# US 8,465,749 B2 · Polysaccharide-protein conjugate vaccines

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 8,465,749 B2
- Document ID: US8465749B2
- Status: Granted
- Kind: B2
- Application number: 13/243,480
- Filed: 2011-09-23
- Published: 2012-03-29
- Granted: 2013-06-18
- Earliest claimed: 2003-08-06
- Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services
- Applicants: Che-Hung Robert Lee; Carl E. Frasch
- Inventors: Che-Hung Robert Lee; Carl E. Frasch
- Web page: https://patentgrep.com/patent/US8465749B2

## CPC

- A61K 39/08: Clostridium, e.g. Clostridium tetani
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 39/0275: {Salmonella}
- A61K 39/092: {Streptococcus}
- A61K 39/095: Neisseria
- A61K 39/102: {Pasteurellales, e.g. Actinobacillus}, Pasteurella; Haemophilus
- A61K 47/61: the organic macromolecular compound being a polysaccharide or a derivative thereof
- A61K 47/6415: {Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins}
- A61K 47/646: {the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines}
- A61P 31/04: Antibacterial agents
- A61P 37/04: Immunostimulants
- A61K 2039/6037: {Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]}
- A61K 2039/6087: {Polysaccharides; Lipopolysaccharides [LPS]}
- A61K 2039/627: {characterised by the linker}
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Methods for synthesis and manufacture of polysaccharide-protein conjugate vaccines at high yield are provided. The methods involve reaction of a hydrazide group on one reactant with an aldehyde or cyanate ester group on the other reactant. The reaction proceeds rapidly with a high conjugation efficiency, such that a simplified purification process can be employed to separate the conjugate product from the unconjugated protein and polysaccharide and other small molecule by-products.

## Claims

### Claim 1

A method for preparing a conjugate vaccine, the method comprising:
reacting a polysaccharide with 1-cyano-4-dimethylammoniumpyridinium tetrafluoroborate, whereby a solution of a cyanate-activated polysaccharide is obtained;
reacting a protein with hydrazine or adipic acid dihydrazide in the presence of 1-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride at a pH of from 6 to 7, whereby a solution of a hydrazide-activated protein is obtained;
raising the pH of the solution of the hydrazide-activated protein to from 7.0 to 11;
buffer exchanging the solution of the hydrazide-activated protein to a pH of from 10.0 to 11.0;
reacting the cyanate-activated polysaccharide with the hydrazide-activated protein at a pH of from 6 to 8 to yield a conjugate vaccine capable of stimulating an immune response.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the step of reacting the cyanate-activated polysaccharide with the hydrazide-activated protein is conducted in the absence of a blocking agent.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the polysaccharide is selected from the group consisting of Meningococcal polysaccharides, Pneumococcus polysaccharides, Hemophilus influenzae type b polysaccharide, Vi polysaccharide of Salmonnella typhi, and group B Streptococcus polysaccharides.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the protein is selected from the group consisting of tetanus toxoid, diptheria toxoid, CRM197, and meningococcal protein.

### Claim 5 (depends on 1)

The method of claim 1, wherein reacting the cyanate-activated polysaccharide with the hydrazide-activated protein is conducted at a temperature of from 0° C. to 5° C. for 36 to 48 hours, followed by an additional 18 to 24 hours at a temperature of from 20° C. to 25° C.

### Claim 6 (depends on 1)

The method of claim 1, wherein the solution of the hydrazide-activated protein is buffer-exchanged at a pH of 10.5.

### Claim 7 (depends on 1)

The method of claim 1, wherein the solution of the hydrazide-activated protein is buffer-exchanged at a pH of 11.

### Claim 8 (depends on 1)

The method of claim 1, wherein the solution of the hydrazide-activated protein is buffer-exchanged with a buffer concentration of 3 mM to 10mM.