Haemophilus influenzae B polysaccharide exotoxoid conjugate vaccine

Claim 2

A water-soluble, heat resistant, covalent polysaccharide-diphtheria or tetanus toxoid conjugate capable of producing T-cell dependent antibody response to polysaccharide from Haemophilus influenzae b, of molecular size above 140,000 and below 4,500,000 dalton as referenced to dextran standard by gel permeation chromatography, and of a ribose/protein ratio between 0.25 and 1.00, prepared by mixing a 4-8 carbon dicarboxylic acid and hydrazide derivatized diphtheria or tetanus toxoid in a substantially cyanogen halide-free solution with a cyanogen halide activated capsular Haemophilus influenzae b polysaccharide consisting of approximately equimolar amounts of ribose, ribitol and phosphate, which had previously been sized by heating until more than 60% of the polysaccharide was adjusted to a molecular size between 200,000 and 2,000,000 dalton as referenced to dextran standard by gel permeation chromatography.

Claim 3

A conjugate of claim 1, wherein said dicarboxylic acid is adipic acid.

Claim 4

A conjugate of claim 1, wherein said toxoid is diphtheria toxoid.

Claim 5

A conjugate of claim 1, wherein said toxoid is tetanus toxoid.

Claim 6

A conjugate of claim 1, wherein said halide is the bromide.

Claim 7

A process for producing a water-soluble covalent polysaccharide-diphtheria or tetanus toxoid conjugate capable of producing T-cell dependent antibody response to polysaccharide from Haemophilus influenzae b of molecular size between 140,000 and 4,500,000 dalton as referenced to dextran standard by gel permeation chromatography, and of a ribose/protein ratio between 0.25 and 1.00, which comprises: (a) heating a capsular polysaccharide of Haemophilus influenzae b consisting of approximately equimolar amounts parts of ribose, ribitol and phosphate until less than 20% is of a molecular size below 200,000 dalton and less than 20% is of a size above 2,000,000 dalton as referenced to dextran standard by gel permeation chromatography; (b) activation of the resulting sized polysaccharide with cyanogen bromide; .(c) removal of substantially all the unreacted cyanogen bromide; and (d) reaction of the activated product with a 4-8 carbon dicarboxylic acid hydrazide derivatized exotoxoid.

Claim 8

A process for producing a water-soluble covalent polysaccharide-diphtheria or tetanus toxoid conjugate capable of producing T-cell dependent antibody response to polysaccharide from Haemophilus influenzae b of molecular size between 140,000 and 4,500,000 dalton as referenced to dextran standard by gel permeation chromatography, and of a ribose/protein ratio between 0.25 and 1.00, which comprises: (a) heating a capsular polysaccharide of Haemophilus influenzae b consisting of approximately equimolar amounts parts of ribose, ribitol and phosphate until less than 20% is of a molecular size below 200,000 dalton and less than 20% is of a size above 2,000,000 dalton as referenced to dextran standard by gel permeation chromatography; (b) activation of the resulting sized polysaccharide with cyanogen bromide; .(c) removal of substantially all the unreacted cyanogen bromide; and (d) reaction of the activated product with a 4-8 carbon dicarboxylic acid hydrazide derivatized exotoxoid.

Claim 9

A process for claim 6, wherein said dicarboxylic acid is adipic acid.

Claim 10

A process of claim 6, wherein said toxoid is diphtheria toxoid.

Claim 11

A process of claim 6, wherein said toxoid is tetanus toxoid.

Claim 12

A hapten substantially free from protein and nucleic acid prepared from capsular Haemophilus influenzae b polysaccharide, consisting of approximately equimolar amounts of ribose, ribitol and phosphate, by heating until less than 20% is of a molecular size is below 200,000 dalton and less than 20% is of molecular size greater than 2,000,000 dalton as referenced to dextran standard by gel permeation chromatography.

Claim 13

A hapten substantially free from protein and nucleic acid prepared from capsular Haemophilus influenzae b polysaccharide, consisting of approximately equimolar amounts of ribose, ribitol and phosphate, by heating until less than 20% is of a molecular size is below 200,000 dalton and less than 20% is of molecular size greater than 2,000,000 dalton as referenced to dextran standard by gel permeation chromatography.

Claim 14

A hapten of claim 10 wherein the polysaccharide is present as the sodium salt.