Title of Invention	A PROCESS OF PRODUCING COMBINED VACCINE
Abstract	The present invention relates to a method for manufacturing a combined vaccine capable of concurrently preventing multiple diseases such as diphtheria, tetanus, pertussis, and hepatitis B which should be prevented in an infant. The method for manufacturing a combined vaccine according to the present invention includes the steps of independently adsorbing each protective antigen to an adsorbent of a aluminum hydroxide gel with respect to various diseases such as diphtheria, tetanus, pertussis, and hepatitis B which should be prevented in the infants, and combining each protective antigen adsorbed to the adsorbent after the adsorption. In the present invention, it is possible to concurrently prevent multiple diseases such as diphtheria, tetanus, pertussis, and hepatitis B which should be prevented in the infant using a combined vaccine manufactured according to the present invention.

Title of Invention

A PROCESS OF PRODUCING COMBINED VACCINE

Abstract

The present invention relates to a method for manufacturing a combined vaccine capable of concurrently preventing multiple diseases such as diphtheria, tetanus, pertussis, and hepatitis B which should be prevented in an infant. The method for manufacturing a combined vaccine according to the present invention includes the steps of independently adsorbing each protective antigen to an adsorbent of a aluminum hydroxide gel with respect to various diseases such as diphtheria, tetanus, pertussis, and hepatitis B which should be prevented in the infants, and combining each protective antigen adsorbed to the adsorbent after the adsorption. In the present invention, it is possible to concurrently prevent multiple diseases such as diphtheria, tetanus, pertussis, and hepatitis B which should be prevented in the infant using a combined vaccine manufactured according to the present invention.

