Process for the preparation and use of a bivalent vaccine against morphine-heroine addiction

A process for preparing a bivalent immunogenic composition against morphine heroin involving a reaction between a carrier protein (“CP”) and a morphinic product, wherein the carrier protein is tetanus toxoid and the morphinic product is selected from the group consisting of EDC-(morphine-6-hemisuccinate) and EDC-3-O-carboxymethylmorphine, wherein the morphinic product is linked to an intermediate conjugate (“CP-TFCS”) in the composition, and wherein the composition is prepared according to the following steps:

• a) reacting 100 mg (0.5 mM) of tetanus toxoid dissolved in 4 ml of a phosphate buffer solution/0.15 mM NaC1, pH 7.2 with 50 μl of a N (ε-trifluoracetylcaproyloxy)-succinimide-ester (“TFCS”) stock solution containing 10-20% DMSO and 90-80% deionized H2O, to begin formation of the intermediate conjugate;
• b) obtaining a final tetanus toxoid concentration of 6.7 mM and 0.5-1% of DMSO;
• c) obtaining a final dilution of 1:10-20 (v:v) of the initial DMSO concentration between tetanus toxoid and TFCS;
• d) incubating the contents of step c) for up to 60-90 minutes at room temperature, wherein synthesis of the CP-TFCS occurs, and wherein TFCS still preserves a reactive amino protected by a protecting trifluoroacetyl chemical group;
• e) incubating the CP-TFCS for 2-3 hours at room temperature and adjusting the pH to 8-8.5, with a concentrated 10N NaOH solution to enhance the protected trifluoroacetyl group in TFCS to be hydrolyzed, so as to generate free reactive amine groups in the CP-TFCS;
• (f) reacting the morphinic product, EDC-(morphine-6-hemisuccinate), with the CP-TFCS, wherein the reaction is carried out in a stoichiometric ratio by reacting mol:mol, 100 μmol of EDC-(M-6-H) per each 0.07 μmol of active free amino groups of the CP-TFCS;
• and
• (g) incubating the reactants obtained in step f) 2-3hours at room temperature under slow and constant stirring to form the immunogenic composition.