Rapid isolation of osteoinductive protein mixtures from mammalian bone tissue

• US 8,829,166 B2
• Filed: 10/20/2009
• Issued: 09/09/2014
• Est. Priority Date: 06/26/2002
• Status: Expired due to Fees

• Current Assignee: Zimmer Orthobiologics, Inc. (Zimmer Biomet Holdings, Inc.)
• Original Assignee: Zimmer Orthobiologics, Inc. (Zimmer Biomet Holdings, Inc.)
• Inventors: Thorne, Kevin, Akella, Rama
• Primary Examiner(s): Afremova, Vera
• Application Number:
  US12/582,476
• Publication Number:
  US 20100041611A1
• Time in Patent Office: 1,785 Days
• Field of Search: 530/840, 530/412, 514/21.92, 424/549
• US Class Current: 530/412
• CPC Class Codes: A61K 35/32 Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
• CPC Class Codes: A61K 38/1875 Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: C07K 1/34 by filtration, ultrafiltration or reverse osmosis
• CPC Class Codes: C07K 1/36 by a combination of two or more processes of different types
• CPC Class Codes: C07K 14/51 Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
• CPC Class Codes: Y10S 530/84 Bones; tendons; teeth; cartilage

Abstract

A method for purifying bone-derived osteoinductive proteins including a demineralization process, a protein extraction process, a high molecular weight ultrafiltration process, a low molecular weight ultrafiltration process, and a recover process. The high and low ultrafiltration processes preferably select proteins having a nominal molecular weight between approximately 8 kilodaltons and approximately 100 kilodaltons. Processes of the present invention may be used to recover osteoinductive proteins from bone demineralization waste streams.

Claims

1. A method of producing an osteoinductive composition, comprising:

contacting bone tissue with a demineralization medium consisting essentially of an acidic solution and optionally a bone mineral solubility enhancer, a defoaming agent or both to provide demineralized bone tissue and a mineralized supernatant;

separating the mineralized supernatant from the demineralized bone tissue;

recovering osteoinductive proteins from the mineralized supernatant; and

combining the osteoinductive proteins with a carrier.

2. The method of claim 1, wherein the carrier comprises a solid.

3. The method of claim 2, wherein the solid carrier is selected from the group consisting of collagen, a bone derivative, hydroxyapatite or a composite device.

4. The method of claim 2, wherein the solid carrier comprises a bone derivative selected from the group consisting of bone chips or bone matrix.

5. The method of claim 2, wherein the solid carrier comprises bone matrix.

6. The method of claim 5, wherein the bone matrix comprises demineralized and devitalized bone matrix (DVBM).

7. The method of claim 1, wherein the carrier comprises a liquid.

8. The method of claim 7, wherein the liquid carrier is selected from the group consisting of saline, a blood-derivative or bone marrow aspirate.

9. The method of claim 7, wherein the liquid carrier comprises a blood derivative selected from the group consisting of whole blood, serum, plasma or platelet rich plasma (PRP).

10. The method of claim 2, wherein the liquid carrier comprises serum.

11. The method of claim 2, wherein the liquid carrier comprises saline.

12. The method of claim 1, wherein the carrier comprises a slurry.

13. The method of claim 1, further comprising storing the osteoinductive proteins as a solution or a solid prior to or after combining with the carrier.

14. The method of claim 13, wherein the osteoinductive proteins are stored sterile.

15. The method of claim 13, wherein the osteoinductive proteins are stored under vacuum or an inert gas atmosphere.

16. The method of claim 15, wherein the inert gas is selected from the group consisting of nitrogen, hydrogen, helium, argon or mixtures thereof.

17. The method of claim 13, wherein the osteoinductive proteins are stored as a solid.

18. The method of claim 17, wherein the osteoinductive proteins are lyophilized.

19. The method of claim 1, further comprising removing at least some mineral from the mineralized supernatant prior to or while recovering osteoinductive proteins.

20. The method of claim 19, wherein removing comprises performing tangential flow filtration of the mineralized supernatant.

21. The method of claim 19, wherein removing comprises contacting the mineralized supernatant with mineral precipitation agent.

22. The method of claim 1, wherein the solubility enhancer is a salt or solvent.

23. The method of claim 22, wherein the salt is CaCl₂ or ethylenediamine tetraacetic acid (EDTA).

24. The method of claim 1, wherein the defoaming agent is an alcohol, hydrocarbon, fatty acid, fatty acid salt of a multivalent cation, ester, amide or silicone oil or silica.

25. The method of claim 24, wherein the alcohol is octyl alcohol.

26. The method of claim 24 wherein the hydrocarbon is mineral oil, white oil or paraffin.

27. The method of claim 24, wherein the ester is a fat, wax or phosphoric ester.

28. The method of claim 24, wherein the amide is an amide wax.

29. The method of claim 1, wherein the defoaming agent is an alcohol.