KR100385711B1 - The quadrivalent combination vaccine including diphtheria toxoid, tetanus toxoid, whole cell pertussis and hepatitis b surface antigen and the preparation thereof - Google Patents

Abstract

The present invention relates to a tetravalent mixed vaccine (hereinafter referred to as "DTwPH mixed vaccine") containing diphtheria, tetanus toxoid and pertussis and hepatitis B surface antigen, and a method for producing the same.

More specifically, diphtheria and tetanus toxoid are adsorbed on aluminum phosphate gel (AlPO₄gel), and hepatitis B surface antigen is adsorbed on aluminum hydroxide gel (Al (OH) ₃gel), and the pH of the final undiluted vaccine of DTwPH is 6.5 to 7.5. The present invention provides a DTwPH mixed vaccine prepared by adjusting the concentration of each component, in particular, aluminum hydroxide gel to 15 to 35 µgAl / mL, and a method of preparing the same.

The DTwPH mixed vaccine of the present invention prevents the aggregation of the final stock solution and adhesion to the vial, and also maintains the immunogenicity of the hepatitis B surface antigen, and the titer of each component can be higher than the biological standard.

In addition, gelatin may be added to the final DTwPH mixed vaccine or stored in a silicone coating bottle to more effectively prevent aggregation and adhesion of the DTwPH mixed vaccine final stock.

Description

QUADRIVALENT COMBINATION VACCINE INCLUDING DIPHTHERIA TOXOID, TETANUS TOXOID, WHOLE CELL PERTUSSIS AND HEPATITIRES AND THE SUPERPARATION THES

The present invention relates to a tetravalent mixed vaccine (hereinafter referred to as "DTwPH mixed vaccine") containing diphtheria, tetanus toxoid and pertussis and hepatitis B surface antigen, and a method for producing the same.

DTP combined vaccine, a combination vaccine to prevent diphtheria, tetanus and pertussis at the same time, is included in the World Health Organization's Expanded Program on Immunization (EPI) program and is used as a basic vaccine in almost all countries around the world. Hepatitis B is a disease that occurs mainly in Southeast Asia, Central Asia, Africa, and South America, including Korea, and is potentially a cause of liver cancer.However, hepatitis B vaccine is currently being used by the World Health Organization. It is part of the EPI program and is engaged in worldwide immunizations.

In recent years, the vaccine injection rate has been reduced by eliminating the inconvenience of having to be immunized twice with different inoculation cycles of the DTP mixed vaccine and hepatitis B vaccine. DTaPH vaccine prepared by mixing DTP vaccine and hepatitis B vaccine has been commercialized and used (European Patent Publication 642355, July 15, 1998; Patent Publication 1992-0009729, Green Cross Co., Ltd.).

Part of active research in the preparation of DTwPH vaccines is the problem of formulation and the maintenance of immunogenicity of hepatitis B surface antigens and the prevention of increased toxicity.

Formulation problems can be divided into two types: coagulation between the final stock solution and adhesion to the storage vial. (Agglutination here refers to the formation of solids that remain unsuspended even if gently shaken by hand several times.Adhesion refers to a state where the DTwPH mixed vaccine particles are firmly adhered to the wall of the storage glass bottle and shaken by hand.) DTwPH mixing Agglutination of the vaccine occurs due to 1) electrostatic attraction between each adsorbent stock and pertussis bacteria, and 2) electrostatic attraction between aluminum hydroxide gel and pertussis bacteria, especially when aluminum hydroxide gel is used as the adsorbent. Is generated by reaction with the glass wall for a long time. Due to this aggregation and adhesion phenomenon, when long-term preservation of the mixed DTwPH vaccine, hard aggregates and deposits are gradually formed over time, seriously inhibiting the stability of the mixed vaccine formulation.

