# US 5,554,730 A · Method and kit for making a polysaccharide-protein conjugate

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- Number: US 5,554,730 A
- Document ID: US5554730A
- Status: Granted
- Kind: A
- Application number: 08/372,820
- Filed: 1994-12-23
- Granted: 1996-09-10
- Earliest claimed: 1993-03-09
- Assignees: Middlesex Sciences, Inc.
- Inventors: James E. Woiszwillo; Jie Di
- Web page: https://patentgrep.com/patent/US5554730A

## CPC

- A61K 9/1658: {Proteins, e.g. albumin, gelatin}
- A61K 9/1694: {resulting in granules or microspheres of the matrix type containing more than 5% of excipient}
- A61K 47/6921: {the form being a particulate, a powder, an adsorbate, a bead or a sphere}
- C07K 1/30: by precipitation
- G01N 33/54313: {the carrier being characterised by its particulate form}
- G01N 33/54326: {Magnetic particles}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 4)

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

### Claim 6 (depends on 4)

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

### Claim 7 (depends on 4)

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

### Claim 8 (depends on 7)

The method of claim 7 further comprising dissolving the microparticle.

### Claim 9 (depends on 8)

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

### Claim 10 (depends on 1)

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

### Claim 11 (depends on 1)

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

### Claim 12 (depends on 1)

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

### Claim 13 (depends on 1)

The method of claim 1 wherein the polysaccharide is derived from a bacteria selected from the group consisting of Haemophilus influenza, pneumococci, meningococci, .beta.-hemolytic streptococci, Escherichia coli, Pseudomonas aeruginosa, Klebsiella, and Vibrio cholerae.

### Claim 14 (depends on 1)

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.

### Claim 15 (depends on 1)

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.

### Claim 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.

### Claim 17 (depends on 16)

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

### Claim 18 (depends on 16)

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

### Claim 19 (depends on 16)

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.

### Claim 20 (depends on 16)

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

### Claim 21 (depends on 16)

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

### Claim 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.

### Claim 23 (depends on 22)

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.

### Claim 24 (depends on 22)

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

### Claim 25 (depends on 1, 22)

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.

### Claim 26 (depends on 1, 22)

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.

### Claim 27 (depends on 23)

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

### Claim 28 (depends on 26)

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

### Claim 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.

### Claim 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.