Full Text	MANUFACTURING METHOD OF COMBINED VACCINE BACKGROUND OF THE INVENTION TECHNICAL FIELD The present invention relates to manufacturing method of combined vaccine capable of preventing concurrently various diseases of infants such as diphtheria, tetanus, pertussis, hepatitis B, and other diseases, which should be prevented in infants. BACKGROUND ART A combined vaccine is directed to a vaccine manufactured by combining protective antigens for each disease with respect to various other infection diseases or a vaccine manufactured by combining of various related antigens to prevent one infection disease. As examples of the former, there are a DTP vaccine capable of enhancing an immunity with respect to diphtheria, tetanus and pertussis, a MMR vaccine capable of enhancing an immunity with respect to measles, mumps, and gennan measles,..and so on. The above vaccines have been used for 20 years. As examples of the latter, there are a pneumococcal vaccine manufactured by combining 14 or 23 pneumococcal polysaccharides capable of enhancing an immunity to pneumonia, and a meningococcus vaccine manufactured by combining 4 meningococcal polysaccharides capable of enhancing an immunity to protect meningitis and so on. The above vaccines have been used for a few years. The above vaccines have been used for many years since its development are well recognized with their high immunogenic effects and less side effect with respect to each infection disease and with their good adaptation to protect various diseases. Vaccine developers are newly developing various types of combined vaccines for the above reasons. As the above combined vaccines, there is a vaccine (Intemational Publication No. WO 99/13906) manufactured by combining a DTP vaccine, bacterial encephalomeningitis vaccine (Hib vaccine), inactivated polio vaccine, and hepatitis B vaccine. A certain combined vaccine is developed by the combination of pneumococcal and meningococcal polysaccharides conjugated with protein. A combined vaccine capable of preventing from an intestinal infection with respect to each causing bacteria of cholera, typhoid, dysentery, and diarrhea will be developed soon. The combined vaccine prepared in the way of combining each antigen for preventing various other infective diseases has advantages in that the number of vaccinations decreases, and the supply of vaccines is simple for thereby decreasing the cost. According to the infant vaccination schedule recommended by Pediatrics Association (1997), the infants gets many times of shot within 1 years after birth, and injection of vaccine is sometimes overiapped with different vaccines due to conveniance or illness. Therefore, it is important to inoculate the infants based on a proper method for thereby providing a certain convenience to both vaccinating persons and inoculated persons. In particular, as the standardized vaccination schedule, the DTP and the hepatitis B vaccinations are guided to perfomi three times of primary shots within the first year of infants and boost shots. In addition, since the vaccination schedules recommended are similar, the problem of overtaping shots can happen. The inventors of the present invention perfonmed a research for providing a certain convenience for vaccination to both the vaccinating persons and the inoculated persons by developing the combined vaccine of DTP and hepatitis B vaccines and stabilizing of the supply of the vaccine for thereby providing a vaccination benefit to more people. Since the research on the combined vaccine is mostly directed to combining each component antigen from the vaccine products which is provided in the immunity and stability, the development of the combining method is important to minimize a variation in the reaction and immunity occuring due to an interaction between each protective antigen and adsorbent is important for the above research. With the completion of development, the homogeneity and stability of the combined vaccine formulations invented have been reviewed. In the present invention, the research has been performed based on the DTP vaccine and hepatitis B vaccine which are proved for their immunity and stability. As a method for combining each component vaccine, there are a method (International Publication Nos. WO 99/13906 and WO 00/7623) of simply combining the products of each component vaccine, a method (International Publication No. WO 99/13906) of administrating simultaneously when the vaccination is perfomied, using the specially designed container, and a method of manufacturing the combined vaccine by mixing each component of vaccine and ingredients in one formulation. Concerning the vaccine supply, the usage of the combined vaccine prepared by the former two methods are similar to the use of each monovalent vaccine singularly it needs more attention to handle vaccines than each monovalent vaccine, so that there is not an advantage in using this type of the combined vaccine. In addition, it is impossible to perform a research with respect to the variation of the immunity and th« occuring of the side effects. Therefore, it is preferable that each vaccine component is mixed in one fomiulation as one product on the research of the combined vaccine. The purpose of the present invention is to provide a manufacturing method of combined vaccine capable of preventing various diseases of infants including diphtheria, tetanus, pertussis and hepatitis B, which should be prevent in infants. DISCLOSURE OF THE INVENTION Accordingly, it is an object of the present invention to provide manufacturing method of combined vaccine for concurrently preventing various diseases of infants such as diphtheria, tetanus, pertussis and hepatitis B, which should be prevent in infants. In order to achieve the above object, there is provided a manufacturing method of a combined vaccine, comprising the steps of: The adsorption step of independently adsorbing each protective antigen to adsorbent respectively with respect to various diseases; and The combination step of mixing each above protective antigen adsorbed to the adsorbent. And there is also provided the method wherein the protective antigen is the antigen selected from the group comprising diphtheria antigen, tetanus antigen, pertussis antigen, heptatitis B antigen, or two or more combination thereof. And there is also provided the method, wherein the adsorbent is aluminum hydroxide gel. And, in order to achieve the above object, there is provided a combined vaccine as produced according to any of the above methods. More specifically, the present invention provides the manufacturing method of a combined vaccine which includes the steps of independently adsorbing each protective antigen to an adsorbent of a aluminum hydroxide gel with respect to various diseases such as diphtheria, tetanus, pertussis, and hepatitis B which should be prevented in the infants, and combining each protective antigen adsorbed to the adsorbent after the adsorption. The antigens of the present invention is not limited by the mentioned diseases but applicated to various antigens from various diseases. BRIEF DESCRIPTION OF THE DRAWINGS The present invention will become better understood with reference to the accompanying drawings which are given only by way of illustration and thus does not limit to the present invention, wherein; Fig. 1 is a view illustrating an adsorption ratio of each component antigen based on concentration of an adsortDent. Diphtheria toxoid, tetanus toxoid, and hepatitis B virus surface antigen are a components of diphtheria antigen, tetanus antigen and hepatitis B antigen, and pertussis toxoid and pertussis FHA antigen(pertussis thready shape blood agglutinin corpuscle) are each component of a purified pertussis antigen; Figs. 2a to 2d are views illustrating an antigenicity and immunity based on an adsorption method wherein sample 1 is a sample in which surplus aluminum hydroxide gel is added after a combination of each component vaccine is completed, and sample 2 is a sample in which the said adsort^ent of the same concentration is previously added before the combination is completed; Figs 2a and 2b are views illustrating the relative antigenicity of samples 1 and 2 respectively and, Figs 2c and 2d are views illustrating the relative level of antibody formation against each antigen of samples 1 and 2, respectively. Fig. 3 is a view illustrating the level of antibody formation against each antigen in the serum obtained from monkeys administered a combined vaccine prepared according to the present invention or conccurently each vaccine product of DTP and hepatitis B based on the date of collecting blood samples from each monkey. Group 1 is designated that each vaccine of DTP and hepatitis B are concurrently administered, end group 2 is designated that a combined vaccine is administered. MODES FOR CARRYING OUT THE PREFERRED EMBODIMENTS As the method for manufacturing the combined vaccine containing each vaccine component as one product, the above method is divided into two steps: a method for the step of combining the antigens which form each component vaccine and perfonning an adsorption using an adsorbent and a method for the step of combining the previously adsorbed antigens, in the case that an adsorbent is used as a component in the said method. However, in the case that each component antigen are first mixed, an adsorption ability with respect to the adsorbent of each antigen may decrease due to the interaction among antigens and ingredients in solution. In addition, the adsorption between the adsorbent and component antigen is optimized under each independent condition. If the above adsorption processes are concurrently performed, the adsorption ratio may be decreased due to a difference in the adsorption condition of each component antigen. Since the interrelationship between the adsorption ratio with respect to the adsorbent of the component antigen and the immunity of the vaccine is in inverse proportion, the immunity of the combined vaccine, which is manufactured in the process performed concurrently, may be decreased compared to the immunity of each monovalent vaccine. Therefore, the inventors of the present invention have judged that it is proper to manufacture a combined vaccine by prepahng a bulk solution of each component antigen, independently performing an adsorption of each antigen and combining the adsorbed antigens together. In addition, in the case that each component antigen is combined in each adsorption type, the final concentration of each component antigen in the combined vaccine should be the same as the concentration of the component antigen in each conventional monovalent vaccine, to compare the immunity and the characteristics of vaccine antigens in vaccine products. Therefore, to manufacture the combined vaccine, the component antigen more concentrated than a single vaccine is adsorbed and then combined. The DTP vaccine is manufactured based on an adsorption using the antigen which is concentrated 1.5-^2 limes, and the hepatitis B vaccine is manufactured using the antigen which is concentrated 2^-3 times. The final concentration of each component antigen of the combined vaccine according to the present invention is coincided with the concentration of each single vaccine adjusting the combining ratio of each antigen. According to the present invention, the inventors performed the various studies of combinations for developing the combined vaccine. In this case, the final content of each immune component in a vaccine product is the same as the content of the monovalent vaccine. In addition, the change of the adsorption ratio of each antigen is monitored by controlling the contents of the adsorbent and other components. Each sample which has a less change in the adsorption ratio is immunized into small animals to compare the level of the antibody formation. A proper combining method was selected by reviewing the result of antibody fomnation in small animals. In addition, as a result of the review with respect to the adsorbents of the currently commercial DTP vaccine and the hepatitis B vaccine, aluminum hydroxide gel; and aluminum phosphate gel were mainly used. It is known that the adsorption of the protein to each aluminum gel is caused by a surface electric charge of each protein and a gel component. And it is also known that the aluminum hydroxide gel has a positive surface electric charge in a physiological pH range, and aluminum phosphate gel has a negative surface electric charge ("Vaccine Design -The subunit and adjuvant approach", ed. By M.F. Powell & M.J. Newman, 1995, p229-239). In the case that the above two types of aluminum gels are concurrently used, a certain interaction occurs, so that the size of particle in the solution may be increased, and the adsorption capacity to protein may be decreased. In addition, the surface electric charge of each component antigen of the hepatitis 8 vaccine and combined vaccine has generally a negative surface electric charge in a physiological pH range. Therefore, the aluminum hydroxide gel is pnDper as an adsorbent. In the conventional art, the component of the adsorbent such as the aluminum hydroxide gel and aluminum phosphate gel was co-used (International Publication No. WO 93/24148) but based on the above reason, the inventors of the present invention judged that the adsorbent of the combined vaccine should have the same salt type and the aluminum hydroxide gel which is used as an adsorbent is an important factor for obtaining a certain adsorption ratio of each component antigen. But it is evident that the adsorbent covers not only the aluminum hydroxide gel but also its equivalents in the present invention. In addition, in the combined vaccine prepared by the present invention, other component antigens except hepatitis B antigen include proteins as a main component, while, in the case of the hepatitis B vaccine, the antigen exists generally in a particle type which includes a phospholipid layer. Therefore, it is preferred that when manufacturing the hepatitis B vaccine for a combined vaccine prepared by the present invention, in order to decrease a certain interference by other antigens with respect to the phospholipid component of the hepatitis B vaccine antigen which includes a phospholipid layer, a neutral surfactant such as Polysorbate 20 (Tween 20). Polysorbate 80 (Tween 80) and Triton X-100 are added. Since the titer of the hepatitis B vaccine tends to decrease in the case that the amount of the neutral surfactant is high, it is preferred that the neutral surfactant is added at the mass ratio below 50% with respect to protein amount of the hepatitis B surface antigen but the above amount is not limited thereto. In the present invention, Corynebacterium diphtheria, PW No. 8 as an antigen of the diphtheria is prepared and cultivated in a proper culture medium. The diphtheria toxoid is preferred, which is obtained by detoxifying the ■ diphtheria toxin which is purified by a conventional method. The amount of the antigen is preferably 10--25 Lf based on the pediatric dose. As a tetanus antigen, Clostridium tetanii. Harvard, is cultivated in a proper culture medium under anaerobic condition. The tetanus toxoid which is obtained by detoxifying the tetanus toxin purified by the conventional method is proper. The amount of the antigen is preferably I'-SLf based on the pediatric dose. In addition, the Pertussis antigen is cultivated in a proper culture medium using Bordetella pertussis, Tohama phase I, and multiple kinds of antigen protein including a Pertussis toxoid are purified by a conventional method in a culture supernatant and detoxified. Purified Pertussis antigens or a whole cell Pertussis antigen is proper. Here, the manufacturing method of a combined vaccine is provided in which in the case of the multiple kinds of antigens, the total amount of the antigens is below 20\|igPN based on the pediatric dose, and in the case of the whole cell Pertussis antigen, the amount of the antigen is preferably below 20 OE based on the pediatric dose. The present invention is further directed to a method for manufacturing a combined vaccine in which purified multiple antigen proteins include a detoxified Pertussis toxoid and filamentous hemagglutinin (FHA) antigen. Preferably, the present invention is directed to a method for manufacturing a combined vaccine in such a manner that the amount of recombinant hepatitis B virus surface antigen, which manufactured by a genetic engineering method as a hepatitis B antigen, is 5-1 Oug based on the pediathc dose. More preferably, the present invention is directed to a method for manufacturing in such a manner that the hepatitis B surface antigen is adsorbed to an adsorbent by mixing with stirring at 2-8°C for 3-^20 hours. In order to find the optimum content of adsortDant for manufacturing of the combined vaccine, the adsorption ratio for each antigen component was analyzed at each concentration of adsortDant. As a result, when the final aluminum ion concentration is below 0.5mg/ml at the time when the final combination is completed, the adsorption ratio of each antigen was relatively decreased (as shown in Fig. 1). In addition, the aluminum ion concentration is stipulated to be under 1.25mg/ml in the aluminum gel used for the vaccine by regulation (WHO, "Requirements for diphtheria, tetanus, pertussis and combined vaccines" in Technical Report Series No. 800, 1990, p87-179). Therefore, as the aluminum hydroxide gel, the concentration of the final aluminum ion is preferably in the range of 0.5'-1.25mg/ml and more preferably in the range of 0.7mg/ml. In addition, in order to prevent a possibility that the absorption ratio of each component antigen is decreased by a repulsive -force caused among each component antigenr even after the combination of each component is completed, the antigenicity and the level of antibody formation of each component antigen are converted to the relative percentage to compare those values between two samples: a sample that the other component except an aluminum hydroxide gel is previously combined and then suplus aluminum hydroxide gel is additionally added and a sample that the adsorbent of the same concentration is previously added before the combination is completed. The change of the antigenicity and the level of antibody formation decreases when the surplus adsorbant is added even after the combination of each component is completed (as shown in Fig. 2). In addition, in the case of the sample in which the polysorbate SO 80 (tween 80) is added, the antigenicity and titer with respect to the hepatitis B were maintained (as shown in Fig. 2). It is expected that a similar result i© may be obtained even when neutral surfactants such as polysorbate 20 and triton X-100 are used whithin proper concentration. As shown in Fig. 2. the sample 1 and sample 2 are prepared in the way of that surplus aluminum hydroxide gel is additionally added after the combination of each component is completed, and that the adsorbent of the same concentration is previously added before the combination is completedr, respectively. Preferably, the present invention is directed to a method for manufacturing a combined vaccine in which the aluminum ion concentration of the.aluminum hydroxide gel is in the range of 0.5'-1.25mg/ml. More preferably, the present invention is directed to a method for manufacturing a combined vaccine which includes a step of adding surplus adsorbent in the range that does not exceed the concentration range of the aluminum ion of 0,5-1.25 mg/ml after protective antigens adsorbed to the adsorbent are combined. In a preferred embodiment of the present invention, it is possible to maintain an inherent adsorption of each component antigen in the combined vaccine and to increase an immunity. As the Pertussis antigen, a purified Pertussis antigen is generally used in Korea and several other countries. Since some American countries and the third world countries use the-a whole cell Pertussis antigen which is detoxified. Therefore, in the present invention, the applicable range of the combined vaccine is widened by the study on the combining method using whole cell pertussis antigen. In this case, the combining was performed in the same method as using the mentioned purified Pertussis antigen for thereby obtaining a sample capable of maintaining an immunity of each component(as shown in Table 1). In the case of the DTP vaccine which includes a whole cell Pertussis antigen, a aluminum phosphate gel is generally used as an adsorbent. However, in order to manufacture a combined vaccine with respect to the hepatitis B antigen, the DTP vaccine was manufactured using the aluminum hydroxide gel. In this case, the immunity with respect to the Pertussis was decreased, but when a surplus aluminum hydroxide gel was additionally added after the combination of each component vaccine was completed (sample A), it was possible to maintain a desired immunity. As a reference, the sample B is prepared in the way of that the adsorbent of the same concentration was previously added before the combination was completed. The safety and efficacy of the combined vaccine manufactured according to the present invention was proved based on the following methods. First, overdosage of combined vaccine was administrated to a rodent, and it was checked that a lesion was not observed as a result of the biopsy (The result of the same is not provided). In addition, the antibody level against each antigen was measured in a group (group 2) in which a combined vaccine was administrated to a monkey and in a group (group 1) in which each vaccine of DTP and hepatitis B was concurrently administrated. As a result of the t-test with respect to the antibody amount between two groups, when comparing the result of the group 2 with the result of the group 1, it was evaluated that the combined vaccine showed the equal or better immunogenecity against each antigen to concurrently injection group of each vaccine (as shown in Fig. 3. The result of the statistic is not provided). In conclusion, the combining method was developed in the present invention, which an interaction among different immune components was minimized, and each immunogenic effect did not decrease. When dividing each immune component used for the combined vaccine based on physical and chemical properties, the immunogenic components may be divided into a protein antigen (diphtheria antigen, tetanus antigen, purified Pertussis antigen), an antigen composed of protein and phospholipid layer (hepatitis B antigen), and an antigen composed of killed whole cell (a whole cell Pertussis antigen), etc. Therefore, it may be predicted that a combination with other antigen of single vaccine may be implemented based on the physical and chemical characteristics of components. A combination of an additional vaccine may be possible with respect to the diseases (hemophilus influenza, polio), which should be prevented in the infants by the combined vaccines prepared according to the present invention. The body vaccination period of the combined vaccine agent according to the present invention may be the previous vaccination periods of the DTP vaccine or the hepatitis B vaccine. In the case that there is the hepatitis B antigen in the mother's body, it is preferred to inoculate three times of second, fourth and sixth months. In the case that there is not the hepatitis B in the mother's body, as a proper vaccination period, the hepatitis B vaccine is singularly inoculated after birth, and then the additional vaccination is performed using a combined vaccine. A proper vaccination with respect to the infants can be perfonned by a muscle or hypodennic injection method. The doze of the antigen of diphtheria, tetanus, Pertussis and hepatitis B respectively in the combined vaccine according to the present invention is the same as the doze for the infant of the antigen of each single vaccine. Example 1 Manufacture of hepatitis B antigen The yeast cell which is capable of expressing the hepatitis B surface antigen by genetic engineering method is intensively cultivated. 