Principles to be followed in vaccine preclinical assay (Part Tw

    1. Vaccine production process research

    (1) Establishing a bacterial strain bank

    Establish a three-level seed bank. The genetic stability of the seed bank was analyzed to determine the number of times the seed bank could be passaged. Production of bacterial strains, cells and process technologies involved in production should pay attention to patents, and relevant patent inquiries should be conducted.

    (2) Cell and/or culture medium for production

    1. The cells for production should meet the requirements of the “Preparation and Verification Protocol for Animal Cell Substrates for the Production of Biological Products” in the current edition of the Chinese Pharmacopoeia. For example, a three-stage cell bank should be established by using the passage cell line;
    2. The production medium is as far as possible avoiding the use of animal sources and raw materials that may cause adverse reactions in humans, and the use of bovine source materials from mad cow disease areas is prohibited.

    (3) Study on the production process of vaccine stock solution

    1. Determination of the main technical parameters of the production process

    (1) Inoculation ratio of virus to cells, optimal parameters of MOI, optimal temperature for cell culture and virus culture, culture time, and harvest time. The inoculation amount, culture and fermentation conditions of the strains are determined by research; (2) selection and basis of inactivating agents; (3) verification of inactivation or lysis conditions and inactivation or detoxification effects, inactivation effects The basis and method of verification; the verification of the inactivation effect should be carried out using cells or media and methods that are as sensitive as possible; (4) Process studies for concentration of the stock solution and/or extraction and purification of active antigens: purification and extraction processes The conditions are optimized, whether the purification and extraction processes have an effect on the active components of the antigen, and a stable purification process, including the recovery rate, antigen activity and purity stability during purification, should be established; and corresponding quality control indicators and detection methods are formulated. Production in accordance with GMP requirements in a production environment that meets GMP requirements; (5) Initially determine theoretical data on the ratio of output of starting materials, stock solutions, semi-finished products and finished products.

    1. Vaccine requirements for adjuvants

    The adjuvants currently available for vaccines are mostly aluminum hydroxide, while aluminum adjuvants typically lag the antibodies produced by the vaccine and increase the chance of side-injection of the injection. If the antigen of the vaccine can meet the needs of immunization, it is advisable not to add an adjuvant. If an adjuvant is used in the final product, the following questions should be investigated. For adjuvants that have been clearly adjuvanted or have been commercialized, only the components or chemical composition of the formulation should be provided, and the class should be used at home and abroad. In the case of preparations, no further toxicological and safety studies are required. If such adjuvants have not been used at home and abroad, detailed study of the principle of action, safety and adjuvant effects must be carried out and practical evaluation methods should be established.

    (4) Formulation study of vaccine

    The formulation of the vaccine is studied, such as whether the stabilizer component, the buffer, the adjuvant, and the excipient component of the lyophilized vaccine added to the vaccine affect the vaccine.

    1. Pharmacology, toxicology and bio-distribution

    The vaccine is different from the general chemical. The pharmacological and toxicological requirements of the experiment are specific. Therefore, the pharmacological test of the product mainly includes the principle of the action, the relationship between the biological titer and the dose, the immunization procedure, and the route and effect of the inoculation. Relationships, etc.; toxicological aspects mainly consider the site of the inoculation and systemic pathological reactions, as well as the body's undesired immune response to the vaccine and the duration of such response. Since all of the above aspects are interrelated, factors such as pharmacology, toxicology, and immunogenicity or bio-potency should be considered.

    Establish appropriate tests and test methods to evaluate the immunogenicity or bio-potency of the vaccine. If there is an animal model or an animal model can be established, the animal model can be used to directly evaluate the bioavailability of the vaccine, such as: for some animal models. Infectious diseases can be evaluated by attack experiments of pathogens. Moreover, the relationship between dose and bio-potency should be established, and the immunization program and the inoculation route should be optimized through experiments. If an animal model cannot be established, an in vitro experiment to verify the effectiveness of the vaccine should be established for evaluation.

    The biological distribution of inactivated vaccines is more difficult to detect and evaluate. The bio-distribution of live attenuated vaccines should establish sensitive animal models for determining viremia (or bacteremia) and duration, vaccination (bacteria) methods and pathways, and for organ reproduction and infection in vivo. Tissue cells should be studied in detail.

    To be continued in Part Three.