Patent DE-102011122891-A1 (DE)
Title: Fermentation medium, used to cultivate strain of Corynebacterium diphtheriae and to prepare diphtheria toxin or its derivative that is used to prepare vaccine for humans, comprises water, nitrogen source, carbon source, and iron additive
Inventor: ISENBERG MANFRED
IPC: C07K14/195
Filed: 2011-11-11
Abstract: Fermentation medium comprises (i) water; (ii) a nitrogen source; (iii) a carbon source; and (iv) an iron additive, where a culture of Corynebacterium diphtheriae yields at least 140 Lf/ml of the diphtheria toxin or its derivative in at least 100 l of the fermentation medium. Independent claims are included for: (1) method-I for preparing the fermentation medium, comprising adding the nitrogen source, the carbon source and the iron additive to water; (2) method-II for preparing the diphtheria toxin or its derivative for the preparation of a vaccine for humans, comprising (i) producing a culture of a strain of Corynebacterium diphtheriae, which expresses the diphtheria toxin or the derivative of the diphtheria toxin 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 the soluble Fe 2> +>/Fe 3> +>, phosphorus and growth factors, (ii) culturing the culture under aerobic conditions to a concentration of at least 140 Lf/ml of the diphtheria toxin or the derivative in the fermentation medium, and (iii) separating the diphtheria toxin or derivative of diphtheria toxin from the fermentation medium, where the separation step comprises a centrifugation step and a filtration step; (3) method-III for preparing a combination vaccine for humans, comprising the steps of (i), (ii) and (iii) as given in the specification, adding formaldehyde to the diphtheria toxin or its derivative obtained in the step (iii) to obtain a diphtheria toxoid, and mixing the diphtheria toxoid with at least one additional protective antigen from a pathogen, where the pathogen does not contain Corynebacterium diphtheriae to obtain the combined vaccine; (4) method-IV for preparing the diphtheria toxoid to prepare the vaccine for humans, comprising (i) culturing the strain of the Corynebacterium diphtheriae, which expresses the diphtheria toxin or its derivative in at least 100 l of the fermentation medium for the cultivation of Corynebacterium diphtheriae culture, where at least 140 Lf/ml of the diphtheria toxin or its derivative is obtained, (ii) separating the diphtheria toxin or its derivative from the fermentation medium to obtain a diphtheria toxin solution, (iii) preparing the diphtheria toxin concentrate from the diphtheria toxin solution, where the concentration of the diphtheria toxin or its derivative is at least 20 times higher in the concentrate than the concentration of the diphtheria toxin or its derivative in the fermentation medium, and (iv) detoxifying the diphtheria-toxin concentrate for the preparation of the diphtheria-toxoid; (5) the diphtheria-toxoid, in the vaccination of humans, where the diphtheria-toxoid is crosslinked by formaldehyde with at least one component of the fermentation medium; (6) a composition for the vaccination of humans, comprising the diphtheria-toxoid, which is purified from the Corynebacterium diphtheriae, is cultivated in the fermentation medium and has a potency of at least 60 IU/ml; (7) method-V for preparing a human vaccine, comprising the mixing the composition with at least one other antigen-containing composition; and (8) the diphtheria toxoid.