In addition, the immunogenicity of hepatitis B surface antigens was increased due to the increased toxicity due to the use of excess adsorbents and proteins and the low compatibility with other antigens. Another problem is reduced compared to the simultaneous administration of the vaccine and hepatitis B vaccine. (WHO headquarters, Informal consultation on quardrivalent diphtheria-tetanus-pertussis-hepatitis B vaccine, 1992)

The present invention effectively prevents the above-mentioned problems of aggregation and adhesion, and furthermore, the geometric average antibody titer and effective antibody formation rate of hepatitis B surface antigen among DTwPH vaccines are DTP mixed vaccine and monovalent vaccine, which are trivalent mixed vaccines. Provided is a novel DTwPH mixed vaccine capable of maintaining immunogenicity by maintaining the same mode of simultaneous administration of hepatitis vaccine, and a method of preparing the same.

Figure 1 is a photograph magnified 100 times the degree of cohesion of aluminum hydroxide gel and pertussis bacterium according to the aluminum hydroxide gel concentration.

Figure 2 is a Western blot picture confirming the adsorption rate of diphtheria adsorption liquid.

Figure 3 is a photograph magnified 100 times the degree of aggregation of the final undiluted DTwPH vaccine with the use of a silicon glass bottle with an optical microscope.

The mixed vaccine of the present invention is a tetravalent mixed vaccine containing diphtheria, tetanus toxoid and pertussis and hepatitis B surface antigen (hereinafter referred to as "DTwPH mixed vaccine"), and diphtheria and tetanus toxoid are adsorbed onto aluminum phosphate gel (AlPO₄gel). , Hepatitis B surface antigen is characterized in that the adsorbed on aluminum hydroxide gel (Al (OH) ₃ gel).

Another feature of the present invention is the above-described method of preparing the DTwPH mixed vaccine (a) before the final preparation of the DTwPH mixed vaccine, each adsorption stock solution (diphtheria, tetanus toxoid aluminum phosphate gel adsorbent, hepatitis B surface antigen aluminum hydroxide gel adsorbent) Adjust the pH of the pertussis stock solution to 6.5 to 7.5, (b) mix the diphtheria and tetanus adsorbent of step (a), and (c) adsorb hepatitis B surface antigen aluminum hydroxide to the mixture of step (b). Mixing the liquid with the inactivated pertussis stock solution.

Another feature of the present invention includes a method of adding gelatin to a general mixed vaccine or the DTwPH mixed vaccine of the present invention described above or storing it in a silicone coated glass bottle.

In the above-described configuration, the concentration of gelatin is preferably 0.1 to 0.7 w / v%.

Hereinafter, the configuration and operation of the present invention will be described in detail.

Diphtheria stock solution used in the present invention is prepared by culturing Corynebacterium diphtheriae Park Willium strain. The diphtheria toxoid stock solution prepared by treating the culture supernatant with formalin without detoxification and then filtration, precipitation and purification is suitable. In addition to diphtheria stock solutions prepared using different strains, diphtheria toxoid stock solutions not based on this method may be used.

Tetanus stock is prepared by culturing Clostridium tetani Harvard strain. A tetanus toxoid stock solution prepared by treating formalin with detoxification and detoxification, followed by urine, precipitation, and purification is suitable. In addition to tetanus stock solutions prepared using different strains, tetanus toxoid stock solutions not based on this method can be used.

Pertussis stock solution is prepared by culturing Bordetella pertussis . Inactivated pertussis bacteria obtained by treating the pertussis bacteria obtained by centrifugation of this culture in physiological saline and then treating them are suitable. In addition, pertussis bacteria not by this production method can also be used.

Hepatitis B surface antigen was cultured with recombinant yeast transformed with hepatitis B surface antigen gene, followed by cell chromatography to extract only hepatitis B surface antigen, followed by ion chromatography, ultrafiltration, diafiltration, ultracentrifugation and gel. It manufactures using a series of methods, such as filtration chromatography. Moreover, hepatitis B surface antigen derived from plasma can be used as well as hepatitis B surface antigen which is not produced by this production method.

