STORAGE-STABLE, FUNCTIONALLY INTACT FIBRINOGEN

• US 20110114524A1
• Filed: 06/22/2009
• Published: 05/19/2011
• Est. Priority Date: 06/23/2008
• Status: Active Grant

• Current Assignee: Bio-Products & Bio-Engineering Aktiengesellschaft
• Original Assignee: Bio-Products & Bio-Engineering Aktiengesellschaft
• Inventors: Eibl, Johann
• Granted Patent: US 8,741,846 B2
• Time in Patent Office: Days
• US Class Current: 206/438
• CPC Class Codes: A61K 47/183 Amino acids, e.g. glycine, EDTA or aspartame
• CPC Class Codes: A61K 47/26 Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• CPC Class Codes: A61K 47/46 Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61K 9/19 lyophilised , i.e. freeze-dried, solutions or dispersions lyophilised products with subsequent particle size reduction A61K9/14; granules or pellets made by lyphilisation A61K9/1682; solid oral dosage forms made by lyophilisation A61K9/2095; lyophilisation additives A61K47/00
• CPC Class Codes: A61P 7/08 Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock artificial tears A61P27/04
• CPC Class Codes: C07K 14/75 Fibrinogen

Abstract

Method for the production of a fibrinogen solution from an aqueous solution that contains functionally intact fibrinogen and is contaminated with profibrin and/or fibrin monomers and/or fibrin monomer complexes and/or fibrin split products, characterized in that the contaminations are precipitated using a non-denaturing precipitating agent at a temperature ranging from −4° to +4° and calcium ion activity not exceeding that of a 1,000 μM CaCl₂ solution and are separated from the solution by means of filtering or centrifugation process.

Claims

1. A method for producing a fibrinogen solution from an aqueous solution containing functionally intact fibrinogen and being contaminated with profibrin and/or fibrin monomers and/or fibrin monomer complexes and/or fibrin split products, wherein the contaminations are precipitated with a non-denaturing precipitating agent at a temperature between −

4°

to +4°

C. and a calcium ion activity not higher than that of a 1,000 μ

M CaCl₂-solution, and are separated by filtration or centrifugation from the aqueous solution.

2. A method according to claim 1, wherein the aqueous solution has a temperature between 0°

and 2°

C. and an electric conductivity of ≦

500 μ

S/cm when it is precipitated.

3. A method according to claim 1, wherein that the calcium ion activity is not exceeding that of a 500 μ

M CaCl₂ solution.

4. A method according to claim 3, wherein the calcium ion activity is not exceeding than that of a 50 μ

M CaCl₂ solution.

5. A method according to claim 2, wherein the solution has a temperature between 0°

and 2°

C., an electric conductivity between 10 to 100 μ

S/cm and a calcium ion activity not exceeding that of a 500 μ

M CaCl₂ solution.

6. A method according to claim 1, wherein the fibrinogen contained in the obtained fibrinogen solution is further purified by at least one more precipitation step.

7. A method according to claim 1, comprising obtaining a fibrinogen solution containing 0.3% to 10% clottable protein, which is storable up to 36 months at a temperature of 2°

to 8°

C., by removal of enzymes and their pro-enzymes which, as such or in activated form, recognize fibrinogen as a substrate, using non-denaturing precipitating agents and, optionally, by adding appropriate virus inactivated, homologous protease inhibitors.

8. A method according to claim 1, wherein that the fibrinogen solution is subjected to a method for virus inactivation or virus removal, which is carried out at temperatures above room temperature, a thrombin activity of ≦

0.03 IE/ml and a calcium ion activity not exceeding that of a 1,000 μ

M CaCl₂ solution.

9. A method according to claim 8, wherein the viruses are removed by means of nanofiltration at temperatures between 20°

and 40°

C. and using nanofilters with a pore size of at most 20 nm, said nanofiltration being carried out in a way so that fibrinopeptide A is not split off.

10. A method for producing a fibrinogen concentrate, wherein a fibrinogen solution produced according to claim 1 is concentrated, deep-frozen or freeze-dried.

11. A fibrinogen concentrate, obtained in the method according to claim 10.

12. A fibrinogen concentrate according to claim 11 in freeze-dried form for use as a pharmaceutical drug substance, wherein the concentrate, when reconstituted with an appropriate solvent to a 0.3 to 10% fibrinogen solution, is storable for at least 10 days between 2°

and 8°

C., without a decrease in clottable protein or without gel formation.

13. A fibrinogen concentrate according to claim 12, which is reconstitutable with a solvent in less than 10 minutes after a storage period of at least 18 months at 2°

to 8°

C.

14. A fibrinogen concentrate according to claim 11, which contains fibrinogen, from which at least 70% fibrinopeptide A, based on clottable protein, is capable of being split off by thrombin.

15. A fibrinogen concentrate according to claim 11, which contains at least 1 arbitrary unit AT-I per 10 mg clottable protein.

16. A fibrinogen concentrate according to claim 11, which does not contain particles having a diameter of more than 20 nm.

17. A fibrinogen concentrate according to claim 11, which was heated to a temperature of 90°

to 145°

C. for 30 to 180 minutes after freeze-drying.

18. A medicinal product containing a fibrinogen concentrate according to claim 11 as a drug substance.

19. A medicinal product according to claim 18 which is contained in ready-to-use syringes or infusion bag.

20. A method for the manufacture of a medicinal product containing a freeze-dried fibrinogen concentrate according to claim 10 and to which basic amino acids up to half of the weight of clottable protein are added, as well as salts, in order to provide osmolality, the freeze-dried fibrinogen concentrates being reconstituted in a solvent containing an amount of calcium salt ensuring that the reconstituted product has a calcium ion activity corresponding to that of a 5 mM CaCl₂ solution.

21. A method according to claim 10, comprising heat inactivation of viruses in final containers for a period of 30 to 100 minutes at temperatures between 90°

and 145°

C.

24. A method according to claim 1, wherein that the calcium ion activity of the aqueous solution is lowered by adding EDTA and that glycine is used as non-denaturing precipitating agent

25. A method according to claim 2, wherein reduction of electric conductivity of the aqueous solution is realized by dialysis, diafiltration or alcohol precipitation in the cold.

26. A fibrinogen concentrate, obtained by a method according to claim 24.

22. (canceled)

23. (canceled)