Method for preparing a stable immunogenic product comprising antigenic heterocomplexes of TNFα and a carrier protein

A method for preparing a stable anti-TNFα immunogenic product for human immunization consisting of heterocomplexes of TNFα and keyhole limpet haemocyanin (KLH) and having an ED50 value of more than 400 ng/ml according to an L929 cell cytotoxicity assay, the method comprising the steps of:

• a) providing a liquid solution containing TNFα at a concentration ranging from 0.1 mg/ml to 50 mg/ml;
• b) adding EDTA and DMSO to said liquid solution containing TNFα of step a) to provide a final concentration of EDTA ranging from 1 mM to 500 mM and a final amount of DMSO ranging from 0.5% v/v to 20% v/v;
• c) adding a KLH carrier protein to the liquid solution obtained at the end of step b) to provide a molar ratio of TNFα to KLH ranging from 20:1 to 80:1;
• d) adding glutaraldehyde to the liquid mixture obtained at the end of step c) to obtain a final glutaraldehyde concentration ranging from 0.05% w/w to 0.5% w/w;
• e) removing glutaraldehyde and free molecules of both TNFα and KLH from the solution obtained at the end of step d);
• f) adding formaldehyde to the liquid solution obtained at the end of step e) and maintaining the presence of formaldehyde for a period of time ranging from 96 hours to 192 hours;
• g) adding a reagent comprising at least one amino group that blocks the reaction with formaldehyde to the liquid solution obtained at the end of step f);
• and
• h) removing formaldehyde and the blocking reagent from the liquid solution obtained at the end of step g), so as to obtain a liquid solution containing said anti-TNFα immunogenic product, the product having an ED50 value of more than 400 ng/ml according to an L929 cell cytotoxicity assay.