Composition, method, and kit for preparing plasmin

• US 8,617,863 B2
• Filed: 06/03/2009
• Issued: 12/31/2013
• Est. Priority Date: 06/04/2008
• Status: Active Grant

• Current Assignee: Grifols Therapeutics LLC (Grifols SA)
• Original Assignee: Grifols Therapeutics Incorporated (Grifols SA)
• Inventors: Koepf, Edward, Zimmerman, Thomas
• Primary Examiner(s): Saidha, Tekchand
• Assistant Examiner(s): Ramanujam, Rama P
• Application Number:
  US12/995,447
• Publication Number:
  US 20110201077A1
• Time in Patent Office: 1,672 Days
• Field of Search: 435/174, 435/217, 435/192
• US Class Current: 435/174
• CPC Class Codes: C07K 14/3153 Streptokinase
• CPC Class Codes: C12N 11/10 the carrier being a carbohydrate
• CPC Class Codes: C12N 9/6435 Plasmin (3.4.21.7), i.e. fibrinolysin
• CPC Class Codes: C12Y 304/21007 Plasmin (3.4.21.7), i.e. fibrinolysin

Abstract

A streptokinase immobilized on a surface, in particular an immobilized plasmin-resistant streptokinase, and compositions, methods and kits of utilizing same for preparing plasmin are provided.

Claims

1. A method for preparing plasmin, the method comprising:

(a) contacting a composition comprising a plasminogen with a streptokinase immobilized on a matrix thereby converting the plasminogen to a plasmin,

wherein the streptokinase is a streptokinase mutant characterized as capable of activating plasminogen to plasmin, yet resistant to plasmin degradation relative to its corresponding wild-type streptokinase; and

wherein the streptokinase comprises an amino acid sequence having an amino acid residue other than lysine at a position corresponding to position 85, 412, or both positions, as shown in SEQ ID NO;

1; and

(b) purifying the plasmin by a method comprising contacting the composition with a plasmin-binding matrix so that the plasmin is retained by the plasmin-binding matrix, the plasmin-binding matrix having a molecule disposed thereon having affinity for the plasmin.

2. The method of claim 1, wherein the streptokinase comprises the amino acid sequence shown as amino acid residues 27 through 440 of SEQ ID NO:

2.

3. The method of claim 1, wherein the streptokinase, optionally, further comprises polar or charged amino acid residues comprising lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, tryosine or cysteine at one or more positions corresponding to positions 406-410 in SEQ ID NO:

1.

4. The method of claim 1, wherein the amino acid residue is glutamine or asparagine.

5. The method of claim 2, wherein the streptokinase, optionally, further comprises polar or charged amino acid residues comprising lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, tryosine or cysteine at one or more positions corresponding to positions 406-410 in SEQ ID NO:

1.

6. The method of claim 1, wherein the streptokinase is recombinant.

7. The method of claim 1, wherein the streptokinase comprises an affinity tag.

8. The method of claim 7, wherein the affinity tag comprises polyhistidine, thioredoxin, or glutathione-S-transferase (GST).

9. The method of claim 7, wherein the affinity tag comprises polyhistidine.

10. The method of claim 1, wherein the streptokinase is immobilized on a matrix comprising immobilized metal ion adsorption chromatography.

11. The method of claim 1, wherein the plasma-binding matrix is benzamidine-coupled to crosslinked beaded agarose.

12. The method of claim 1, wherein the plasminogen is plasma-derived.

13. The method of claim 1, wherein the plasminogen is recombinant.

14. A method for preparing plasmin, the method comprising:

(a) contacting a composition comprising a plasminogen with a streptokinase immobilized on a matrix thereby converting the plasminogen to a plasmin,

wherein the streptokinase is a streptokinase mutant characterized as capable of activating plasminogen to plasmin, yet resistant to plasmin degradation relative to its corresponding wild-type streptokinase;

wherein the streptokinase comprises an amino acid sequence having an amino acid residue other than lysine at a position corresponding to position 85, 412, or both positions, as shown in SEQ ID NO;

1; and

wherein the streptokinase comprises a polyhistidine affinity tag; and

(b) purifying the plasmin by a method comprising contacting the composition with a plasmin-binding matrix so that the plasmin is retained by the plasmin-binding matrix;

wherein the plasma-binding matrix is benzamidine-coupled to crosslinked beaded agarose.

15. The method of claim 14, wherein the streptokinase comprises the amino acid sequence shown as amino acid residues 27 through 440 of SEQ ID NO:

2.

16. The method of claim 14, wherein the streptokinase, optionally, further comprises polar or charged amino acid residues comprising lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, tryosine or cysteine at one or more positions corresponding to positions 406-410 in SEQ ID NO:

1.

17. The method of claim 14, wherein the amino acid residue is glutamine or asparagine.

18. The method of claim 15, wherein the streptokinase, optionally, further comprises polar or charged amino acid residues comprising lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, tryosine or cysteine at one or more positions corresponding to positions 406-410 in SEQ ID NO:

1.

19. The method of claim 14, wherein the plasminogen is plasma-derived.

20. The method of claim 14, wherein the plasminogen is recombinant.