As the adsorbent used in the present invention, aluminum hydroxide gel and aluminum phosphate gel are suitable. These aluminum gels may be purchased commercially, or may be manufactured by themselves through known methods.

The silicon coating bottle used in the present invention is preferably prepared by soaking the glass bottle in 1/40 dilution of 35% polymethylsiloxane (Dow corning 365 silicon emulsion) for a while and then drying it at 310 ° C. for 10 minutes. It is also possible to use silicone coating bottles not commercially available or commercially available.

The DTwPH mixed vaccine of the present invention first adsorbs each stock solution of diphtheria, tetanus toxoid and hepatitis B surface antigen to aluminum gel, and then adjusts each of the adsorption stock solution and the pertussis stock solution so that the monovalent vaccine has the appropriate concentration. It manufactures by making. At this time, diphtheria and tetanus toxoid stock solution are adsorbed on the aluminum phosphate gel, and hepatitis B surface antigen is adsorbed on the aluminum hydroxide gel.

The concentrations of the aforementioned diphtheria, tetanus toxoid, pertussis and hepatitis B surface antigens are 20-50 Lf / mL, 4-20 Lf / mL, 10-24 OU / mL, and 10-30 μg / mL, respectively. The gel concentration is 15 to 35 μg Al / mL, and the total aluminum gel concentration is 550 to 1000 μg Al / mL. (The concentration of the Al gel means the concentration of Al in the aluminum gel.)

1 is a magnified photograph of the aluminum hydroxide gel with pertussis bacterium according to the concentration of aluminum hydroxide gel 100 times magnified by an optical microscope (Axioplan, Carl Zeiss co., Germany). When aluminum hydroxide gel and pertussis bacteria are mixed, they cause aggregation by the electrostatic attraction. Therefore, in order to minimize this aggregation, the concentration determination of aluminum hydroxide gel acts as an important factor. When the final concentration of pertussis bacteria was 24 OU / mL, when the concentration of aluminum hydroxide gel was less than 35 µgAl / mL (Fig. 1, a), aggregation did not occur, but when it was higher, aggregation began to occur and hydroxide When the concentration of the aluminum gel is 200 μg Al / mL or more (Fig. 2, b), the aggregation is very severe.

Therefore, the concentration of aluminum hydroxide gel used for the adsorption of hepatitis B surface antigen should be 15 to 35 μg Al / mL based on the final stock solution.

Diphtheria toxoid adsorption stock solution has a zero charge or a negative charge at neutral pH, but a positive charge at weakly acidic pH, whereas the surface charge of pertussis bacteria is negative at neutral pH, so the pH of the pertussis stock solution and diphtheria toxoid adsorbent are adjusted to 6.5 to 7.5. In case of mixing, aggregation can be prevented by electrostatic repulsive force between each other (pH measuring device: Zeta-Meter System 3.0, NJB PACIFIC. INC, USA)

Therefore, when the pH of each adsorbed stock solution (diphtheria, tetanus toxoid adsorbed solution and hepatitis B surface antigen adsorbed solution) and pertussis stock solution are adjusted to 6.5 to 7.5, the pH of the final stock solution of DTwPH mixed vaccine is about 6.5 to 7.5. To prevent aggregation.

The DTwPH mixed vaccine of the present invention prepared by the above-described method, the titer of each vaccine component not only meets the biological standards (Food and Drug Administration, 1999), but also reduces toxicity and maintains stable immunogenicity of hepatitis B surface antigen. It becomes possible. (Stable means that when stored for 3 months at 4 ° C after preparation of the combined vaccine, the immunogenicity is not lowered and is maintained at the previous or initial level.)

When mixed with a general mixed vaccine or the above-described DTwpH mixed vaccine of the present invention to a high viscosity material such as gelatin to 0.1 to 0.7 w / v% or stored in a silicone-coated glass bottle does not cause aggregation and adhesion to the glass wall It becomes a stable white suspension. (The stable white suspension means that aggregation and adhesion do not occur even when stored for 3 months in a cold room at 2 to 8 ° C.)

