Process for preparing an attenuated tetravalent dengue vaccine
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Publication Number
US-10004795-B2
Publication Date
2018-06-26
Expiration Date
Abstract
The present invention refers to a process for preparing an attenuated tetravalent dengue vaccine and its product. The present invention also refers to a process for preparing a tetravalent dengue vaccine for administration to a subject, to a method for inducing an immune response to virus dengue serotype 1, 2, 3 and 4 in a patient and to a tetravalent dengue vaccine kit.
Core Innovation
The invention relates to a process for preparing an attenuated tetravalent dengue vaccine by producing Master and Working banks of Vero cells adapted for growth in serum-free medium and sub-culturing with trypsin of non-animal origin. Vero cells are grown in serum-free medium and later sub-cultured in 225 cm2 Tissue Culture flasks and in a multi-layered cell culture system, and the Master and Working banks serve as the source for dengue virus preparation.
The process infects Vero cells independently with dengue virus serotypes 1, 2, 3 and 4 from a Seed or Working bank of each virus, using separate cultures with serum free medium. The infected cultures are incubated at 36.5°C (±1°C) for 10 to 20 days, the supernatants are harvested, and each dengue virus suspension is filtered through a membrane with 0.2 μm porosity before storage at ≤80°C (±5°C).
Dengue virus bulks are used to formulate monovalent vaccines, and a tetravalent vaccine is formulated by mixing the monovalent vaccines. The tetravalent vaccine is filled into vials, lyophilized, sealed, and stored as a lyophilized and sealed product at 2-8°C.
The described production approach is centered on serum-free Vero cell cultivation with trypsin of non-animal origin and the independent preparation of each dengue serotype prior to monovalent-to-tetravalent mixing. The summary further reports a lyophilized formulation with stability at 2-8°C over time and human phase II testing for preliminary safety and immunogenicity.
Claims Coverage
Two independent claims are provided. Across these claims, the coverage includes serum-free adapted Vero cell culture with non-animal origin trypsin, independent infection of Vero cells with dengue virus serotypes 1-4 in separate cultures, harvesting and filtering of dengue virus, preparation of dengue virus bulks, formulation of monovalent vaccines and mixing into a tetravalent vaccine, vial filling, lyophilization, sealing, and 2-8°C storage.
Serum-free Vero cell banks adapted for non-animal origin trypsin
Amplifying Vero cells in culture to produce Master and Working banks of Vero cells adapted for growth in serum-free medium, grown in serum-free medium, and sub-cultured with trypsin of non-animal origin.
Separate infection of serum-free Vero cultures with dengue serotypes 1-4
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.
Harvesting and filtering dengue virus from independent cultures
Harvesting the supernatants of each culture and filtering each dengue virus suspension through a membrane with 0.2 μm of porosity, followed by storing the filtered dengue virus at ≤80°C (±5°C).
Monovalent formulation and tetravalent mixing
Preparing dengue virus bulks of the serotypes 1, 2, 3 and 4, formulating monovalent vaccines, and formulating tetravalent vaccine by mixing the monovalent vaccines.
Vial filling, lyophilization, sealing, and 2-8°C storage
Filling vials with the tetravalent vaccine, lyophilizing the tetravalent vaccine in the vials, sealing the lyophilized tetravalent vaccine in the vials, and storing the lyophilized and sealed product at 2-8°C thereby preparing an attenuated tetravalent dengue vaccine.
The independent claims broadly cover a serum-free Vero cell-based process in which Vero cell Master/Working banks are used to independently infect separate cultures with dengue virus serotypes 1-4, after which supernatants are harvested and filtered (0.2 μm), dengue virus bulks are prepared, monovalent vaccines are formulated and mixed into a tetravalent vaccine, and the product is filled, lyophilized, sealed, and stored at 2-8°C.
Stated Advantages
Provides reduced animal-origin contaminants.
Improves yield/reproducibility.
Documented Applications
A tetravalent dengue vaccine for administration, including a tetravalent dengue vaccine kit and reconstitution with a defined sodium phosphate composition for administration to induce an immune response.
Human phase II testing reporting preliminary safety and immunogenicity.
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