1kg of the precipitate of yeast cells which is obtained by the centrifugation is diluted at a ratio of 1:2 in a buffer solution (0.5M NaCI, lOmM EDTA, 0.01% Thimerosal, 0.1M Phosphate. pH 7.0). The diluted solution flows through a glass bead beator (or Dynomil) for thereby breaking cell walls and so on. The obtained solution is added with a neutral surfactant (Tween group or Triton group) by 0.5% and is unifonmly nnixed by stirring at 4°C. Sodium hydroxide was added to the resultant solution for thereby obtaining pH 11 and is then mixed by stirring at 4°C for 5 hours. A diluted hydrochloric acid was added to the resultant solution for thereby obtaining pH 4. The precipitate was removed by a centrifugation at 6000rpm for 15 minutes using the centrifugal machine (ROTOR: JA-14, Beckman Inc. USA), and the upper solution Including the hepatitis B surface antigen was obtained. The pH of the upper solution was made at 7, and then silica was added thereto and was mixed at 4-25°C for 3^-16 hours, so that the hepatitis B surface antigen was adsorbed to the silica. The silica gel which is preferably used in the present invention is Aerosil 380(Degussa, USA) which includes fine hydration silica or anhydrous silica having a valid surface area of 100-"500mm^/g. In order to remove contaminants from the silica to which the hepatitis B surface antigen is adsorbed, the resultant solution was washed two times using sodium phosphate - sodium chloride buffer solution of pH 7. The washed silica was contacted in the sodium carbonate buffer solution of pH 9.6 for about 2 hours for thereby desorption of a surface antigen. The thus separated surface antigen had a protein purity of above 90% in the solution. The resultant solution^ was flown in the DEAE-Sepharose (PHAMACIA, Sweden) which was balanced using the said sodium buffer solution for thereby specifically attaching a surface antigen and removing the substances separated in the column using the said buffer solution. Contaminants slightly attached on the column was eluted using the buffer solution including sodium chloride of 0.05-0.1 M. Thereafter, the hepatitis B surface antigen was eluted using the said buffer solution including the sodium chloride of 0.2M. The thus eluted solution was concentrated using a ultrafiltration membrane which is capable of separating the substance of molecular weight above than 100,000. and the precipitate was separated by centrifugation and removed, and the upper solution was collected, and the gel permeation chromatography (Sepharose CL-4B, PHAMACIA, Sweden) was perfomied with respect to the collected upper solution. The fractions which included a pure surface antigen were confirmed by electrophorsis and were used as the hepatitis B antigen. In addition, in order to decrease an interaction with each of the hepatitis surface antigen, the tween 80 was added to 0, 5, 10\|ig per 1ml, and then the effect of tween 80 was observed (as shown in Fig. 2). Example 2 Manufacture of diphtheria toxoid The corynebacterium diphtheria PW No. 8 was cultured at 35°C for 24 hours in the nutrition agar culture medium (DIFCO. USA) and was subcultured two times. One of the colony was cultured at 35°C for 24 hours in 2ml of the brain heart infusion culture medium (DIFCO, USA), and 1.2ml of it was inoculated to the modified Muller culture medium (refer to: Stainer, et. al, Canadian J, Microbiol.. 14:155, 1968) of 300ml and was cultured at 35°C for 36 hours. After the culture, cells were removed from the culturing solution, and the toxin solution was collected and it was added with ammonium sulfate at 4°C. The final concentration was made in 25(w/v)%. and pH was made in 8.0. The culturing solution having adjusted pH and concentration' of salt was dropped into the phenyl-sepharose column which was previously balanced using lOmM tris buffer solution pH 8.0 including 25(w/v)% ammonium sulfate. The buffer solution same as the column balance solution and lOmM tris buffer solution(pH 8.0) including 15(w/v)% ammonium sulfate were sequentially flown to the column for thereby removing impurities. The toxin was eluted using 10mM tris buffer solution(pH 8.0) which did not include salt. The eluted diphtheria toxin solution was collected and was dialyzed to lOmM phosphate buffer solution(pH 7.4) including sodium chloride of 150mM. After the dialyzing process was completed, fonnalin was added, so that the final concentration was 0.05(w/v)%, and the resultant solution was reacted at 37°C for 1 hour, and lysine was added for thereby obtaining the final concentration of 0.05M and then, the resultant solution was detoxified at 37°C for 4 weeks. Thejoxoid solution was dialyzed into 10mM phosphate buffer solution(pH 7.4) with sodium chloride for thereby fully removing the fomnalin, and thimerosal was added to have 0.01(w/v)% of the final concentration. The resultant solution was used as diphtheria toxoid solution. Example 3 Manufacture of tetanus toxoid The Clostridium tetanii (Harvard strain) was cultured by molten agar method with sterilized liver-bile agar medium (DIFCO, USA). A few colonies from above culture were inoculated into brain heart infusion culture medium (DIFCO, USA) including 0.3(w/v)% yeast extraction (DIFCO, USA) of 2ml having decreased oxygen level and were cultured at 35°C for 24 hours in anaerobic state. And then, the culture solution was inoculated into fully nitrogen-satured brain heart infusion culture medium (DIFCO, USA) 500ml including 0.3(w/v)% yeast extraction (DIFCO, USA) and were cultured at 35°C for 7 days in anaerobic state. After the culture, cells were removed from the culture solution, the toxin solution was collected and added with ammonium sulfate at 4°C for thereby obtaining the final concentration of 60(w/v)% and was mixed for over 24 hours and was fully mixed for thereby obtaining a precipitate by centrifugation. The precipitate was dissolved in a small amount of distilled water, and the insoluble material was removed. The resultant solution was dropped into the sephagrill S-IOO column which was previously balanced using 10mM tris buffer solution(pH 8.0) including 0.5M sodium chloride. The same buffer solution was flown for thereby eluting the separated toxin. The tetanus toxin solution was collected and dialyzed into 10mM phosphate buffer solutJon(pH 7.4) including 150mM sodium chloride. After the dialysis, formalin was added to have the final concentration of 0.025(w/v)% and was reacted at 37°C for 1 hour. Thereafter, lysine and sodium hydrogencarbonate were added to have the final concentration of O.OSmM and 0.04M, respectively, and was matured at 37°C for 4 weeks and was detoxified. After the detoxification, the toxoid solution was dialyzed into 10mM phosphate buffer solution (pH 7,4) including 150mM sodium chloride, and formalin was fully removed. Thimerosal was added to have the final concentration of 0.01(w/v)% and was used as tetanus toxoid solution. Example 4 Manufacture of purified Pertussis antigen protein Bordetella pertussis, Tohama phase I was cultured in Bodet-Gengo agar culture medium(DIFCO, USA) including 15% rabbit blood at SS^C for 72 hours and was passaged two times. A few colonies were inoculated into 50ml of stanor-sholte culture medium and was cultured at 35°C for 48 hours. The thusly cultured solution were re-inoculated in 500ml of changed stanor-sholte culture medium(refer to Imazumi et a!., INFECT.-IMMUN.. 1983, vol 41, pp. 1138) and was cultured at 35^0 for 36 hours. 10% thimerosal aqueous solution was added to the cultured solution to have the final concentration of 0.01% for thereby preventing the growth of the Pertussis bacteria, and then the cells were removed by centhfugation. Three times volume of distilled water was added to the resultant solution, and was mixed well. The current fDH was decreased to pH 6.0 using IN sulfuric acid. The resultant solution was dropped Into CM-sepharose column which was previously balanced using lOmM sodium phosphate buffer of pH 6.0. The buffer solution same as the column balance solution was flown to the column for thereby removing the substances which were not coupled to the column, and the impurities which were slightly coupled to the column were removed using the buffer solution same as the column balance solution including lOOmM sodium chloride. When the impurities were not eluted anymore through the column, the bound materials were eluted with linear gradient formed by the column balance solution including lOOmM sodium chloride and 600mM sodium chloride which have the same volume, so that a fraction including Pertussis antigen protein such as Pertussis toxin and FHA (filamentous hemagglutinin) was separated. The antigen fraction was diluted using lOmM sodium phosphate buffer solution(pH 8.0) of two times volume to have pH 8.0. The resultant solution was dropped into hydroxy apatite column which was previously balanced using lOmM sodium phosphate buffer solution of pH 8.0. The buffer solution same as the column balance solution including lOOmM sodium chloride was flown to the column for thereby removing the substances which were not coupled to the column. 80mM sodium phosphate buffer solution of pH 8.0 including lOOmM sodium chloride was flown at the same rate as the dropping rate for thereby separating Pertussis toxin fraction. When the Pertussis toxin was separated, 250mM sodium phosphate buffer solution of pH 8.0 including lOOmM sodium chloride was flown at the same rate as the dropping rate for thereby separating FHA(filamentous hemagglutinin) fraction. Separated Pertussis toxin and FHA(filamentous hemagglutinin) were dialyzed in lOmM sodium phosphate buffer solution(pH 7.4) including 0.15% soaium chloride at 4°C. Glycerol and glutal aldehyde were added to dialyzed Pertussis toxin sample solution to have the final concentration of 50(w/v)% and 0.05(w/v)% and were detoxified at 37°C for 4 hours. Sodium aspartate was added to have the final concentration of 0.025M for thereby completing the detoxifying process. Glycerol and formalin were added fnto the dialyzed FHA (filamentous hemagglutinin) sample solution to have 50(w/v)% and 0.025^w/v)% and were detoxified at 37^C for 24 hours. Lysine was added to have the final concentration of 0.025M, so that the detoxifying process was completed. Each sample solution was dialyzed into 10mM sodium phosphate buffer solution (pH 7.4) including 0.15% sodium chloride at room temperature. Thimerosal was added to have the final concentration of 0.01(w/v)%. Thereafter, detoxified Pertussis toxin sample solution and FHA (filamentous hemagglutinin) sample solution were mixed at a ratio of 1:4 and were used as a Pertussis antigen protein. Example 5 Manufacture of a whole cell Pertussis antigen Pertussis bacteria. Bordetella pertussis, Tohama phase I was cultured in Bodet-Gengo agar culture medium(DIFCO, USA) including 15% rabbit blood at SS^'C for 72 hours and was passaged two times. Some of the colonies were inoculated into 50ml of Stanor-sholte culture medium and were cultured at 35°C for 48 hours. The resultant solution was re-inoculated in the changed stanor-sholte culture medium(refer to Imazumi et al.. INFECT.-IMMUN., 1983, vol 41, pp. 1138) of 500ml and were cultured at 35°C for 36 hours. 10% thimerosal aqueous solution was added into the culture solution to have the final concentration of 0.01% for thereby preventing the growth of the Pertussis bacteria and collecting somatic based on the centrifugation. The collected somatic solution was dialyzed in lOmM sodium phosphate buffer solution(pH 7.4) including 0.15% sodium chloride at 4°C. Fonnalin was added into the dialyzed Pertussis somatic solution to have the final concentration of 0.025(w/v)% and was detoxified at 37^C for 4 weeks. Lysine was added to have the final concentration of 0.025M for thereby completing the detoxifying process. The sample solution was dialyzed into lOmM sodium phosphate buffer solution(pH 7.4) including sodium chloride of 0.15%, and thimerosal was added to have the final concentration of 0.01(w/v)% and was used as the whole cell Pertussis antigen. Example 6 Manufacture of combined vaccine including purified Pertussis antigen Step 1. Manufacture of hepatitis B vaccine The hepatitis B vaccine was manufactured using the hepatitis B surface ' antigen manufactured in Example 1. aluminum hydroxide solution was manufactured by adding aluminum hydroxide gel to the phosphate buffer solution. The resultant solution was slowly mixed, and the above antigen solution was dropped and was mixed by stirring slowly at 4^C for 15 hours for thereby manufacturing hepatitis B vaccine. At this time, the amount of the hepatitis surface antigen was 60\|ig/ml which was high concentrated three times compared to the amount of the common hepatitis B surface antigen. In the case of the first sample, the amount of the aluminum ion in the aluminum hydroxide gel was 0.9mg/ml. In the case of the second sample, the amount of the aluminum ion in the aluminum hydroxide gel was 1.5mg/ml(as shown in Fig. 2). Step 2. Manufacture of DTP combined vaccine The DTP combined vaccine was manufactured using each antigen manufactured in the second, third and fourth examples. The aluminum hydroxide solution was manufactured by adding aluminum hydroxide gel to the phosphate buffer solution in the conventional method. As the resultant solution was slowly mixed, each antigen solution was dropped thereto and was mixed by stirring slowly for thereby implementing a uniform adsorption, so that the DTP combined vaccine was manufactured. At this time, the amount of each antigen was concentrated 1.5 times high compared to the amount of each component antigen of common DTP vaccine. The amount of the aluminum ion in the aluminum hydroxide gel was 0.3mg/ml. Step 3. Manufacture of DTP-hepatitis B combined vaccine Each DTP vaccine and hepatitis B vaccine manufactured in Steps 1 and 2 were slowly mixed at the volume ratio of 2:1. In the case of the first sample, surplus aluminum hydroxide gel was added so that the amount of the aluminum ion in the aluminum hydroxide gel was finally 0.7mg/ml, and was continuously mixed by stirring at 25°C for 1 hour for thereby manufacturing a stable combined vaccine which does not interact with other components. In the case of the second sample, surplus aluminum hydroxide gel was not added and was continuously mixed by stirring at 25°C for 1 hour for thereby manufacturing combined vaccine. The amounts of the aluminum ions in the aluminum hydroxide gel of the combined vaccines of the first and second samples were 0.7mg/ml. For the test of antibody titer, the composition of the combined vaccine was 20\|ig of the hepatitis B surface antigen, 25Lf of diphtheria toxoid, 3Lf of tetanus toxoid, 2.5^gPN of Pertussis toxoid, and lOi^gPN of Pertussis FHA antigen(Pertussis FHA(filamentous hemagglutinin) antigen based on 1ml(as shown in Fig. 2). Example 7 Manufacture of combined vaccine including a whole cell Pertussis antigen Step 1. Manufacture of hepatitis B vaccine The hepatitis B vaccine was manufactured using the hepatitis B surface antigen manufactured in Example 1. An aluminum hydroxide gel was added to phosphate buffer solution for thereby manufacturing the aluminum hydroxide solution. As the solution was slowly mixed, the above antigen solution was dropped and was fully mixed at 4°C for 15 hours for thereby manufacturing the hepatitis B vaccine. At this time, the amount of the hepatitis B surface antigen was 60^g/ml which was concentrated three times high compared to the amount of the common hepatitis B vaccine. In the case of the first sample, the amount of the aluminum ion was 0.9mg/ml in the aluminum hydroxide gel. In the case of the second sample, the amount of the aluminum ion was 1.5mg/ml in the aluminum hydroxide gel(as shown in Fig. 2). Step 2. Manufacture of DTP combined vaccine The DTP combined vaccine was manufactured using each antigen manufactured in the Examples 2, 3 and 5. The aluminum hydroxide gel was added to the phosphate buffer solution in the conventional method for thereby manufacturing an aluminum hydroxide solution. As the resultant solution was slowly mixed, the above antigen solution was dropped and was fully mixed. A uniform adsorption was implemented for thereby manufacturing a DTP combined vaccine. At this time, the amount of each antigen was concentrated 1.5 times high compared to the amount of each component antigen of the common DTP vaccine. The amount of the aluminum ion was 0.3mg/ml in the aluminum hydroxide gel. Step 3. Manufacture of DTP-hepatitis B combined vaccine Each DTP vaccine and hepatitis B vaccine manufactured in Steps 1 and 2 were slowly mixed at a volume ratio of 2:1. In the case of the first sample, a surplus aluminum hydroxide gel was added so that the amount of the aluminum ion was finally 0.7mg/ml in the aluminum hydroxide gel, and the resultant solution was continuously mixed at 25°C for 1 hour for thereby manufacturing a stable combined vaccine which does not interact with other components In the case of the second sample, surplus aluminum hydroxide gel was not added, and the solution was continuously mixed at 25'C for 1 hour for thereby manufacturing a combined vaccine. The amounts of the aluminum ions in the aluminum hydroxide gel of the combined vaccines of the samples 1 and 2 were 0.7mg/ml. For the test of antibody titer, the composition of the combined vaccine was 20/ig of the hepatitis B surface antigen, 50Lf of diphtheria toxoid, lOLf of tetanus toxoid, and 20OE of a whole cell Pertussis antigen based on 1ml(as shown in Fig. 2). Example 8, Test of antigenicity in combined vaccine The test of antigenicity in combined vaccine manufactured by the examples 6 and 7 was performed by the Enzyme Immune Assay (EIA) using monoclonal antibody and polyclonal antibody by comparison to the standard of . each antigen with respect to each antigen content. In addition, the antigen adsort^ed to the aluminum salt was removed by the centrifugation for measuring the adsorption ratio of each antigen, and the amount of the separated antigen was measured using the upper solution. The average of each result were shown in Figs. 1 and 2 shows relative antigenicity of each antigen. Example 9. Test of antibody formation by combined vaccine Test of antibody formation by hepatitis B surface antigen The combined vaccine and hepatitis B vaccine manufactured in Examples 6 and 7 were inoculated to ICR mice in which 16 mice were grouped as the group 1, and a blood was collected from the ICR mice after 18 days. Serum was separated from the blood. The antibody level against the hepatitis B surface antigen was obtained based on the unit of lU/ml using the EIA(Enzyme Immune Assay) and calculated as in geomean antibody titer. The titer of the combined vaccine was computed with respect to the hepatitis B vaccine. Fig. 2c and 2d shows relative antibody titer of combined vaccine with repect to each antigen and Table 1 shows the values of each titer. Test of antibody formation by diphtheria antigen The combined vaccine and DTP vaccine manufactured in Examples 6 and 7 were inoculated to the ICR mice in which 16 mice are grouped as the group 1. The blood was collected from the CIR mice after 28 days. Serum was separated from the bicod. The antibody level against the diphtheria antigen of the serum was measured using the EIA(Enzyme Immune Assay) based on the unit of lU/ml and calculated as in geomean antibody titer. Thereafter, the titer of the combined vaccine was measured compared to the DTP vaccine. Fig. 2c and 2d shows relative antibody titer of combined vaccine with repect to each antige, and Table 1 shows the values of each titer. Test of antibody formation by tetanus antigen The combined vaccine and DTP vaccine manufactured in Examples 6 and 7 were inoculated to the ICR mice in which 16 mice are grouped as the group 1. The blood was collected from the ICR mice after 28 days. Serum was separated from the blood. The antibody level against the tetanus antigen of the serum was measured using the EIA(En2yme Immuno Assay) based on the unit of lU/ml and calculated as in geomean antibody titer. Thereafter, the titer of the combined vaccine was measured compared to the DTP vaccine. Fig. 2c and 2d shows relative antibody titer of combined vaccine with repect to each antigen, and Table 1 shows the values of each titer. Test of antibody formation by Pertussis antigen The combined vaccine and DTP vaccine manufactured in Examples 6 and 7 were inoculated to the ICR mice in which 16 mice are grouped as the group 1. The blood was collected from the ICR mice after 28 days. Serum was separated from the blood. The antibody level against pertussis antigen of the serum was measured using the EIA(Enzyme Immuno Assay) based on the unit of lU/ml and as in geomean antibody titer Thereafter, the titer of the combined vaccine was measured compared to the DTP vaccine. Fig. 2c and 2d shows relative antibody titer of com.bined vaccine with repect to each antigen, and Table 1 shows the values of each titer. Example 10. Comparative effectiveness test with monkey A test was performed for proving the immunogenicity in a primate of the combined vaccine and comparing with the conventional single vaccine. The combined vaccine manufactured in Example 6 was vaccinated to six monkeys (group 2) formed of the same numbers of male and female monkeys, and each component vaccine was vaccinated to 6 monkeys(group 1) formed of the same numbers of male and female monkeys simultaneously. The same products as the products vaccinated to each group was re-vaccinated 30^^ and 60^ days. The blood was collected from each experimental animal on 1^\ 29^^, 58^, and 86^ days from the initial vaccination date, and the antibody titer against each disease was measured based on the ELISA method proper to the serum of the monkey. Fig. 3 shows the geomean antibody titer against each antigen of each group. INDUSTRIAL APPLICABILITY According to the present invention, it is directed to a method for manufacturing a combined vaccine for concurrently preventing the diseases such as diphtheria, tetanus, pertussis, and hepatitis B, etc. which should be prevented in an infant. By the present invention, it is possible to concurrently prevent the diseases such as diphtheria, tetanus, pertussis, and hepatitis B which should be prevented in an infant using the combined vaccine according to the present invention. As the present invention may be embodied in several forms- without departing from the spirit or essential characteristics thereof, it should also be understood that the above-described embodiments are not limited by any of the details of the foregoing description, unless otherwise specified, but rather should be constaied broadly within its spirit and scope as defined in the appended claims, and therefore all changes and modifications that fall within the meets and bounds of the claims, or equivalences of such meets and bounds are therefore intended to be embraced by the appended claims. CLAIMS 1. A manufacturing method of a combined vaccine, comprising the steps of; adsorption step of independently adsorbing each protective antigen to adsorbent respectively with respect to various diseases; and combination step of mixing each mentioned protective antigen adsorbed to the adsorbent. 2. The method of claim 1. wherein the protective antigen is the antigen selected from the group comprising diphtheria antigen, tetanus antigen, pertussis antigen, heptatitis B antigen, or two or more combination thereof. 3. The method of claim 1, wherein the adsorbent is aluminum hydroxide gel. 4. The method of claim 3, wherein aluminum ion concentration of the aluminum hydroxide gel after the manufacture of a combined vaccine is in the range of O.S-'1.25mg/ml. 5. The method of either claim 3 or 4, further comprising a step of: adding the adsorbent in which the final aluminum ion concentration does not exceed in the range of 0.5 -1.25mg/ml in aluminum hydroxide gel. 6. The method of claim 2, wherein a recombinant hepatitis B surface antigen as the hepatitis B antigen is combined by the amount of 5-10\|ig based on the pediatric dose. 7. The method of claim 6, further comprising a step of: adding a neutral surfactant such as polysorbate 20, polysorbate 80 and tritonX-100. 8. The method of claim 6, wherein said hepatitis B antigen is mixed by stirring with an adsorbent at 2-8^C for 3-'20 hours and is matured and adsorbed. 9. The method of claim 2, wherein the diphtheria toxoid detoxified as a diphtheria antigen is combined by the amount of 10-25Lf based on the pediatric dose. 10. The method of claim 2. wherein the tetanus toxoid detoxified as a tetanus antigen is combined by the amount of 1--5Lf based on the pediatric dose. 11. The method of claim 2, wherein the pertussis antigen including the purified antigens is combined by the amount below 20^gPN based on the pediatric dose or the whole cell pertussis antigen is combined by the amount below 20OE based on the pediatric dose. 12. The method of claim 11, wherein mentioned purified pertussis antigens include pertussis toxoid and pertussis FHA (filamentous hemagglutinin). 13. A combined vaccine as produced according to any of the methods claimed in Claims 1 to 12. A manufacturing method of a combined vaccine substantially as herein described with reference to the accompanying drawings. A combined vaccine substantially as herein described with reference to the accompanying drawings.

