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(ll)PatentNumber: KE 380

(45) Dato of grant' 23/03/2010
       
(51) Int.C1.7:   

(73)0wner: A 61K 39/29    LG LIFE SCIENCES LTD of  20, Yeoido-dong, Youngdeungpo-ku, 150-721 Seoul, Rep.ofKorea

(21)Application Number:KE/P/2002/ 000228           

(72) Inventor:KIM, Kyu-wan;; JI, Hyi-jeong;; KIM, Wan-kyu;; LEE,

(22) Filing Date: Youn- kyeong;; LEE, Hee-ku; and KIM, Won-kyurn;09/01/2002           

(74) Agent/address for correspondence:

(30) Priority data:    KINOT1 & KillE CO ADVOCATES, P. 0. BOX 29871- 2001-0001290  10/0112001  KR    00202, NAIROBI

(86) PCT data PCT/KR02/00034 09/01/2002 wo 02/055105 18/07/2002

(54)Tit1e: MANUFACTURING METHOD OF COMBINED VACCINE

(57) 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 infant<>, 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.

MANUFACTURING METHOD OF COMBINED VACCINE

BACKGROUND OF THE INVENTION

TECHNICAL FIELD

The present inventon relates to manufacturtng method of combined

vaccine capable of preventing concurrently various diseases of infants such as diphtherta, tetanus, pertussis, hepatitis 8, and other dis~ases, which should be

prevented in infants.

10    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.

15    As  examples  at the fonmer,  there  are  a  DTP  vaccine  capable  at

enhancing an immunity with  respect to diphtheria, tetanus and  pertussis,  a

MMR vaccine capable of enhancing  an  immunity wtth  respect to measles,

mumps, and gennan measles, and so on. The above vaccines have been used

for 20 years. As examples at the latter, there are a pneumococcal vaccine

20    manufactured by combining 14 or 23 pneumococcal polysaccharides capable of enhancing an immunity to pneumonia, and a meningococcus vaccine manufactured by combining 4 meningococcal polysacchartdes capable of enhancing an immunity to protect meningitis and so on. The above vaccines

have been used for a few years.

2 5    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

30    above combined vaccines, there Is a vaccine (lntemational Publication No. WO
 
99113906) manufactured by combining a DTP vaccine, bacterial encephalomeningitis vaccine (Hib vaccine), inactivated polio vaccine, and hepatitis B veccine. A certain combined vaccine is developed by me 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

1a   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 overlapped

with different vaccines due to convenience or illness. Therefore, it is important

15    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

8 vaccinations are guided to perfonn three times of primary shots within the

ftrst  year  of  infants  and boost shots.   In addition,  since  the  vaccination

20    schedules recommended are similar, the problem of overtaping shots can happen.
The  inventors  of the  present invention  parfonned  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

25    hepatitis 8 vaccines and stab~izing 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 eacn c:omponent antigen from the vaccine products which ls

provided in the  immun~y and  stability,  the development of the  combining

30    method  is  important  to  minimize  a variation  in  the  reaction  and  immunity

occunng due to an interaction between each protective antigen and adSoribent is important for the above research. With the completion of development, the homogenetty and stability of the combined vaccine formulations invented have

been reviewed.  In the present invention,  the researdl has been perfonned

based on the DTP vaccine and hepatitis B vaccine which are proved for their

immunity and stabiltty.

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

10    (International Publication No. WO 99/13906) of administrating simultaneously when the vaccination is performed, using the specially designed container, and a method of manufacturing the combined vaccine by mixing each component

of vaccine and ingredients in one formulatJon. Conceming the vaccine supply,

the usage of the combined vaccine prepared by the former two methods are

15    similar to the use of each monovalent vacdne singularty 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 pertorm a research with respect to the variation Of the immunity

and the  occuring  of the  side  effects.  Therefore,  it is preferable that each

20    vaccine component is mixed in one formulation 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

2 5    prevent in infants.

DISCLOSURE OF THE INVENTION

Accordingly,  it  is  an  object  of  the  present  invention  to  provide

manufacturing method of combined vaccine for concurrenuy preventing various

30    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 anUgen is the antigen selected from the group comprising diphtheria antigen, tetanus

10    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 15 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

20    hepatitis 8 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.