Hereinafter, the present invention will be described in detail through Examples and Test Examples of the present invention, but the scope of the present invention is not limited thereto.

Example 1 Preparation of Diphtheria Toxoid Stock Solution

Diphtheria stock solution used in the present invention was prepared by culturing Corynebacterium diphtheriae Park Willium strain. Formalin was added to 0.9% of the culture supernatant and left to detoxify at 36 ° C. for 28 days, followed by ultrafiltration and diafiltration, ammonium sulfate precipitation, gel filtration chromatography, and dialysis to prepare a diphtheria toxoid stock solution. .

Example 2 Preparation of Tetanus Toxoid Stock Solution

Tetanus stock was prepared by culturing Clostridium tetani Harvard strain. Formalin was added to the final culture solution at a final 0.45%, left at 36 ° C for 28 days for detoxification, and then purified by microfiltration, ultrafiltration and diafiltration, ammonium sulfate precipitation, gel filtration chromatography, and dialysis to prepare tetanus toxoid stock solution. Prepared.

Example 3 Preparation of Pertussis Stock Solution

Pertussis stock solution was prepared by culturing Bordetella pertussis. The pertussis bacteria obtained by centrifugation of the culture solution were suspended in physiological saline and then inactivated by treatment at 56 ° C. for 30 minutes to prepare pertussis stock solution.

Example 4 Preparation of Hepatitis B Surface Antigen

Hepatitis B surface antigen was prepared by culturing recombinant Hansenula polymorpha , followed by cell disruption, ion chromatography, ultrafiltration, diafiltration, ultracentrifugation, and gel filtration chromatography.

Example 5 Preparation of Diphtheria, Tetanus Toxoid Adsorbent

Diphtheria and tetanus toxoid stock solution were mixed with aluminum phosphate gel of pH 5.5, stirred and adsorbed, and diluted with isotonic sodium chloride solution if necessary. At this time, the concentration of protein and toxoid in the final adsorbent can be appropriately adjusted, but the concentration of the diphtheria toxoid stock solution is largely adjusted to 280 Lf / mL and the concentration of the tetanus toxoid stock solution to 50 Lf / mL. In addition, the pH of each adsorption stock solution was 5.5 to 6.0, it was confirmed by HPLC and western blot (Fig. 2) that the diphtheria toxoid was adsorbed on the aluminum phosphate gel. (In Figure 2, Lane 1 is the molecular weight marker, Lane 2, 3, 4 is the case where 0.025 Lf, 0.05 Lf, 0.10 Lf developed diphtheria stock solution, respectively, Lane 5 is 0.4 Lf supernatant after centrifugation of diphtheria adsorption liquid) (A substantial amount when diphtheria toxoid was desorbed at 100%). This was a case of development.

Example 6 Preparation of Hepatitis B Surface Antigen Adsorbent

Hepatitis B surface antigen was adsorbed onto aluminum hydroxide gel to enhance immunogenicity. The aluminum hydroxide gel and the hepatitis B surface antigen stock solution were mixed, stirred and adsorbed, and diluted with isotonic sodium chloride solution if necessary. At this time, the concentration of aluminum hydroxide gel was adjusted to 60 µg Al / mL and the concentration of hepatitis B surface antigen stock solution was 72 µg / mL. It was confirmed by HPLC and western blot that the hepatitis B surface antigen was adsorbed on the aluminum hydroxide gel. The prepared adsorption liquid was stored at 2 to 8 ° C. cold room.

Example 7 Preparation of DTwPH Combined Vaccine Final Stock

Each of the adsorbents prepared in Example 5 and Example 6 and the pertussis stock solution (192 OU / mL) prepared in Example 3 were mixed to prepare a final stock solution of the DTwPH mixed vaccine. At this time, in order to prevent aggregation due to electrostatic attraction between each adsorbent and pertussis stock solution, the pH was adjusted to 7.1 and mixed.