Full Text

MANUFACTURING METHOD OF COMBINED VACCINE
BACKGROUND OF THE INVENTION TECHNICAL FIELD
The present invention relates to manufacturing method of combined vaccine capable of preventing concurrently various diseases of infants such as diphtheria, tetanus, pertussis, hepatitis B, and other diseases, which should be prevented in infants.
BACKGROUND ART
A combined vaccine is directed to a vaccine manufactured by combining protective antigens for each disease with respect to various other infection diseases or a vaccine manufactured by combining of various related antigens to prevent one infection disease.
As examples of the former, there are a DTP vaccine capable of enhancing an immunity with respect to diphtheria, tetanus and pertussis, a MMR vaccine capable of enhancing an immunity with respect to measles, mumps, and gennan measles,..and so on. The above vaccines have been used for 20 years. As examples of the latter, there are a pneumococcal vaccine manufactured by combining 14 or 23 pneumococcal polysaccharides capable of enhancing an immunity to pneumonia, and a meningococcus vaccine manufactured by combining 4 meningococcal polysaccharides capable of enhancing an immunity to protect meningitis and so on. The above vaccines have been used for a few years.
The above vaccines have been used for many years since its development are well recognized with their high immunogenic effects and less side effect with respect to each infection disease and with their good adaptation to protect various diseases. Vaccine developers are newly developing various types of combined vaccines for the above reasons. As the above combined vaccines, there is a vaccine (Intemational Publication No. WO

