Immobilization of active protein by cross-linking to inactive protein

• US 4,970,156 A
• Filed: 09/13/1985
• Issued: 11/13/1990
• Est. Priority Date: 03/29/1968
• Status: Expired due to Fees

• Current Assignee: Agence Nationale de Valorisation de la Recherche (Bpifrance SA /Old/)
• Original Assignee: Agence Nationale de Valorisation de la Recherche (Bpifrance SA /Old/)
• Inventors: Thomas, Daniel, Broun, Georges, Selegny, Eric, Avrameas, Statis
• Primary Examiner(s): Naff, David M.
• Application Number:
  US06/775,956
• Time in Patent Office: 1,887 Days
• Field of Search: 435/174, 435/176, 435/177, 435/178, 435/179, 435/181, 435/182, 260/112 R, 260/121, 530/810, 530/811, 530/812, 530/813, 530/814, 530/815, 436/518, 436/519, 436/524, 436/528, 436/529, 436/530, 436/531
• US Class Current: 435/174
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 38/38 Albumins
• CPC Class Codes: A61K 38/443 acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
• CPC Class Codes: A61K 38/47 acting on glycosyl compounds (3.2), e.g. cellulases, lactases
• CPC Class Codes: A61K 38/4826 Trypsin (3.4.21.4) Chymotrypsin (3.4.21.1)
• CPC Class Codes: A61K 38/51 Lyases (4)
• CPC Class Codes: A61K 39/44 Antibodies bound to carriers
• CPC Class Codes: C12N 11/00 Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
• CPC Class Codes: C12N 11/02 Enzymes or microbial cells immobilised on or in an organic carrier
• CPC Class Codes: C12N 11/14 Enzymes or microbial cells immobilised on or in an inorganic carrier
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: D06M 15/11 Starch or derivatives thereof
• CPC Class Codes: D06M 16/00 Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
• CPC Class Codes: Y10S 530/81 Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
• CPC Class Codes: Y10S 530/811 Peptides or proteins is immobilized on, or in, an inorganic carrier
• CPC Class Codes: Y10S 530/812 Peptides or proteins is immobilized on, or in, an organic carrier
• CPC Class Codes: Y10S 530/814 Cellulose or derivatives thereof
• CPC Class Codes: Y10S 530/815 Carrier is a synthetic polymer

Abstract

A solution of an active protein substance and an inactive protein substance is reacted with a cross-linking agent, optionally in the presence of an inert carrier, under cross-linking conditions to produce articles comprising both active and inactive protein substances. The active protein substance comprises up to about 20 percent, e.g. from 1 to 20 percent by weight, based on the final weight of the total protein substance, whereas the cross-linking agent comprises from 0.5 to 8 percent by weight, based on the weight of the total treated mixture. The obtained articles are in the form of a solution or a suspension in aqueous medium, in the form of a film, in the form of a membrane, in the form of a fabric, in the form of a porous material, or in the form of a mass, such as granules, pills or tablets.

Claims

1. A product which consists essentially of immobilized active protein in the form of a compound in which the active protein is homogeneously distributed and correlated with and thus chemically bound to inactive protein through glutaraldehyde-based bridges, the weight ratio of active protein to inactive protein in the compound being at most 1 to 4 and the proportion of glutarladehyde bridges in said compound being sufficient to immobilize the active protein.

2. A product according to claim 1 wherein the active protein is a member selected from the group consisting of antigen, allergen, antibody, hormone, enzyme and proteinic part of a virus or of a cell.

3. A product according to claim 1 wherein the active protein is a member selected from the group consisting of oxidase, hydroxylase, isomerase, transferase, lyase and oxidoreductase.

4. A product according to claim 1 wherein the active protein is a member selected from the group consisting of pepsin, subtilisin, trypsin, chymotrypsin and papain.

