Immobilization of active protein by cross-linking to inactive protein

• US 4,464,468 A
• Filed: 04/16/1982
• Issued: 08/07/1984
• 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, Avrameas, Stratis, Broun, Georges, Selegny, Eric
• Primary Examiner(s): Naff, David M.
• Application Number:
  US06/369,253
• Time in Patent Office: 844 Days
• Field of Search: 435/174, 435/177, 435/179, 435/181, 260/112 R, 260/121
• US Class Current: 435/177
• 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 35/74 Bacteria therapeutic use of a bacterial protein A61K38/00
• CPC Class Codes: A61K 35/742 Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
• CPC Class Codes: A61K 38/363 Fibrinogen
• 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 11/02 Enzymes or microbial cells immobilised on or in an organic carrier
• CPC Class Codes: Y10S 530/816 Attached to the carrier via a bridging agent

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 Self-supporting open-cell foam or sponge consisting essentially of a homogenous distribution of active protein and inactive protein cross-linked together with glutaraldehyde having the following properties:

(a) surface area--10³ cm² /cm³ or, when dry, 1 m² /g;

(b) specific gravity--0.1 when dry (including open cells);

(c) free volume--90 percent;

(d) open cell sizes--from 1 to 200 microns;

(e) capacity for water absorption--10 times its volume when starting with dry material;

(f) absorption capacity--30 percent when considering only the insoluble phase;

(g) elasticity (tension modulus)--0.4 to 0.7 kg/mm² ;

(h) compression--0.2 to 0.9 kg/mm², said active protein comprising up to 20 percent by weight of the total active and inactive proteins.

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

2. A process for producing an immobilized active protein in the form of a self-supporting flexible sheet or film or a self-supporting open-cell foam or sponge consisting essentially of mixing

(a) active protein and

(b) inactive protein simultaneously with

(c) glutaraldehyde

in a solvent-containing reaction medium to form a reaction mixture and reacting with active and inactive proteins with said glutaraldehyde under cross-linking conditions until the active protein is cross-linked with the inactive protein through bridges provided by the glutaraldehyde to form said sheet, film, foam or sponge, said active protein comprising up to 20 percent by weight of total active and inactive proteinic substance and said glutaraldehyde being in the range of from 0.5 to 8 percent by weight, based on the weight of total reaction mixture, said sheet or film being formed by spreading said reaction mixture on a surface before cross-linking and said foam or sponge being formed by freezing and slowly thawing said reaction mixture during said cross-linking.

3. A process according to claim 2 wherein cross-linking is effected while reducing the amount of solvent in the reaction medium to form said sheet or film.

4. A process according to claim 2 wherein said foam or sponge is formed.

5. A process according to claim 2 wherein the inactive protein is an albumin and the amount of solvent in the reaction medium is reduced during cross-linking.

6. A process according to claim 2 wherein said sheet or film is formed, the inactive protein is gelatin and the amount of solvent in the reaction medium is reduced during cross-linking.

7. An immobilized active protein produced by the process of claim 2.

8. Immobilized active protein according to claim 7 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.

9. Immobilized active protein according to claim 7 wherein the active protein is a member selected from the group consisting of oxidase, hydroxylase, isomerase, transferase, lyase and oxidoreductase.

10. Immobilized active protein according to claim 7 wherein the active protein is a member selected from the group consisting of pepsin, subtilisin, trypsin, chymotrypsin and papain.

11. Immobilized active protein according to claim 7 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.

12. Immobilized active protein according to claim 7 wherein the active protein substantially retains both its activity and specificity.

13. Immobilized active protein according to claim 7 wherein the inactive protein is a member selected from the group consisting of albumin, plasma protein, ovalbumin, fibrinogen, gelatin and hemoglobin.

14. Immobilized active protein according to claim 7 wherein the inactive protein is that found in microbes.

15. Immobilized active protein according to claim 7 wherein the inactive protein is albumin.

16. Immobilized active protein according to claim 8 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.

17. Immobilized active protein according to claim 9 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.

18. Immobilized active protein according to claim 10 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.

19. Immobilized active protein according to claim 11 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.

20. Immobilized active protein according to claim 7 wherein the inactive protein is gelatin.

21. Immobilized active protein according to claim 7 wherein the active protein is an antigen.

22. Immobilized active protein according to claim 7 wherein the active protein is an antibody.

23. Immobilized active protein according to claim 7 wherein the active protein is an allergen.

24. Immobilized active protein according to claim 7 wherein the active protein is a part of a virus.

25. Immobilized active protein according to claim 12 wherein the inactive protein is that of a microbe.

26. Immobilized active protein according to claim 7 wherein the active protein and the inactive protein are derived from a single impure source.

27. Immobilized active protein according to claim 26 wherein the single impure source is a microbe source.

29. A sheet or film of immobilized active protein according to claim 7 in combination with an inert non-proteinic carrier which imparts form and strength thereto.

30. A sheet or film of immobilized active protein according to claim 29 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.

31. Immobilized active protein according to claim 7 in the form of an open-cell foam or sponge.

32. Immobilized active protein according to claim 7 wherein the active protein is hydrolytic enzyme.

33. Immobilized active protein according to claim 7 wherein the active protein is proteolytic hydrolase.

34. Immobilized active protein according to claim 8 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.