99/13906) manufactured by combining a DTP vaccine, bacterial encephalomeningitis vaccine (Hib vaccine), inactivated polio vaccine, and hepatitis B vaccine. A certain combined vaccine is developed by the combination of pneumococcal and meningococcal polysaccharides conjugated with protein. A combined vaccine capable of preventing from an intestinal infection with respect to each causing bacteria of cholera, typhoid, dysentery, and diarrhea will be developed soon.
The combined vaccine prepared in the way of combining each antigen for preventing various other infective diseases has advantages in that the number of vaccinations decreases, and the supply of vaccines is simple for thereby decreasing the cost. According to the infant vaccination schedule recommended by Pediatrics Association (1997), the infants gets many times of shot within 1 years after birth, and injection of vaccine is sometimes overiapped with different vaccines due to conveniance or illness. Therefore, it is important to inoculate the infants based on a proper method for thereby providing a certain convenience to both vaccinating persons and inoculated persons. In particular, as the standardized vaccination schedule, the DTP and the hepatitis B vaccinations are guided to perfomi three times of primary shots within the first year of infants and boost shots. In addition, since the vaccination schedules recommended are similar, the problem of overtaping shots can happen.
The inventors of the present invention perfonmed a research for providing a certain convenience for vaccination to both the vaccinating persons and the inoculated persons by developing the combined vaccine of DTP and hepatitis B vaccines and stabilizing of the supply of the vaccine for thereby providing a vaccination benefit to more people.
Since the research on the combined vaccine is mostly directed to combining each component antigen from the vaccine products which is provided in the immunity and stability, the development of the combining method is important to minimize a variation in the reaction and immunity

occuring due to an interaction between each protective antigen and adsorbent is important for the above research. With the completion of development, the homogeneity and stability of the combined vaccine formulations invented have been reviewed. In the present invention, the research has been performed based on the DTP vaccine and hepatitis B vaccine which are proved for their immunity and stability.
As a method for combining each component vaccine, there are a method (International Publication Nos. WO 99/13906 and WO 00/7623) of simply combining the products of each component vaccine, a method (International Publication No. WO 99/13906) of administrating simultaneously when the vaccination is perfomied, using the specially designed container, and a method of manufacturing the combined vaccine by mixing each component of vaccine and ingredients in one formulation. Concerning the vaccine supply, the usage of the combined vaccine prepared by the former two methods are similar to the use of each monovalent vaccine singularly it needs more attention to handle vaccines than each monovalent vaccine, so that there is not an advantage in using this type of the combined vaccine. In addition, it is impossible to perform a research with respect to the variation of the immunity and th« occuring of the side effects. Therefore, it is preferable that each vaccine component is mixed in one fomiulation as one product on the research of the combined vaccine.
The purpose of the present invention is to provide a manufacturing method of combined vaccine capable of preventing various diseases of infants including diphtheria, tetanus, pertussis and hepatitis B, which should be prevent in infants.
DISCLOSURE OF THE INVENTION
Accordingly, it is an object of the present invention to provide manufacturing method of combined vaccine for concurrently preventing various diseases of infants such as diphtheria, tetanus, pertussis and hepatitis B, which

should be prevent in infants.
In order to achieve the above object, there is provided a manufacturing method of a combined vaccine, comprising the steps of:
The adsorption step of independently adsorbing each protective antigen to adsorbent respectively with respect to various diseases; and
The combination step of mixing each above protective antigen adsorbed to the adsorbent.
And there is also provided the method wherein the protective antigen is the antigen selected from the group comprising diphtheria antigen, tetanus antigen, pertussis antigen, heptatitis B antigen, or two or more combination thereof.
And there is also provided the method, wherein the adsorbent is aluminum hydroxide gel.
And, in order to achieve the above object, there is provided a combined vaccine as produced according to any of the above methods.
More specifically, the present invention provides the manufacturing method of a combined vaccine which includes the steps of independently adsorbing each protective antigen to an adsorbent of a aluminum hydroxide gel with respect to various diseases such as diphtheria, tetanus, pertussis, and hepatitis B which should be prevented in the infants, and combining each protective antigen adsorbed to the adsorbent after the adsorption.
The antigens of the present invention is not limited by the mentioned diseases but applicated to various antigens from various diseases.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will become better understood with reference to the accompanying drawings which are given only by way of illustration and thus does not limit to the present invention, wherein;
Fig. 1 is a view illustrating an adsorption ratio of each component antigen based on concentration of an adsortDent. Diphtheria toxoid, tetanus

toxoid, and hepatitis B virus surface antigen are a components of diphtheria antigen, tetanus antigen and hepatitis B antigen, and pertussis toxoid and pertussis FHA antigen(pertussis thready shape blood agglutinin corpuscle) are each component of a purified pertussis antigen;
Figs. 2a to 2d are views illustrating an antigenicity and immunity based on an adsorption method wherein sample 1 is a sample in which surplus aluminum hydroxide gel is added after a combination of each component vaccine is completed, and sample 2 is a sample in which the said adsort^ent of the same concentration is previously added before the combination is completed; Figs 2a and 2b are views illustrating the relative antigenicity of samples 1 and 2 respectively and, Figs 2c and 2d are views illustrating the relative level of antibody formation against each antigen of samples 1 and 2, respectively.
Fig. 3 is a view illustrating the level of antibody formation against each antigen in the serum obtained from monkeys administered a combined vaccine prepared according to the present invention or conccurently each vaccine product of DTP and hepatitis B based on the date of collecting blood samples from each monkey. Group 1 is designated that each vaccine of DTP and hepatitis B are concurrently administered, end group 2 is designated that a combined vaccine is administered.
MODES FOR CARRYING OUT THE PREFERRED EMBODIMENTS
As the method for manufacturing the combined vaccine containing each vaccine component as one product, the above method is divided into two steps: a method for the step of combining the antigens which form each component vaccine and perfonning an adsorption using an adsorbent and a method for the step of combining the previously adsorbed antigens, in the case that an adsorbent is used as a component in the said method. However, in the case that each component antigen are first mixed, an adsorption ability with respect to the adsorbent of each antigen may decrease due to the interaction

among antigens and ingredients in solution. In addition, the adsorption between the adsorbent and component antigen is optimized under each independent condition. If the above adsorption processes are concurrently performed, the adsorption ratio may be decreased due to a difference in the adsorption condition of each component antigen. Since the interrelationship between the adsorption ratio with respect to the adsorbent of the component antigen and the immunity of the vaccine is in inverse proportion, the immunity of the combined vaccine, which is manufactured in the process performed concurrently, may be decreased compared to the immunity of each monovalent vaccine.
Therefore, the inventors of the present invention have judged that it is proper to manufacture a combined vaccine by prepahng a bulk solution of each component antigen, independently performing an adsorption of each antigen and combining the adsorbed antigens together.
In addition, in the case that each component antigen is combined in each adsorption type, the final concentration of each component antigen in the combined vaccine should be the same as the concentration of the component antigen in each conventional monovalent vaccine, to compare the immunity and the characteristics of vaccine antigens in vaccine products. Therefore, to manufacture the combined vaccine, the component antigen more concentrated than a single vaccine is adsorbed and then combined. The DTP vaccine is manufactured based on an adsorption using the antigen which is concentrated 1.5-^2 limes, and the hepatitis B vaccine is manufactured using the antigen which is concentrated 2^-3 times. The final concentration of each component antigen of the combined vaccine according to the present invention is coincided with the concentration of each single vaccine adjusting the combining ratio of each antigen.
According to the present invention, the inventors performed the various studies of combinations for developing the combined vaccine. In this case, the final content of each immune component in a vaccine product is the same as

the content of the monovalent vaccine. In addition, the change of the adsorption ratio of each antigen is monitored by controlling the contents of the adsorbent and other components. Each sample which has a less change in the adsorption ratio is immunized into small animals to compare the level of the antibody formation. A proper combining method was selected by reviewing the result of antibody fomnation in small animals.
In addition, as a result of the review with respect to the adsorbents of the currently commercial DTP vaccine and the hepatitis B vaccine, aluminum hydroxide gel; and aluminum phosphate gel were mainly used. It is known that the adsorption of the protein to each aluminum gel is caused by a surface electric charge of each protein and a gel component. And it is also known that the aluminum hydroxide gel has a positive surface electric charge in a physiological pH range, and aluminum phosphate gel has a negative surface electric charge ("Vaccine Design -The subunit and adjuvant approach", ed. By M.F. Powell & M.J. Newman, 1995, p229-239). In the case that the above two types of aluminum gels are concurrently used, a certain interaction occurs, so that the size of particle in the solution may be increased, and the adsorption capacity to protein may be decreased. In addition, the surface electric charge of each component antigen of the hepatitis 8 vaccine and combined vaccine has generally a negative surface electric charge in a physiological pH range. Therefore, the aluminum hydroxide gel is pnDper as an adsorbent.
In the conventional art, the component of the adsorbent such as the aluminum hydroxide gel and aluminum phosphate gel was co-used (International Publication No. WO 93/24148) but based on the above reason, the inventors of the present invention judged that the adsorbent of the combined vaccine should have the same salt type and the aluminum hydroxide gel which is used as an adsorbent is an important factor for obtaining a certain adsorption ratio of each component antigen.
But it is evident that the adsorbent covers not only the aluminum hydroxide gel but also its equivalents in the present invention.

