RU2542393C2 - METHOD OF PREPARING CONJUGATE OF CAPSULAR POLYSACCHARIDE Haemophilus influenzae TYPE b (Hib) C ANTIGENS FOR PREPARING IMMUNOBIOLOGICAL PREPARATIONS - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method comprises preparing a mixture of tetanus and diphtherial anatoxins adsorbed on the gel of hydroxide or aluminium phosphate which is used as an antigen. The said mixture is centrifuged at 3000 rpm for 5 min, the resulting precipitate is bonded with the carbodiimide method through adipic acid dihydrazide with 3 mg of capsular polysaccharide Hib dissolved in 1 ml of distilled water. The conjugate is washed with 0.9% sodium chloride solution by centrifugation 3000 rpm for 5 min and adjusted to 0.9% with sodium chloride solution to the volume of 100 ml.

EFFECT: method enables to eliminate lyophilisation of obtained conjugates and thereby to reduce the antigen load on the immunised organism.

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Description

The invention relates to biotechnology and relates to a method for binding capsular polysaccharide Haemophilus influenzae type b (Hib) with antigens, which are used as tetanus and diphtheria toxoids.

There is a method of binding the Hib capsular polysaccharide, in which the Hib bromine-modified capsular polysaccharide Hib and the diphtheria toxoid derivative are washed ten times from the chemical reaction products by ultrafiltration, concentrated, mixed in equal volumes and incubated for 18 hours at 4 ° C until the conjugate is obtained (US 44965) .

There is a conjugation method in which the Hib capsular polysaccharide is covalently coupled to a tetanus toxoid via adipic acid dihydrazide and purified by ultrafiltration of unbound components. The resulting conjugate is adsorbed on an aluminum phosphate gel and lyophilized. Before use, the drug is mixed with water for injection or with another liquid multicomponent vaccine (DTP) adsorbed on an aluminum phosphate gel (US20020182226).

There is also a binding method in which the capsular polysaccharides Hib and meningococcus are conjugated to tetanus and diphtheria toxoids, respectively. The resulting preparations were washed and concentrated using ultrafiltration. Each of the conjugates is separately adsorbed on an aluminum phosphate gel, combined and lyophilized in the presence of sucrose. Before administration, the lyophilisate is mixed with the liquid components of the vaccine (hepatitis B surface antigen, whole whooping cough cells) adsorbed on an aluminum phosphate gel (US20030180316).

In these methods, the conjugation of the capsular polysaccharide Hib with protein antigens is carried out in aqueous media. To remove chemicals and unbound components, the membrane ultrafiltration method was used. This technology requires high concentrations of antigens, large volumes of buffer solutions and leads to significant drug losses at various stages of production. To use the obtained conjugate as a component of a multicomponent vaccine, its lyophilization in the presence of a stabilizer (sucrose, lactose, etc.) is necessary, which significantly affects the properties and cost of the final product. In the case of mixing the conjugate with the DTP vaccine, antigenic overload of the immunized organism with diphtheria or tetanus toxoids occurs.

The aim of the present invention was to develop a method of binding capsular polysaccharide Hib with protein antigens, which would eliminate the lyophilization of conjugates and reduce the antigenic load on the immunized organism.

The goal is achieved in that the chemical binding of the capsular polysaccharide Haemophilus influenzae type b is carried out with a mixture of tetanus and diphtheria toxoids adsorbed on an aluminum hydroxide gel or aluminum phosphate gel.

The method is as follows.

Use the capsular polysaccharide Haemophilus influenzae type b obtained by the technology described in patent RU 2185191 (2002). The bacteria Haemophilus influenzae type b is grown on solid nutrient medium (chocolate nutrient agar), the culture is washed off with saline and the inoculum is seeded on a liquid synthetic nutrient medium. The grown biomass is treated with a 0.1% aqueous formalin solution for 1-2 hours, and the cells are separated by ultrafiltration on molecular fiber filters. The Hib capsular polysaccharide in the ultrafiltrate is concentrated on molecular fiber filters and lyophilized.

100 ml of a mixture of tetanus and diphtheria toxoids adsorbed on an aluminum hydroxide or aluminum phosphate gel are centrifuged at 3000 rpm for 5 minutes. The precipitate was chemically coupled by the carbodiimide method via adipic acid dihydrazide with 3 mg of Hib capsular polysaccharide dissolved in 1 ml of distilled water. The resulting conjugate is washed with 0.9% sodium chloride solution by centrifugation at 3000 rpm for 5 minutes and adjusted with a 0.9% sodium chloride solution to a volume of 100 ml.

The study of the immunogenicity of the drug was carried out on four groups of outbred white mice. Animals were vaccinated once intraperitoneally in a volume of 0.5 ml (one human dose).

The first group was inoculated with an experimental adsorbed liquid diphtheria-tetanus-hemophilic conjugate, the second with a commercial ADS vaccine mixed with one human dose of dry conjugated with tetanus toxoid hemophilic vaccine (AKT-Hib), the third was immunized with a commercial ADS vaccine, the fourth group of animals were not immunized used as a control. After a month, the content of antibodies to the Hib capsular polysaccharide, as well as to diphtheria and tetanus toxoids, was determined in the blood serum of mice by enzyme-linked immunosorbent assay, comparing the optical density of the reaction mixture. The test results are presented in table No. 1.

Table 1 The results of ELISA of blood serum of white mice after immunization with vaccines №№ Group name Number of animals Average optical density λ 450 nm / 630 nm Hib tetanus toxoid diphtheria toxoid one. ADS-Hib conjugate (experimental) 10 1.72 1.41 0.62 2. a mixture of ADS and AKT-Hib (prototype, commercial) 10 0.84 1,62 0.66 3. control group immunized with ADS 10 - 1.38 0.84 four. control (non-immunized) 10 0,03 0,03 0.05

As can be seen from the table, the drug obtained by the proposed method has a higher immunogenic activity of the Hib component compared to the commercial ADS vaccine mixed with lyophilized hemophilic-tetanus conjugate and does not cause antigenic overload with tetanus toxoid. The data obtained indicate that the proposed method of binding capsular polysaccharide Hib does not require a high concentration of antigens, a large volume of buffer solutions and does not lead to loss of hemophilic component. Other immunogenic polymers may also be used as antigens. The developed technology does not require lyophilization and does not lead to antigenic overload by the T-dependent component used as a carrier.

Claims (1)

A method for producing a Haemophilus influenzae type b capsular polysaccharide conjugate with antigens by the carbodiimide method via adipic acid dihydrazide, characterized in that a mixture of tetanus and diphtheria toxoids adsorbed on an aluminum hydroxide or phosphate gel is centrifuged at 3000 rpm. within 5 min, the resulting precipitate was coupled by the carbodiimide method via adipic acid dihydrazide with 3 mg of Hib capsular polysaccharide dissolved in 1 ml of distilled water, washing the conjugate 0.9% by the centrifugation 3000 rpm / min with a solution of sodium chloride for 5 minutes and adjusted to 0.9% sodium chloride solution to a volume of 100 ml.