First, the Tf teria adsorption solution and the tetanus adsorption solution were mixed, and the final stock solution of DTwPH vaccine was prepared by adding the hepatitis B surface antigen adsorption solution and the inactivated pertussis stock solution. An additional aluminum phosphate gel was added to adjust the concentration of the final aluminum gel to a desired level, and the concentration of each stock solution can be adjusted to a desired level by adding saline or phosphate buffer solution. Chimerosal (0.05 to 0.1 mg / mL) was added as a preservative, and 2-phenoxyethanol (3 to 6 mg / mL) may be added if necessary. At this time, the pH of the final stock solution is 7.1.

The composition of the final undiluted DTwPH vaccine prepared in this Example is shown in the table below.

ingredient Furtherance Diphtheria Toxoid (Lf / mL) 40 Tetanus Toxoid (Lf / mL) 15 Pertussis disinfection (OU / mL) 24 Hepatitis B surface antigen (㎍ / mL) 24 Aluminum Hydroxide Gel (µgAl / mL) 20 Aluminum Phosphate Gel (µgAl / mL) 780

Test Example 1: Determination of immunogenicity against hepatitis B surface antigen of DTwPH mixed vaccine final solution

In order to determine the immunogenicity of hepatitis B surface antigen of the DTwPH mixed vaccine prepared in Example 7, 30 mL of 4 to 5 weeks old ICR mice were subcutaneously injected in each abdomen, and blood was collected 4 weeks later. Abbott's Ausab IMx kit Immunogenicity, ie, geometric mean antibody titer (Ab GMT) and effective antibody titer, was measured using. (The geometric mean antibody titer is the geometric mean value of the antibody titer of each mouse of 1 mIU / mL or more, and the effective antibody titer rate refers to the fraction of mice of 10 mIU / mL or more of all mice.)

In addition, as a control, DTwP mixed vaccine (diphtheria toxoid 40 Lf / mL, tetanus toxoid 15 Lf / mL, pertussis bacteria 24 OU / mL), which is a trivalent vaccine having the same antigenic composition as Example 7, and hepatitis B vaccine, which is a monovalent vaccine (B Hepatitis B surface antigen was measured by subcutaneous injection (simultaneous administration method) of 0.5 mL each of hepatitis B antigen (24 ㎍ / mL), and the immunogenicity of hepatitis B surface antigen was measured by the same method as described above.

As shown in the table below, the stability of hepatitis B surface antigen immunogenicity (geometric mean antibody formation rate, effective antibody formation rate) showed almost similar results to the simultaneous administration.

division DTwPH Mixed Vaccine Concurrent Dosing Method Hepatitis B surface antigen Ab GMT (mIU / mL) 372 354 Hepatitis B surface antigen effective antibody formation rate (%) 90 93

Test Example 2: Activity test of DTwPH mixed vaccine

The titers of diphtheria, tetanus, pertussis and hepatitis B surface antigens among the DTwPH mixed vaccines prepared in Example 7 are the "biological formulation criteria" (adsorption diphtheria, tetanus toxoid and pertussis mixed vaccines) proposed by the Food and Drug Administration. 130, 1999; hepatitis b vaccine, 310 to 313, 1999).

Results of diphtheria, tetanus toxoid, pertussis, and hepatitis B surface antigens of 2-4 units / ml, 4 units / ml or more, 10 IU / mL or more, and the standard product (Green Cross Vaccine Co., Ltd. standard) It has been shown to meet the biological standards.

Example 8 Use of Gelatin

Gelatin, which is a substance that increases the viscosity of the liquid, was added to the final undiluted DTwPH vaccine prepared in Example 7 so that the final 0.6 w / v%. As a result, agglomeration and adhesion to the glass wall were not found at all even when stored for 2 months or more at 2 to 8 ℃.