In addition, in the combined vaccine prepared by the present invention, other component antigens except hepatitis B antigen include proteins as a main component, while, in the case of the hepatitis B vaccine, the antigen exists generally in a particle type which includes a phospholipid layer. Therefore, it is preferred that when manufacturing the hepatitis B vaccine for a combined vaccine prepared by the present invention, in order to decrease a certain interference by other antigens with respect to the phospholipid component of the hepatitis B vaccine antigen which includes a phospholipid layer, a neutral surfactant such as Polysorbate 20 (Tween 20). Polysorbate 80 (Tween 80) and Triton X-100 are added.
Since the titer of the hepatitis B vaccine tends to decrease in the case that the amount of the neutral surfactant is high, it is preferred that the neutral surfactant is added at the mass ratio below 50% with respect to protein amount of the hepatitis B surface antigen but the above amount is not limited thereto.
In the present invention, Corynebacterium diphtheria, PW No. 8 as an antigen of the diphtheria is prepared and cultivated in a proper culture medium. The diphtheria toxoid is preferred, which is obtained by detoxifying the ■ diphtheria toxin which is purified by a conventional method. The amount of the antigen is preferably 10--25 Lf based on the pediatric dose. As a tetanus antigen, Clostridium tetanii. Harvard, is cultivated in a proper culture medium under anaerobic condition. The tetanus toxoid which is obtained by detoxifying the tetanus toxin purified by the conventional method is proper. The amount of the antigen is preferably I'-SLf based on the pediatric dose. In addition, the Pertussis antigen is cultivated in a proper culture medium using Bordetella pertussis, Tohama phase I, and multiple kinds of antigen protein including a Pertussis toxoid are purified by a conventional method in a culture supernatant and detoxified. Purified Pertussis antigens or a whole cell Pertussis antigen is proper. Here, the manufacturing method of a combined vaccine is provided in which in the case of the multiple kinds of antigens, the total amount of the antigens is below 20|igPN based on the pediatric dose, and in the case of the

whole cell Pertussis antigen, the amount of the antigen is preferably below 20 OE based on the pediatric dose. The present invention is further directed to a method for manufacturing a combined vaccine in which purified multiple antigen proteins include a detoxified Pertussis toxoid and filamentous hemagglutinin (FHA) antigen.
Preferably, the present invention is directed to a method for manufacturing a combined vaccine in such a manner that the amount of recombinant hepatitis B virus surface antigen, which manufactured by a genetic engineering method as a hepatitis B antigen, is 5-1 Oug based on the pediathc dose. More preferably, the present invention is directed to a method for manufacturing in such a manner that the hepatitis B surface antigen is adsorbed to an adsorbent by mixing with stirring at 2-8°C for 3-^20 hours. In order to find the optimum content of adsortDant for manufacturing of the combined vaccine, the adsorption ratio for each antigen component was analyzed at each concentration of adsortDant. As a result, when the final aluminum ion concentration is below 0.5mg/ml at the time when the final combination is completed, the adsorption ratio of each antigen was relatively decreased (as shown in Fig. 1). In addition, the aluminum ion concentration is stipulated to be under 1.25mg/ml in the aluminum gel used for the vaccine by regulation (WHO, "Requirements for diphtheria, tetanus, pertussis and combined vaccines" in Technical Report Series No. 800, 1990, p87-179). Therefore, as the aluminum hydroxide gel, the concentration of the final aluminum ion is preferably in the range of 0.5'-1.25mg/ml and more preferably in the range of 0.7mg/ml.
In addition, in order to prevent a possibility that the absorption ratio of each component antigen is decreased by a repulsive -force caused among each component antigenr even after the combination of each component is completed, the antigenicity and the level of antibody formation of each component antigen are converted to the relative percentage to compare those values between two samples: a sample that the other component except an

aluminum hydroxide gel is previously combined and then suplus aluminum hydroxide gel is additionally added and a sample that the adsorbent of the same concentration is previously added before the combination is completed. The change of the antigenicity and the level of antibody formation decreases when the surplus adsorbant is added even after the combination of each component is completed (as shown in Fig. 2).
In addition, in the case of the sample in which the polysorbate SO 80 (tween 80) is added, the antigenicity and titer with respect to the hepatitis B were maintained (as shown in Fig. 2). It is expected that a similar result i© may be obtained even when neutral surfactants such as polysorbate 20 and triton X-100 are used whithin proper concentration. As shown in Fig. 2. the sample 1 and sample 2 are prepared in the way of that surplus aluminum hydroxide gel is additionally added after the combination of each component is completed, and that the adsorbent of the same concentration is previously added before the combination is completedr, respectively.
Preferably, the present invention is directed to a method for manufacturing a combined vaccine in which the aluminum ion concentration of the.aluminum hydroxide gel is in the range of 0.5'-1.25mg/ml. More preferably, the present invention is directed to a method for manufacturing a combined vaccine which includes a step of adding surplus adsorbent in the range that does not exceed the concentration range of the aluminum ion of 0,5-1.25 mg/ml after protective antigens adsorbed to the adsorbent are combined. In a preferred embodiment of the present invention, it is possible to maintain an inherent adsorption of each component antigen in the combined vaccine and to increase an immunity.
As the Pertussis antigen, a purified Pertussis antigen is generally used in Korea and several other countries. Since some American countries and the third world countries use the-a whole cell Pertussis antigen which is detoxified. Therefore, in the present invention, the applicable range of the combined vaccine is widened by the study on the combining method using whole cell

pertussis antigen. In this case, the combining was performed in the same method as using the mentioned purified Pertussis antigen for thereby obtaining a sample capable of maintaining an immunity of each component(as shown in Table 1).
In the case of the DTP vaccine which includes a whole cell Pertussis antigen, a aluminum phosphate gel is generally used as an adsorbent. However, in order to manufacture a combined vaccine with respect to the hepatitis B antigen, the DTP vaccine was manufactured using the aluminum hydroxide gel. In this case, the immunity with respect to the Pertussis was decreased, but when a surplus aluminum hydroxide gel was additionally added after the combination of each component vaccine was completed (sample A), it was possible to maintain a desired immunity. As a reference, the sample B is prepared in the way of that the adsorbent of the same concentration was previously added before the combination was completed.
The safety and efficacy of the combined vaccine manufactured according to the present invention was proved based on the following methods. First, overdosage of combined vaccine was administrated to a rodent, and it was checked that a lesion was not observed as a result of the biopsy (The result of the same is not provided). In addition, the antibody level against each antigen was measured in a group (group 2) in which a combined vaccine was administrated to a monkey and in a group (group 1) in which each vaccine of DTP and hepatitis B was concurrently administrated. As a result of the t-test with respect to the antibody amount between two groups, when comparing the result of the group 2 with the result of the group 1, it was evaluated that the combined vaccine showed the equal or better immunogenecity against each antigen to concurrently injection group of each vaccine (as shown in Fig. 3. The result of the statistic is not provided).
In conclusion, the combining method was developed in the present invention, which an interaction among different immune components was minimized, and each immunogenic effect did not decrease. When dividing each

immune component used for the combined vaccine based on physical and chemical properties, the immunogenic components may be divided into a protein antigen (diphtheria antigen, tetanus antigen, purified Pertussis antigen), an antigen composed of protein and phospholipid layer (hepatitis B antigen), and an antigen composed of killed whole cell (a whole cell Pertussis antigen), etc. Therefore, it may be predicted that a combination with other antigen of single vaccine may be implemented based on the physical and chemical characteristics of components. A combination of an additional vaccine may be possible with respect to the diseases (hemophilus influenza, polio), which should be prevented in the infants by the combined vaccines prepared according to the present invention.
The body vaccination period of the combined vaccine agent according to the present invention may be the previous vaccination periods of the DTP vaccine or the hepatitis B vaccine. In the case that there is the hepatitis B antigen in the mother's body, it is preferred to inoculate three times of second, fourth and sixth months. In the case that there is not the hepatitis B in the mother's body, as a proper vaccination period, the hepatitis B vaccine is singularly inoculated after birth, and then the additional vaccination is performed using a combined vaccine. A proper vaccination with respect to the infants can be perfonned by a muscle or hypodennic injection method. The doze of the antigen of diphtheria, tetanus, Pertussis and hepatitis B respectively in the combined vaccine according to the present invention is the same as the doze for the infant of the antigen of each single vaccine.
Example 1
Manufacture of hepatitis B antigen
The yeast cell which is capable of expressing the hepatitis B surface antigen by genetic engineering method is intensively cultivated. 1kg of the precipitate of yeast cells which is obtained by the centrifugation is diluted at a ratio of 1:2 in a buffer solution (0.5M NaCI, lOmM EDTA, 0.01% Thimerosal,

0.1M Phosphate. pH 7.0). The diluted solution flows through a glass bead beator (or Dynomil) for thereby breaking cell walls and so on. The obtained solution is added with a neutral surfactant (Tween group or Triton group) by 0.5% and is unifonmly nnixed by stirring at 4°C. Sodium hydroxide was added to the resultant solution for thereby obtaining pH 11 and is then mixed by stirring at 4°C for 5 hours. A diluted hydrochloric acid was added to the resultant solution for thereby obtaining pH 4. The precipitate was removed by a centrifugation at 6000rpm for 15 minutes using the centrifugal machine (ROTOR: JA-14, Beckman Inc. USA), and the upper solution Including the hepatitis B surface antigen was obtained. The pH of the upper solution was made at 7, and then silica was added thereto and was mixed at 4-25°C for 3^-16 hours, so that the hepatitis B surface antigen was adsorbed to the silica. The silica gel which is preferably used in the present invention is Aerosil 380(Degussa, USA) which includes fine hydration silica or anhydrous silica having a valid surface area of 100-"500mm^/g. In order to remove contaminants from the silica to which the hepatitis B surface antigen is adsorbed, the resultant solution was washed two times using sodium phosphate - sodium chloride buffer solution of pH 7. The washed silica was contacted in the sodium carbonate buffer solution of pH 9.6 for about 2 hours for thereby desorption of a surface antigen. The thus separated surface antigen had a protein purity of above 90% in the solution. The resultant solution^ was flown in the DEAE-Sepharose (PHAMACIA, Sweden) which was balanced using the said sodium buffer solution for thereby specifically attaching a surface antigen and removing the substances separated in the column using the said buffer solution. Contaminants slightly attached on the column was eluted using the buffer solution including sodium chloride of 0.05-0.1 M. Thereafter, the hepatitis B surface antigen was eluted using the said buffer solution including the sodium chloride of 0.2M. The thus eluted solution was concentrated using a ultrafiltration membrane which is capable of separating the substance of molecular weight above than 100,000. and the precipitate was separated by

centrifugation and removed, and the upper solution was collected, and the gel permeation chromatography (Sepharose CL-4B, PHAMACIA, Sweden) was perfomied with respect to the collected upper solution. The fractions which included a pure surface antigen were confirmed by electrophorsis and were used as the hepatitis B antigen. In addition, in order to decrease an interaction with each of the hepatitis surface antigen, the tween 80 was added to 0, 5, 10|ig per 1ml, and then the effect of tween 80 was observed (as shown in Fig.
2).
Example 2
Manufacture of diphtheria toxoid
The corynebacterium diphtheria PW No. 8 was cultured at 35°C for 24 hours in the nutrition agar culture medium (DIFCO. USA) and was subcultured two times. One of the colony was cultured at 35°C for 24 hours in 2ml of the brain heart infusion culture medium (DIFCO, USA), and 1.2ml of it was inoculated to the modified Muller culture medium (refer to: Stainer, et. al, Canadian J, Microbiol.. 14:155, 1968) of 300ml and was cultured at 35°C for 36 hours. After the culture, cells were removed from the culturing solution, and the toxin solution was collected and it was added with ammonium sulfate at 4°C. The final concentration was made in 25(w/v)%. and pH was made in 8.0. The culturing solution having adjusted pH and concentration' of salt was dropped into the phenyl-sepharose column which was previously balanced using lOmM tris buffer solution pH 8.0 including 25(w/v)% ammonium sulfate. The buffer solution same as the column balance solution and lOmM tris buffer solution(pH 8.0) including 15(w/v)% ammonium sulfate were sequentially flown to the column for thereby removing impurities. The toxin was eluted using 10mM tris buffer solution(pH 8.0) which did not include salt. The eluted diphtheria toxin solution was collected and was dialyzed to lOmM phosphate buffer solution(pH 7.4) including sodium chloride of 150mM. After the dialyzing process was completed, fonnalin was added, so that the final concentration was 0.05(w/v)%,

