| 主权项 |
1. A process for preparing an attenuated tetravalent dengue vaccine comprising:
(i) amplifying Vero cells in culture to produce Master and Working banks of Vero cells, wherein the Vero cells are adapted for growth in serum-free medium, are grown in serum-free medium, and are sub-cultured with trypsin of non-animal origin of this cell in 225 cm2 Tissue Culture (TC)-flasks and later in a Cell Factory System™ (CFS); (ii) infecting Vero cells from the Master or Working bank with dengue virus serotypes 1, 2, 3 and 4 from a Seed or Working bank of each virus, wherein the Vero cells are independently infected with dengue virus serotypes 1, 2, 3, and 4 in separate cultures with serum free medium; (iii) incubating the 225 cm2 TC-flasks or Cell Factory System™ (CFS) containing the Vero cells infected with each dengue virus at 36.5° C. (±1° C.) for 10 to 20 days; (iv) harvesting the supernatants of each culture; (v) filtering each dengue virus suspension from step (iv) through a membrane with 0.2 μm of porosity and storing the filtered dengue virus at −80° C. (±5° C.); (vi) preparing dengue virus bulks of the serotypes 1, 2, 3 and 4; (vii) formulating monovalent vaccines; (viii) formulating tetravalent vaccine by mixing the monovalent vaccines; (ix) filling vials with the tetravalent vaccine; (x) lyophilizing the tetravalent vaccine in the vials; (xi) sealing the lyophilized tetravalent vaccine in the vials; and (xii) storing the lyophilized and sealed product at 2-8° C., thereby preparing an attenuated tetravalent dengue vaccine. |