Example 9 Use of Silicone Coating Bottles

The final undiluted DTwPH vaccine prepared in Example 7 was dispensed 0.5 mL each into a silicone coated bottle (3 mL glass bottle) and the same bottle not coated with silicone, and then stored at 2 to 8 ° C. for at least 3 months. As a result, no adhesion phenomenon on the glass wall was found when the silicone coating bottle was used. In addition, after the two glass bottles were vigorously stirred by hand and magnified 100 times with a microscope (Axioplan, Carl Zeiss co, Germany) to observe their properties, when the liquid was stored in a silicon-coated bottle, the liquid had the same uniform properties as the initial one. On the other hand, in the case of the glass bottle not treated with silicon, it was found that the DTwPH mixed vaccine particles were agglomerated as a whole (FIG. 3).

According to the present invention described above, DTwPH by adjusting the concentration of the aluminum hydroxide gel and the pH of each adsorption stock solution (diphtheria, tetanus toxoid adsorbent, hepatitis B surface antigen adsorbent) and pertussis stock solution before preparation of DTwPH mixed vaccine final stock solution The final preparation of the mixed vaccine and the addition of gelatin or storage in a silicone coating bottle can prevent aggregation and adhesion of the DTwPH mixed vaccine final stock.

In addition, diphtheria and tetanus toxoid are adsorbed on aluminum phosphate gel, hepatitis B surface antigen is adsorbed on aluminum hydroxide gel, and each component is adjusted to an appropriate concentration to prepare DTwPH mixed vaccine final stock solution for hepatitis B of mixed DTwPH vaccine. In addition to maintaining the immunogenicity of surface antigens, the potency of each component can be made higher than the biological standards.

In addition, the present invention is applicable to the case of immunization after additionally mixing monovalent vaccines, such as Haemophilus influenza non-influenza vaccine (Hib), inactivated poly vaccine (IPV), or just before injection.

Claims (13)

In mixed vaccine of diphtheria, tetanus, pertussis and hepatitis B, diphtheria and tetanus toxoid are adsorbed on aluminum phosphate gel, hepatitis B surface antigen is adsorbed on aluminum hydroxide gel, concentration of diphtheria, tetanus toxoid, and pertussis bacteria Concentrations and hepatitis B surface antigen concentrations of 20 to 50 Lf / mL, 4 to 20 Lf / mL, 10 to 24 OU / mL, and 10 to 30 µg / mL, respectively. , DTwPH mixed vaccine comprising a total aluminum gel concentration of 15 to 35 μg Al / mL and 550 to 1000 μg Al / mL, respectively. The DTwPH mixed vaccine according to claim 1, wherein the pH of the final DTwPH mixed vaccine is 6.5 to 7.5. delete delete The DTwPH mixed vaccine according to claim 1 or 2, further comprising 0.5 to 0.7 w / v% gelatin. delete delete In the method for producing a mixed vaccine of diphtheria, tetanus, pertussis and hepatitis B, (a) 20 to 50 Lf / mL diphtheria toxoid aluminum phosphate adsorption liquid and 4 to 20 Lf / mL tetanus toxoid aluminum phosphate adsorption liquid are mixed, (b) Mix the adsorbed solution mixed in step (a) with 10-30 μg / mL hepatitis B surface antigen aluminum hydroxide adsorbed solution and 10-24 OU / mL inactivated pertussis stock solution, but the DTwPH mixed vaccine final A method of producing a mixed DTwPH vaccine comprising adjusting the aluminum hydroxide gel concentration of the stock solution and the total aluminum gel concentration to 15 to 35 µg Al / mL and 550 to 1000 µg Al / mL, respectively. The method for producing a mixed DTwPH vaccine according to claim 8, wherein the final pH of the mixed DTwPH vaccine is adjusted to 6.5 to 7.5. delete delete A method for storing a mixed DTwPH vaccine, wherein the final undiluted vaccine of the DTwPH mixed vaccine of claim 1, 2 and 5 is dispensed and stored in a glass jar coated with silicone. delete