and the resultant solution was reacted at 37°C for 1 hour, and lysine was added for thereby obtaining the final concentration of 0.05M and then, the resultant solution was detoxified at 37°C for 4 weeks. Thejoxoid solution was dialyzed into 10mM phosphate buffer solution(pH 7.4) with sodium chloride for thereby fully removing the fomnalin, and thimerosal was added to have 0.01(w/v)% of the final concentration. The resultant solution was used as diphtheria toxoid solution.
Example 3
Manufacture of tetanus toxoid
The Clostridium tetanii (Harvard strain) was cultured by molten agar method with sterilized liver-bile agar medium (DIFCO, USA). A few colonies from above culture were inoculated into brain heart infusion culture medium (DIFCO, USA) including 0.3(w/v)% yeast extraction (DIFCO, USA) of 2ml having decreased oxygen level and were cultured at 35°C for 24 hours in anaerobic state. And then, the culture solution was inoculated into fully nitrogen-satured brain heart infusion culture medium (DIFCO, USA) 500ml including 0.3(w/v)% yeast extraction (DIFCO, USA) and were cultured at 35°C for 7 days in anaerobic state. After the culture, cells were removed from the culture solution, the toxin solution was collected and added with ammonium sulfate at 4°C for thereby obtaining the final concentration of 60(w/v)% and was mixed for over 24 hours and was fully mixed for thereby obtaining a precipitate by centrifugation. The precipitate was dissolved in a small amount of distilled water, and the insoluble material was removed. The resultant solution was dropped into the sephagrill S-IOO column which was previously balanced using 10mM tris buffer solution(pH 8.0) including 0.5M sodium chloride. The same buffer solution was flown for thereby eluting the separated toxin. The tetanus toxin solution was collected and dialyzed into 10mM phosphate buffer solutJon(pH 7.4) including 150mM sodium chloride. After the dialysis, formalin was added to have the final concentration of 0.025(w/v)% and was reacted at

37°C for 1 hour. Thereafter, lysine and sodium hydrogencarbonate were added to have the final concentration of O.OSmM and 0.04M, respectively, and was matured at 37°C for 4 weeks and was detoxified. After the detoxification, the toxoid solution was dialyzed into 10mM phosphate buffer solution (pH 7,4) including 150mM sodium chloride, and formalin was fully removed. Thimerosal was added to have the final concentration of 0.01(w/v)% and was used as tetanus toxoid solution.
Example 4
Manufacture of purified Pertussis antigen protein
Bordetella pertussis, Tohama phase I was cultured in Bodet-Gengo agar culture medium(DIFCO, USA) including 15% rabbit blood at SS^C for 72 hours and was passaged two times. A few colonies were inoculated into 50ml of stanor-sholte culture medium and was cultured at 35°C for 48 hours. The thusly cultured solution were re-inoculated in 500ml of changed stanor-sholte culture medium(refer to Imazumi et a!., INFECT.-IMMUN.. 1983, vol 41, pp. 1138) and was cultured at 35*^0 for 36 hours. 10% thimerosal aqueous solution was added to the cultured solution to have the final concentration of 0.01% for thereby preventing the growth of the Pertussis bacteria, and then the cells were removed by centhfugation. Three times volume of distilled water was added to the resultant solution, and was mixed well. The current fDH was decreased to pH 6.0 using IN sulfuric acid. The resultant solution was dropped Into CM-sepharose column which was previously balanced using lOmM sodium phosphate buffer of pH 6.0. The buffer solution same as the column balance solution was flown to the column for thereby removing the substances which were not coupled to the column, and the impurities which were slightly coupled to the column were removed using the buffer solution same as the column balance solution including lOOmM sodium chloride. When the impurities were not eluted anymore through the column, the bound materials were eluted with linear gradient formed by the column balance solution including lOOmM sodium

chloride and 600mM sodium chloride which have the same volume, so that a fraction including Pertussis antigen protein such as Pertussis toxin and FHA (filamentous hemagglutinin) was separated. The antigen fraction was diluted using lOmM sodium phosphate buffer solution(pH 8.0) of two times volume to have pH 8.0. The resultant solution was dropped into hydroxy apatite column which was previously balanced using lOmM sodium phosphate buffer solution of pH 8.0. The buffer solution same as the column balance solution including lOOmM sodium chloride was flown to the column for thereby removing the substances which were not coupled to the column. 80mM sodium phosphate buffer solution of pH 8.0 including lOOmM sodium chloride was flown at the same rate as the dropping rate for thereby separating Pertussis toxin fraction. When the Pertussis toxin was separated, 250mM sodium phosphate buffer solution of pH 8.0 including lOOmM sodium chloride was flown at the same rate as the dropping rate for thereby separating FHA(filamentous hemagglutinin) fraction. Separated Pertussis toxin and FHA(filamentous hemagglutinin) were dialyzed in lOmM sodium phosphate buffer solution(pH 7.4) including 0.15% soaium chloride at 4°C. Glycerol and glutal aldehyde were added to dialyzed Pertussis toxin sample solution to have the final concentration of 50(w/v)% and 0.05(w/v)% and were detoxified at 37°C for 4 hours. Sodium aspartate was added to have the final concentration of 0.025M for thereby completing the detoxifying process. Glycerol and formalin were added fnto the dialyzed FHA (filamentous hemagglutinin) sample solution to have 50(w/v)% and 0.025^w/v)% and were detoxified at 37^C for 24 hours. Lysine was added to have the final concentration of 0.025M, so that the detoxifying process was completed. Each sample solution was dialyzed into 10mM sodium phosphate buffer solution (pH 7.4) including 0.15% sodium chloride at room temperature. Thimerosal was added to have the final concentration of 0.01(w/v)%. Thereafter, detoxified Pertussis toxin sample solution and FHA (filamentous hemagglutinin) sample solution were mixed at a ratio of 1:4 and were used as a Pertussis antigen protein.

Example 5
Manufacture of a whole cell Pertussis antigen
Pertussis bacteria. Bordetella pertussis, Tohama phase I was cultured in Bodet-Gengo agar culture medium(DIFCO, USA) including 15% rabbit blood at SS^'C for 72 hours and was passaged two times. Some of the colonies were inoculated into 50ml of Stanor-sholte culture medium and were cultured at 35°C for 48 hours. The resultant solution was re-inoculated in the changed stanor-sholte culture medium(refer to Imazumi et al.. INFECT.-IMMUN., 1983, vol 41, pp. 1138) of 500ml and were cultured at 35°C for 36 hours. 10% thimerosal aqueous solution was added into the culture solution to have the final concentration of 0.01% for thereby preventing the growth of the Pertussis bacteria and collecting somatic based on the centrifugation. The collected somatic solution was dialyzed in lOmM sodium phosphate buffer solution(pH 7.4) including 0.15% sodium chloride at 4°C. Fonnalin was added into the dialyzed Pertussis somatic solution to have the final concentration of 0.025(w/v)% and was detoxified at 37^C for 4 weeks. Lysine was added to have the final concentration of 0.025M for thereby completing the detoxifying process. The sample solution was dialyzed into lOmM sodium phosphate buffer solution(pH 7.4) including sodium chloride of 0.15%, and thimerosal was added to have the final concentration of 0.01(w/v)% and was used as the whole cell Pertussis antigen.
Example 6
Manufacture of combined vaccine including purified Pertussis antigen
Step 1. Manufacture of hepatitis B vaccine
The hepatitis B vaccine was manufactured using the hepatitis B surface ' antigen manufactured in Example 1. aluminum hydroxide solution was manufactured by adding aluminum hydroxide gel to the phosphate buffer solution. The resultant solution was slowly mixed, and the above antigen

solution was dropped and was mixed by stirring slowly at 4^C for 15 hours for thereby manufacturing hepatitis B vaccine. At this time, the amount of the hepatitis surface antigen was 60|ig/ml which was high concentrated three times compared to the amount of the common hepatitis B surface antigen. In the case of the first sample, the amount of the aluminum ion in the aluminum hydroxide gel was 0.9mg/ml. In the case of the second sample, the amount of the aluminum ion in the aluminum hydroxide gel was 1.5mg/ml(as shown in Fig.
2).
Step 2. Manufacture of DTP combined vaccine
The DTP combined vaccine was manufactured using each antigen manufactured in the second, third and fourth examples. The aluminum hydroxide solution was manufactured by adding aluminum hydroxide gel to the phosphate buffer solution in the conventional method. As the resultant solution was slowly mixed, each antigen solution was dropped thereto and was mixed by stirring slowly for thereby implementing a uniform adsorption, so that the DTP combined vaccine was manufactured. At this time, the amount of each antigen was concentrated 1.5 times high compared to the amount of each component antigen of common DTP vaccine. The amount of the aluminum ion in the aluminum hydroxide gel was 0.3mg/ml.
Step 3. Manufacture of DTP-hepatitis B combined vaccine Each DTP vaccine and hepatitis B vaccine manufactured in Steps 1 and 2 were slowly mixed at the volume ratio of 2:1. In the case of the first sample, surplus aluminum hydroxide gel was added so that the amount of the aluminum ion in the aluminum hydroxide gel was finally 0.7mg/ml, and was continuously mixed by stirring at 25°C for 1 hour for thereby manufacturing a stable combined vaccine which does not interact with other components. In the case of the second sample, surplus aluminum hydroxide gel was not added and was continuously mixed by stirring at 25°C for 1 hour for thereby

manufacturing combined vaccine. The amounts of the aluminum ions in the aluminum hydroxide gel of the combined vaccines of the first and second samples were 0.7mg/ml. For the test of antibody titer, the composition of the combined vaccine was 20|ig of the hepatitis B surface antigen, 25Lf of diphtheria toxoid, 3Lf of tetanus toxoid, 2.5^gPN of Pertussis toxoid, and lOi^gPN of Pertussis FHA antigen(Pertussis FHA(filamentous hemagglutinin) antigen based on 1ml(as shown in Fig. 2).
Example 7
Manufacture of combined vaccine including a whole cell Pertussis antigen
Step 1. Manufacture of hepatitis B vaccine
The hepatitis B vaccine was manufactured using the hepatitis B surface antigen manufactured in Example 1. An aluminum hydroxide gel was added to phosphate buffer solution for thereby manufacturing the aluminum hydroxide solution. As the solution was slowly mixed, the above antigen solution was dropped and was fully mixed at 4°C for 15 hours for thereby manufacturing the hepatitis B vaccine. At this time, the amount of the hepatitis B surface antigen was 60^g/ml which was concentrated three times high compared to the amount of the common hepatitis B vaccine. In the case of the first sample, the amount of the aluminum ion was 0.9mg/ml in the aluminum hydroxide gel. In the case of the second sample, the amount of the aluminum ion was 1.5mg/ml in the aluminum hydroxide gel(as shown in Fig. 2).
Step 2. Manufacture of DTP combined vaccine
The DTP combined vaccine was manufactured using each antigen manufactured in the Examples 2, 3 and 5. The aluminum hydroxide gel was added to the phosphate buffer solution in the conventional method for thereby manufacturing an aluminum hydroxide solution. As the resultant solution was slowly mixed, the above antigen solution was dropped and was fully mixed. A

