Method and kit for making a polysaccharide-protein conjugate

• US 5,554,730 A
• Filed: 12/23/1994
• Issued: 09/10/1996
• Est. Priority Date: 03/09/1993
• Status: Expired due to Term

• Current Assignee: Baxter Healthcare SA (Baxter International Inc.), Baxter International Inc.
• Original Assignee: Epic Therapeutics, Inc. (Baxter International Inc.)
• Inventors: Di, Jie, Woiszwillo, James E.
• Primary Examiner(s): Elliott, George C.
• Assistant Examiner(s): LEITH, NANCY J
• Application Number:
  US08/372,820
• Time in Patent Office: 627 Days
• Field of Search: 530/410, 530/350, 424/256.1, 424/245.1, 424/250.1, 424/236.1, 424/234.1, 424/241.1, 424/257.1, 424/244.1, 424/259.1, 424/261.1, 424/260.1
• US Class Current: 530/410
• CPC Class Codes: A61K 47/6921 the form being a particulate, a powder, an adsorbate, a bead or a sphere
• CPC Class Codes: A61K 9/1658 Proteins, e.g. albumin, gelatin
• CPC Class Codes: A61K 9/1694 resulting in granules or microspheres of the matrix type containing more than 5% of excipient
• CPC Class Codes: C07K 1/30 by precipitation
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/54326 Magnetic particles

Abstract

A method and kit for making a polysaccharide-protein Schiff base conjugate. A polysaccharide is oxidized with an oxidizing agent and combined with a protein in the presence of a macromolecular crowding agent to form a Schiff base. Preferably, the macromolecular crowding agent is a soluble linear polymer selected from the group consisting of polyvinylpyrrolidone, polyethylene glycol, dextran, nonylphenol-ethoxylates, polyvinyl alcohol, and mixtures thereof. Most preferably, the macromolecular crowding agent is a mixture of polyvinylpyrrolidone and polyethylene glycol. The microparticles or substantially dissolved microparticles are immunogenic and are useful for inducing an immune response when administered to humans or animals.

Claims

1. A method for making a polysaccharide-protein conjugate comprising:

a) oxidizing a polysaccharide with an oxidizing agent to form an oxidized polysaccharide, and

b) combining the oxidized polysaccharide with a protein in the presence of a macromolecular crowding agent

wherein the polysaccharide and protein form a Schiff base conjugate in the absence of a reducing agent.

2. The method of claim 1 wherein the macromolecular crowding agent is a soluble linear polymer.

3. The method of claim 1 wherein the soluble linear polymer is a compound selected from the group consisting of polyvinylpyrrolidone, polyethylene glycol, dextran, nonylphenol-ethoxylates, polyvinyl alcohol, and mixtures thereof.

4. The method of claim 1 wherein the macromolecular crowding agent is a mixture of polyvinylpyrrolidone and polyethylene glycol.

5. The method of claim 4 wherein the molecular weight of the polyvinylpyrrolidone is approximately 40,000.

6. The method of claim 4 wherein the molecular weight of the polyethylene glycol is 3500.

7. The method of claim 4 wherein the polysaccharide and protein form a Schiff base microparticle.

8. The method of claim 7 further comprising dissolving the microparticle.

9. The method of claim 8 wherein the microparticle is dissolved with a base.

10. The method of claim 1 wherein the oxidizing agent is selected from the group consisting of lead tetra-acetate, periodic acid, and sodium periodate.

11. The method of claim 1 further comprising quenching the oxidizing agent with a quenching reagent.

12. The method of claim 1 wherein the quenching reagent is an alcohol.

13. The method of claim 1 wherein the polysaccharide is derived from a bacteria selected from the group consisting of Haemophilus influenza, pneumococci, meningococci, β

-hemolytic streptococci, Escherichia coli, Pseudomonas aeruginosa, Klebsiella, and Vibrio cholerae.

14. The method of claim 1 wherein the protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid, Neisseria meningitidis outer membrane protein, a nontoxic cross-reacting mutant of diphtheria toxin, a protein isolated from bacteria, and a synthetic protein containing lysine residues.

15. The method of claim 1 wherein the polysaccharide and protein form a Schiff base conjugate microparticle that yields solubilized conjugate when the microparticle is dissolved.

25. The conjugate of claim 22 of claim 1 prepared by a method wherein the soluble linear polymer is a compound selected from the group consisting of polyvinylpyrrolidone, polyethylene glycol, dextran, nonylphenol-ethoxylates, polyvinyl alcohol, and mixtures thereof.

26. The conjugate of claim 22 of claim 1 prepared by a method wherein the macromolecular crowding agent is a mixture of polyvinylpyrrolidone and polyethylene glycol.

28. The conjugate of claim 26 prepared by a method wherein the microparticle is dissolved with a base.

16. A kit for making a polysaccharide-protein conjugate consisting essentially of:

a) an oxidizing agent for oxidizing a polysaccharide,

b) a protein,

c) a soluble linear polymer macromolecular crowding agent, and,

d) a base.

17. The kit of claim 16 wherein the oxidizing agent is selected from the group consisting of lead tetra-acetate, periodic acid, and sodium periodate.

18. The kit of claim 16 further comprising quenching reagent for quenching the oxidizing agent.

19. The kit of claim 16 wherein the protein is a carrier protein selected from the group consisting of tetanus toxoid, diphtheria toxoid, Neisseria meningitidis outer membrane protein, a nontoxic cross-reacting mutant of diphtheria toxin, a protein isolated from bacteria, and a synthetic protein containing lysine residues.

20. The kit of claim 16 wherein the macromolecular crowding agent is a mixture of polyvinylpyrrolidone and polyethylene glycol.

21. The kit of claim 16 further comprising a base for dissolving polysaccharide-protein conjugate microparticles.

22. A polysaccharide-protein conjugate prepared by a method comprising:

a) oxidizing a polysaccharide with an oxidizing agent to form an oxidized polysaccharide and

b) combining the oxidized polysaccharide with a protein in the presence of a macromolecular crowding agent,

wherein the polysaccharide and protein form a Schiff base conjugate in the absence of a reducing agent.

23. The conjugate of claim 22 prepared by a method wherein the polysaccharide and protein form a Schiff base conjugate microparticle that yields solubilized conjugate when the microparticle is dissolved.

24. The conjugate of claim 22 prepared by a method wherein the macromolecular crowding agent is a soluble linear polymer.

27. The conjugate of claim 23 wherein the method further comprises dissolving the microparticle.

29. A method for making a polysaccharide-protein conjugate microparticle comprising:

a) oxidizing a polysaccharide with an oxidizing agent to form an oxidized polysaccharide, and

b) combining the oxidized polysaccharide with a protein in the presence of a macromolecular crowding agent

wherein the polysaccharide and protein form a Schiff base conjugate microparticle.

30. A polysaccharide-protein conjugate microparticle prepared by a method comprising:

a) oxidizing a polysaccharide with an oxidizing agent, and

b) combining the oxidized polysaccharide with a protein in the presence of a macromolecular crowding agent,

wherein the polysaccharide and protein form a Schiff base conjugate.