Fermentation medium, useful e.g. to cultivate strain of Corynebacterium diphtheriae to prepare diphtheria toxin, comprises water, nitrogen source, carbon source and iron additive, where medium is free of ingredients from animal origin

摘要

Fermentation medium comprises water, a nitrogen source, a carbon source and an iron additive, where a culture of Corynebacterium diphtheriaein at least 100 l of the fermentation medium yields at least 140 Lf/ml of diphtheria toxin or its derivative and the medium is free of ingredients from animal origin. Independent claims are included for: (1) preparing the fermentation medium comprising adding the nitrogen source, carbon source and iron additive into water; (2) preparing diphtheria toxin or its derivative to prepare a vaccine for humans comprising (a) preparing a culture of strain of Corynebacterium diphtheriae, which prepares the diphtheria toxin or its derivative, in at least 100 l of the fermentation medium containing the nitrogen source, at least 0.08 M of the carbon source, 1.5-30 mu M of soluble iron 2 +/iron 3 +, phosphorus and growth factors, (b) culturing the strain under aerobic conditions to form a concentration of at least 140 Lf/ml of the diphtheria toxin or its derivative and (c) separating the diphtheria toxin from the fermentation medium, where the separation step comprises a centrifugation step and a filtration step; (3) preparing a combination vaccine comprising carrying out the steps (a)-(c) as above per se, (d) adding formaldehyde to the diphtheria toxin or its derivative to obtain diphtheria toxoid and (e) mixing the diphtheria toxoid with at least one supplementary, protective antigen of a pathogen, which is not Corynebacterium diphtheriae; (4) preparing a diphtheria toxoid comprising either (a1) culturing the strain of Corynebacterium diphtheriae, expressing diphtheria toxin or its derivative, in at least 100 l of the fermentation medium, (b1) separating the diphtheria toxin or its derivative from the fermentation medium to obtain a diphtheria toxin solution, (c1) preparing a diphtheria toxin concentrate from the diphtheria toxin solution, where the concentration of the diphtheria toxin or its derivative in the concentrate is at least 20 times higher than the concentration of the diphtheria toxin or its derivative in the fermentation medium at the end of the step (a1) and (d1) detoxifying the diphtheria toxin concentrate, or (a2) culturing the strain of Corynebacterium diphtheriaein at least 100 l of the fermentation medium, which is free of ingredients from animal origin and optionally contains yeast extract, (b2) purifying the diphtheria toxin or its derivative from the fermentation medium to obtain purified diphtheria toxin or its derivative, where the purified diphtheria toxin has a purity of at least 85% and a purity of = 1500 Lf/mg of nitrogen, (c2) adding the formaldehyde to the purified diphtheria toxin or its derivative and (d2) incubating the purified diphtheria toxin or its derivative, or (a3) preparing a solution of the diphtheria toxin or its derivative in a concentration of at least 2000 Lf/ml, (b3) adding a solution containing an amide in a final concentration of = 0.025 M and formaldehyde in a final concentration of 0.75-1% and (c3) incubating the solution; (5) the diphtheria toxoid obtained by the above method, where the diphtheria toxoid is crosslinked by formaldehyde with at least one ingredient of the fermentation medium; (6) a composition (TT) comprising the diphtheria toxoid with a power of at least 60 international unit/ml; (7) a composition comprising a formaldehyde-crosslinked diphtheria toxoid with an isoelectric point of 4-5, where at least 70% of the toxoid is in monomer form; (8) a composition comprising the formaldehyde-crosslinked diphtheria toxoid and the protective antigen of a pathogen, which is not Corynebacterium diphtheriae; (9) a composition (AA) comprising 100-250 Lf/ml of the diphtheria toxoid and 40-100 Lf/ml of tetanus toxoid, where the ratio of the diphtheria toxoid to tetanus toxoid is 2:1-3:1; (10) preparing a human vaccine comprising mixing the composition (AA) with at least one further antigen-containing composition; and (11) a composition (BB) comprising the diphtheria toxoid, where (BB) is adsorbed in insoluble aluminum salt adjuvant (e.g. aluminum hydroxide). ACTIVITY : Antibacterial. MECHANISM OF ACTION : Vaccine.