5. A product according to claim 1 wherein the active protein is a member selected from the group consisting of glucose-oxidase, carbonic anhydrase, lipolytic hydrolase, amylolytic hydrolase, urease, asparaginase, uricase, peroxidase, catalase, phenylalanine-hydroxylase, galactose phosphate uridyl transferase, pronase, collagenase, keratinase, elastase, urate oxidase, tyrosine decarboxylase, hexokinase, phosphatase, L-amino-acid-oxidase, xanthine oxidase, decarboxylase, ribonuclease, α

-amylase and β

-galactosidase.

6. A product according to claim 1 wherein the active protein substantially retains both its activity and specificity.

7. A shaped solid product having useful mechanical strength and consisting essentially of immobilized active protein according to claim 1.

8. A product according to claim 1 wherein the inactive protein is a member selected from the group consisting of albumin, plasma protein, ovalbumin, fibrinogen, gelatin and hemoglobin.

9. A product according to claim 1 wherein the inactive protein is that found in microbes.

10. A product according to claim 1 wherein the inactive protein is albumin.

11. A product according to claim 2 wherein the inactive protein is a member selected from the group consisting of albumin, plasma protein, ovalbumin, fibrinogen, gelatin, hemoglobin and inactive protein found in microbes.

12. A product according to claim 3 wherein the inactive protein is a member selected from the group consisting of albumin, plasma protein, ovalbumin, fibrinogen, gelatin, hemoglobin and inactive protein found in microbes.

13. A product according to claim 4 wherein the inactive protein is a member selected from the group consisting of albumin, plasma protein, ovalbumin, fibrinogen, gelatin, hemoglobin and inactive protein found in microbes.

14. A product according to claim 5 wherein the inactive protein is a member selected from the group consisting of albumin, plasma protein, ovalbumin, fibrinogen, gelatin, hemoglobin and inactive protein found in microbes.

15. A product according to claim 7 wherein the inactive protein is a member selected from the group consisting of albumin, plasma protein, ovalbumin, fibrinogen, gelatin, hemoglobin and inactive protein found in microbes.

16. A product according to claim 1 wherein the inactive protein is gelatin.

17. A product according to claim 1 wherein the active protein is an antigen.

18. A product according to claim 1 wherein the active protein is an antibody.

19. A product according to claim 1 wherein the active protein is an allergen.

20. A product according to claim 1 wherein the active protein is a part of a virus.

21. A product according to claim 6 wherein the inactive protein is that of a microbe.

22. A product according to claim 1 wherein the active protein and the inactive protein are derived from a single impure source.

23. A product according to claim 22 wherein the single impure source is a microbe source.

24. A self-sustaining flexible sheet or film of immobilized active protein according to claim 1 which is free from a separate or distinct carrier.

25. A sheet or film of immobilized active protein according to claim 1 in combination and integral with an inert non-proteinic carrier which imparts form and strength thereto.

26. A sheet or film according to claim 25 wherein the immobilized active protein is diffused throughout the carrier, which is macroporgus.

27. A sheet or film of immobilized active protein according to claim 25 wherein the carrier is of a material selected from the group consisting of cellulose, regenerated cellulose, amylose, an alginate, a polysilane, polyvinyl alcohol, aminated paper, a polyacrylamide, silicone sheet, activated carbon and silk.

28. Open-cell foam or sponge which consists essentially of immobilized active protein according to claim 1.

29. A product according to claim 1 wherein the active protein is hydrolytic enzyme.

30. A product according to claim 1 wherein the active protein is proteolytic hydrolase.

31. A product according to claim 2 wherein the active protein substantially retains both its activity and specificity, and the inactive protein is a member selected from the group consisting of albumin, plasma protein, ovalbumin, fibrinogen, gelatin and hemoglobin.

32. A product according to claim 1 which, when free from a separate or distinct carrier, is capable of being in a shaped self-supporting form selected from the group consisting of flexible sheet, flexible film, open-cell foam and sponge.

33. A product according to claim 32 which is free from a separate or distinct carrier.

34. A product according to claim 33 which is in one of the stated shaped self-supporting forms.

35. A product according to claim 1 which consists essentially of self-supporting solid.