Pneumococcal polysaccharide conjugate vaccine

A pneumococcal polysaccharide-immunogenic protein conjugate produced by the process of:

• (a) Culturing a pneumococcus and isolating crude pneumococcal polysaccharide or solubilizing pneumococcal polysaccharide powder;
• (b) Purifying and partially-hydrolyzing the polysaccharide of step (a) to an endpoint predetermined to generate a polysaccharide amenable to conjugation having no more than a 30% reduction of the polysaccharide's type-specific antigenicity as compared with the crude polysaccharide of step (a);
• and
• (c) Conjugating the product of step (b) with OMPC or MIEP;
• wherein the pneumococcus cultured in step (a) is selected from one or more of the subtypes:
• 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F, and further, wherein the Pn-Ps retains its antigenic integrity as measured by Ouchterlony double immunodiffusion or rate nephelometry assay using an anti-Pn-Ps type-specific antibody, said Pn-Ps prior to conjugation being physically sheared in a Gaulin press at a pressure between about 2000 and 15000 PSI or hydrolyzed by heating at 100.degree. C. for 24 hours or by sonicating, to a viscosity for a 1 mg/ml solution in 0.9M sodium chloride or K.sub.d (peak) endpoint as follows for each listed Pn-Ps subtype:
• ______________________________________ Target Endpoint Pn-Ps Viscosity Target Endpoint Subtype (centistokes) K.sub.d (peak) ______________________________________ Pn4-Ps 1.5-1.00 0.65 .+-. 0.05 Pn6B-Ps 1.3-1.00 0.60 .+-. 0.05 Pn6B-Ps 1.3-1.00 0.60 .+-. 0.05 Pn9V-Ps 1.3-1.00 0.65 .+-. 0.05 Pn14-Ps 1.1-0.95 0.60 .+-. 0.05 Pn18C-Ps 1.5-1.00 0.65 .+-. 0.05 Pn19F-Ps 1.3-1.00 0.65 .+-. 0.05 Pn23F-Ps 1.5-1.00 0.54 .+-. 0.05;
• ______________________________________
• optionally followed by chromatographic or alcohol fractionation to select material having a polydispersity below 1.4.

A process for making a Pn-Ps-PRO conjugate which comprises:

• a) Isolating crude pneumococcal polysaccharide, Pn-Ps;
• b) i-Optionally purifying the crude Pn-Ps by ion exchange adsorption of impurities;
• ii-Partially-hydrolyzing or mechanically-shearing the crude Pn-Ps;
• Fractionating the partially-hydrolyzed Pn-Ps according to size and purity;
• d) Derivatizing the fractionated Pn-Ps, derived from one or more pneumococcal subtypes according to steps (a)-(c), to display pendant nucleophilic or electrophilic moieties;
• e) Isolating Neisseria meningitidis b OMPC, or MIEP;
• f) Functionalizing the OMPC or MIEP to exhibit reactive electrophilic or nucleophilic moieties;
• g) Conjugating the polysaccharide of step (d) with the protein of step (f);
• h) Capping the conjugate to remove residual functional groups;
• and
• i) Isolating the conjugate product, wherein steps (b) and (c) further comprise:
• (b) 1-Optionally, adsorbing onto Whatman DE52 anionic impurities at a solution pH of about 5;
• 2-Partially hydrolyzing the Pn-Ps in solution to an endpoint viscosity predetermined to diminish the Pn-Ps binding to anti-pneumococcal type specific antibody by no more than 30% as compared with crude Pn-Ps by:
• 1. heating at 50.degree. to 150.degree. C. for between 1 to 48 hours;
• or
• 2. sonicating for periods of 5 seconds to 5 minutes, depending on the power setting of the sonication probe, followed by periods of cooling and additional sonication;
• or
• 3. shearing in a Gaulin press at pressures between about 2000 and 15000 PSI;
• and
• c) Fractionating the partially-hydrolyzed Pn-Ps according to size and purity wherein step (c) comprises:
• Fractionating the hydrolyzed Pn-Ps and selecting a fraction having a molecular weight in the range between 1.times.10.sup.5 and 1.times.10.sup.6 by:
• i-differential alcohol solubility using isopropanol at concentrations predetermined to precipitate the desired Pn-Ps size range, or
• ii-fractionation on a size-exclusion liquid chromatography column capable of including and fractionating polysaccharides in the size range between 5.times.10.sup.4 and 1.times.10.sup.6 wherein the
• endpoint for hydrolysis or shear is determined by viscometry of a 1 mg/ml solution in 0.1M sodium phosphate, pH 7.2, or chromatography for each of the listed polysaccharides according to the end-point for that subtype Pn-Ps:
• ______________________________________ Target Endpoint Viscosity Target Endpoint Pn-Ps Subtype (centistokes) K.sub.d (peak) ______________________________________ Pn4-Ps 1.5-1.00 0.65 .+-. 0.05 Pn6B-Ps 1.3-1.00 0.60 .+-. 0.05 Pn9V-Ps 1.3-1.00 0.65 .+-. 0.05 Pn14-Ps 1.1-0.95 0.60 .+-. 0.05 Pn18C-Ps 1.5-1.00 0.65 .+-. 0.05 Pn19F-Ps 1.3-1.00 0.65 .+-. 0.05 Pn23F-Ps 1.5-1.00 0.54 .+-. 0.05. ______________________________________