipid-enveloped virus contaminant, wherein said solvent-detergent mixture comprises a non-ionic detergent and an organic solvent and

wherein said method results in a reduction in formation of aggregates of said therapeutic protein as compared to a comparable method, wherein said comparable method comprises incubation with said solvent-detergent mixture while said therapeutic protein is not immobilized.

24 . The method according to claim 23 , wherein said therapeutic protein is at least one selected from ADAMTS13, Factor II, Factor VIII, Factor VIIa, Factor IX, von Willebrand factor, and anti-MIF antibody.

25 . The method according to claim 23 , wherein said therapeutic protein is ADAMTS13.

26 . The method according to claim 23 , wherein said solvent-detergent mixture comprises 0.3% Tri-N-butyl phosphate, 0.3% Polysorbate 80, and 1% of a detergent composition comprising octylphenol ethoxylates of general formula:

wherein the average value of N is approximately 9.5.

27 . The method according to claim 23 , wherein incubating with said solvent-detergent mixture is from about 30 minutes to about 1 hour.

28 . The method according to claim 23 , further comprising eluting said therapeutic protein from said anion exchange resin or cation exchange resin with a storage buffer.

29 . The method according to claim 28 , wherein said storage buffer has a pH of greater than 7.0 and comprises less than 10 mM calcium ions, a buffering compound, 0.05% non-ionic detergent, and a salt.

30 . The method according to claim 28 , wherein said therapeutic protein is immobilized on the cation exchange resin; and

wherein there is an ultrafiltration, diafiltration, or buffer exchange step subsequent to the eluting step from the cation exchange resin with the storage buffer.

31 . The method of claim 23 , wherein said therapeutic protein is immobilized on a cation exchange resin.

32 . The method of claim 23 , wherein said therapeutic protein is immobilized on an anion exchange resin.

33 . The method of claim 23 , wherein said solvent-detergent mixture comprises at least 0.1% solvent and at least 0.1% detergent.

34 . The method of claim 23 , wherein said method results in a reduction of at least about 50% in the formation of aggregates of said therapeutic protein as compared to solvent-detergent treatment while said therapeutic protein is not immobilized.

35 . The method of claim 23 , wherein said method results in a reduction of at least about 10% in the formation of aggregates of said therapeutic protein as compared to solvent-detergent treatment while said therapeutic protein is not immobilized.

Assignments (5)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 12, 2025
From: HASSLACHER, MEINHARD; FIEDLER, CHRISTIAN; MAYER, CHRISTA; MITTERER, ARTUR
To: BAXTER INTERNATIONAL INC.; BAXTER HEALTHCARE S.A.
Reel/Frame 072871/0668 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 15, 2024
From: BAXTER HEALTHCARE S.A.
To: BAXALTA GMBH; BAXALTA INCORPORATED
Reel/Frame 069166/0416 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 15, 2024
From: BAXALTA GMBH; BAXALTA INCORPORATED
To: TAKEDA PHARMACEUTICAL COMPANY LIMITED
Reel/Frame 069172/0813 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 14, 2024
From: BAXTER INTERNATIONAL INC.
To: BAXALTA GMBH; BAXALTA INCORPORATED
Reel/Frame 069169/0001 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 14, 2024
From: HASSLACHER, MEINHARD; FIEDLER, CHRISTIAN; MAYER, CHRISTA; MITTERER, ARTUR
To: BAXTER INTERNATIONAL INC.; BAXTER HEALTHCARE S.A.
Reel/Frame 068885/0988 →
Continuity (4)
Continuation 14571670 · Dec 16, 2014
Continuation 12847956 · Jul 30, 2010
Provisional Application 61230308 · Jul 31, 2009
Related Publication 20230242897A1 · Aug 3, 2023
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