uniform adsorption was implemented for thereby manufacturing a DTP combined vaccine. At this time, the amount of each antigen was concentrated 1.5 times high compared to the amount of each component antigen of the common DTP vaccine. The amount of the aluminum ion was 0.3mg/ml in the aluminum hydroxide gel.
Step 3. Manufacture of DTP-hepatitis B combined vaccine Each DTP vaccine and hepatitis B vaccine manufactured in Steps 1 and 2 were slowly mixed at a volume ratio of 2:1. In the case of the first sample, a surplus aluminum hydroxide gel was added so that the amount of the aluminum ion was finally 0.7mg/ml in the aluminum hydroxide gel, and the resultant solution was continuously mixed at 25°C for 1 hour for thereby manufacturing a stable combined vaccine which does not interact with other components In the case of the second sample, surplus aluminum hydroxide gel was not added, and the solution was continuously mixed at 25'C for 1 hour for thereby manufacturing a combined vaccine. The amounts of the aluminum ions in the aluminum hydroxide gel of the combined vaccines of the samples 1 and 2 were 0.7mg/ml. For the test of antibody titer, the composition of the combined vaccine was 20/ig of the hepatitis B surface antigen, 50Lf of
diphtheria toxoid, lOLf of tetanus toxoid, and 20OE of a whole cell Pertussis antigen based on 1ml(as shown in Fig. 2).
Example 8, Test of antigenicity in combined vaccine
The test of antigenicity in combined vaccine manufactured by the examples 6 and 7 was performed by the Enzyme Immune Assay (EIA) using monoclonal antibody and polyclonal antibody by comparison to the standard of . each antigen with respect to each antigen content.
In addition, the antigen adsort^ed to the aluminum salt was removed by the centrifugation for measuring the adsorption ratio of each antigen, and the amount of the separated antigen was measured using the upper solution.

The average of each result were shown in Figs. 1 and 2 shows relative antigenicity of each antigen.
Example 9. Test of antibody formation by combined vaccine
Test of antibody formation by hepatitis B surface antigen The combined vaccine and hepatitis B vaccine manufactured in Examples 6 and 7 were inoculated to ICR mice in which 16 mice were grouped as the group 1, and a blood was collected from the ICR mice after 18 days. Serum was separated from the blood. The antibody level against the hepatitis B surface antigen was obtained based on the unit of lU/ml using the EIA(Enzyme Immune Assay) and calculated as in geomean antibody titer. The titer of the combined vaccine was computed with respect to the hepatitis B vaccine.
Fig. 2c and 2d shows relative antibody titer of combined vaccine with repect to each antigen and Table 1 shows the values of each titer.
Test of antibody formation by diphtheria antigen
The combined vaccine and DTP vaccine manufactured in Examples 6 and 7 were inoculated to the ICR mice in which 16 mice are grouped as the group 1. The blood was collected from the CIR mice after 28 days. Serum was separated from the bicod. The antibody level against the diphtheria antigen of the serum was measured using the EIA(Enzyme Immune Assay) based on the unit of lU/ml and calculated as in geomean antibody titer. Thereafter, the titer of the combined vaccine was measured compared to the DTP vaccine.
Fig. 2c and 2d shows relative antibody titer of combined vaccine with repect to each antige, and Table 1 shows the values of each titer.
Test of antibody formation by tetanus antigen
The combined vaccine and DTP vaccine manufactured in Examples 6 and 7 were inoculated to the ICR mice in which 16 mice are grouped as the

group 1. The blood was collected from the ICR mice after 28 days. Serum was separated from the blood. The antibody level against the tetanus antigen of the serum was measured using the EIA(En2yme Immuno Assay) based on the unit of lU/ml and calculated as in geomean antibody titer. Thereafter, the titer of the combined vaccine was measured compared to the DTP vaccine.
Fig. 2c and 2d shows relative antibody titer of combined vaccine with repect to each antigen, and Table 1 shows the values of each titer.
Test of antibody formation by Pertussis antigen
The combined vaccine and DTP vaccine manufactured in Examples 6 and 7 were inoculated to the ICR mice in which 16 mice are grouped as the group 1. The blood was collected from the ICR mice after 28 days. Serum was separated from the blood. The antibody level against pertussis antigen of the serum was measured using the EIA(Enzyme Immuno Assay) based on the unit of lU/ml and as in geomean antibody titer Thereafter, the titer of the combined vaccine was measured compared to the DTP vaccine.
Fig. 2c and 2d shows relative antibody titer of com.bined vaccine with repect to each antigen, and Table 1 shows the values of each titer.

Example 10.
Comparative effectiveness test with monkey
A test was performed for proving the immunogenicity in a primate of the combined vaccine and comparing with the conventional single vaccine. The combined vaccine manufactured in Example 6 was vaccinated to six monkeys (group 2) formed of the same numbers of male and female monkeys, and each component vaccine was vaccinated to 6 monkeys(group 1) formed of the same numbers of male and female monkeys simultaneously. The same products as the products vaccinated to each group was re-vaccinated 30^^ and 60*^ days. The blood was collected from each experimental animal on 1^\ 29^^, 58*^, and 86*^ days from the initial vaccination date, and the antibody titer against each disease was measured based on the ELISA method proper to the serum of the monkey.
Fig. 3 shows the geomean antibody titer against each antigen of each group.

INDUSTRIAL APPLICABILITY
According to the present invention, it is directed to a method for manufacturing a combined vaccine for concurrently preventing the diseases such as diphtheria, tetanus, pertussis, and hepatitis B, etc. which should be prevented in an infant. By the present invention, it is possible to concurrently prevent the diseases such as diphtheria, tetanus, pertussis, and hepatitis B which should be prevented in an infant using the combined vaccine according to the present invention.
As the present invention may be embodied in several forms- without departing from the spirit or essential characteristics thereof, it should also be understood that the above-described embodiments are not limited by any of the details of the foregoing description, unless otherwise specified, but rather should be constaied broadly within its spirit and scope as defined in the appended claims, and therefore all changes and modifications that fall within the meets and bounds of the claims, or equivalences of such meets and bounds are therefore intended to be embraced by the appended claims.

CLAIMS
1. A manufacturing method of a combined vaccine, comprising the steps
of;
adsorption step of independently adsorbing each protective antigen to adsorbent respectively with respect to various diseases; and
combination step of mixing each mentioned protective antigen adsorbed to the adsorbent.
2. The method of claim 1. wherein the protective antigen is the antigen
selected from the group comprising diphtheria antigen, tetanus antigen,
pertussis antigen, heptatitis B antigen, or two or more combination thereof.
3. The method of claim 1, wherein the adsorbent is aluminum hydroxide gel.
4. The method of claim 3, wherein aluminum ion concentration of the aluminum hydroxide gel after the manufacture of a combined vaccine is in the range of O.S-'1.25mg/ml.
5. The method of either claim 3 or 4, further comprising a step of:
adding the adsorbent in which the final aluminum ion concentration does not exceed in the range of 0.5 -1.25mg/ml in aluminum hydroxide gel.
6. The method of claim 2, wherein a recombinant hepatitis B surface antigen as the hepatitis B antigen is combined by the amount of 5-10|ig based on the pediatric dose.
7. The method of claim 6, further comprising a step of:
adding a neutral surfactant such as polysorbate 20, polysorbate 80 and tritonX-100.

8. The method of claim 6, wherein said hepatitis B antigen is mixed by stirring with an adsorbent at 2-8^C for 3-'20 hours and is matured and adsorbed.
9. The method of claim 2, wherein the diphtheria toxoid detoxified as a diphtheria antigen is combined by the amount of 10-25Lf based on the pediatric dose.
10. The method of claim 2. wherein the tetanus toxoid detoxified as a tetanus antigen is combined by the amount of 1--5Lf based on the pediatric dose.
11. The method of claim 2, wherein the pertussis antigen including the purified antigens is combined by the amount below 20^gPN based on the pediatric dose or the whole cell pertussis antigen is combined by the amount below 20OE based on the pediatric dose.
12. The method of claim 11, wherein mentioned purified pertussis antigens include pertussis toxoid and pertussis FHA (filamentous hemagglutinin).
13. A combined vaccine as produced according to any of the methods
claimed in Claims 1 to 12.

A manufacturing method of a combined vaccine substantially as herein described with reference to the accompanying drawings. A combined vaccine substantially as herein described with reference to the accompanying drawings.

Documents:

1056-chenp-2003-abstract.pdf

1056-chenp-2003-claims duplicate.pdf

1056-chenp-2003-claims original.pdf

1056-chenp-2003-correspondnece-others.pdf

1056-chenp-2003-correspondnece-po.pdf

1056-chenp-2003-description(complete) duplicate.pdf

1056-chenp-2003-description(complete) original.pdf

1056-chenp-2003-drawings.pdf

1056-chenp-2003-form 1.pdf

1056-chenp-2003-form 19.pdf

1056-chenp-2003-form 26.pdf

1056-chenp-2003-form 3.pdf

1056-chenp-2003-form 5.pdf

1056-chenp-2003-pct.pdf

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Patent Number

201519

Indian Patent Application Number

1056/CHENP/2003

PG Journal Number

08/2007

Publication Date

23-Feb-2007

Grant Date

26-Jul-2006

Date of Filing

08-Jul-2003

Name of Patentee

M/S. LG LIFE SCIENCES, LTD

Applicant Address

20, Yeoido-dong, Youngdeungpo-ku, 150-721 Seoul

Inventors:

#	Inventor's Name	Inventor's Address
1	KIM, Kyu-wan	8-110, LG Dormitory, Doryong-Dong, Yuseong-Gu, 305-340 Daejeon
2	JI, Hyi-jeong	6-302, LG Dormitory, Doryong-Dong, Yuseong-Gu, 305-340 Daejeon
3	LEE, Youn-kyeong	202-1705, Saemmeori Apt., Dunsan 2-Dong, Seo-Gu, 302-777 Daejeon
4	KIM, Wan-kyu	106-401, Chung-gu Apt., Jeonmin-Dong, Yuseong-Gu, 305-729 Da
5	LEE, Hee-ku	305-403, Songganggreen Apt., Songgang-Dong Yuseong-Gu, 305-751 Daejeon
6	KIM, Won-kyum	109-709, Nokwon Apt., Dunsan 2-Dong, Seo-Gu, 302-732 Daejeon

PCT International Classification Number

A61K39/29

PCT International Application Number

PCT/KR2002/000034

PCT International Filing date

2002-01-09

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	2001-0001290	2001-01-10	Republic of Korea