25    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 30 antigen based on concentration of an adsorbent. Diphtheria toxoid, tetanus

toxoid, and hepatitis 8 virus surface antigen are a components of diphtheria antigen, tetanus antigen and hepatitis 8 antigen, and periussis toxoid and pertussis FHA antigen(pertussls thready shape blood agglutinin corpuscle) are each component of a purified pertussis antigen;

Figs. 2a to 2d are views illustrating an antigenicrty and immunrty based on an adsorption method wherein sample 1 is a sample in whidl 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 adsorbent of the same concentrration is previously added before the combination is

10    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 fonmation against each

15 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 8 based on the date of collecting blood samples from each monkey. Group 1 is designated that each vaccine of DTP and hepatitis 8 are concurrently administered, and group 2 is designated that a
2 o   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 2 s   steps:  a method  for the  step of combining  the antigens which tonm  each component vaccine and performing 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 abil~y wrth
30    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 immuntty 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

10    vaccine.

Therefore, the inventors of the present invention have judged that it is

proper to manufacture a combined vaccine by preparing a bulk solution of each

component antigen, independently performing an adsorption of each antigen

and combining the adsortled antigens together.

15    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 compone~t antigen in each conventional monovalent vaccine, to compare the immunity and the characteristics of vaccine antigens in vaccine products. Therefore, to

2 o manufacture the combined vaccine, the component antigen more concentrated than a single vaccine is adsortled and then combin6d. •The DTP vaccine is

manufactured based on an adsorption using the antigen which is concentrated

1.5-2 times,  and the hepatitis 8 vaccine is manufactured using the antigen

which is concentrated 2-3 times. The final concentration of each component 2 5 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 perfonned uie various

studies of combinations for developing the combined vaccine. In this case, the

30    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 adsortlent 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 formation 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 8 vaccine, aluminum

hydroxide gel, and aluminum phosphate gel were mainly used. It is known that

10    the adsorption of the protein to each aluminum gel is caused by a surface electric charge of each protein and a gel component. And ~ 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

electrtc charge ("Vaccne Design -The subunit and adjuvant approach", ed. By

15    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 B vaccine and combined vaccine

20    has generally a negative surface electnc charge in a physiological pH range. Therefore, the aluminum hydroxide gel is proper as an adsorbent.
In the conventional art, the component of the adsortJent such as the aluminum hydroxide gel and aluminum phosphate gel was co-used
(International Publication No. WO 93124146) but based on the above reason,

2 5 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 adsortlent covers not only the aluminum 30 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 8 vaccine, the antigen
exists  generally  in  a  particle  type  which  includes  a  phospholipid  layer.

Therefore, ~ is prelenred that when manufacturing the hepatitis B vaccine lor 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 8 vaccine antigen Ylhich includes a phospholipid

layer, a neutral surfactant such as Polysorbate 20 (Tween 20), Polysorbate 80 10 (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.

15    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 1Q--25 Lf based on the pediatric dose. As a tetanus
20    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 purWied by the conventional method is proper. The amount of the antigen is preferably 1-5Lf based on the pediatric dose. In addition, the

Pertussis antigen  is cultivated  in  a proper culture  medium  using 8ordetella

2 5    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 Jo   antigens is below 20~gPN based on the pediatric dose, and in the case of the

whole cell Pertussis antigen, the amounl 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  6  virus  surface  antigen,  which  manufactured  by  a

genetic engineering method as a hepatitis B antigen, is 5-10ug based on the

10    pediatric dose. More preferably, the present invention is directed to a method for manufacturing in such a manner that the hepatitis 8 surface antigen is

adsorbed Ia an adsortlent by mixing with stirring al 2-s•c tor 3--20 hours. In order to find the optimum content of adscrtlant for manufacturing of the

combined  vaccine,  the  adsorption  ratio  for  each  antigen  component  was

15    analyzed at each concentration of adsorbent. 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

20    regulation (WHO, "Requirements tor diphtheria, tetanus, pertussis and combined vaccines" in Technical Report Series No. 800, 1990, p87-179).
Therefore, as the aluminum hydroxide gel, lhe concentration of the final aluminum ion is preferably in lhe range of 0.5-1 .25mg/ml and more preferably in the range of 0. 7mg/ml.
25    In addition, in order to prevent a possibility that the abscrplion ratio of

each component antigen  is decreased by a repulsive force caused among

each component antigen~" even after the combination of each component is

completed,  the  antigenic~y and  the  level  of  antibody  formation  of  each

component antigen are converted to the relative percentage to compare those

30    values between lwo samples: a sample that the other component except an
 
    10   
    aluminum  hydroxide gel  is previously combined  and then  sup/us aluminum
    hydroxide gel is eddHionally 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 Fg. 2).   
    In addition, in the case of the sample in which the polysorbate :!Q 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 e similar result i&
1 o    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 addttionally added after the combination of each component is
    completed, and that the adsorbent of the same concentration  is previously
15    added before the combination is completed,, 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
20    vaccine which includes a step of adding surplus adsorbent in the range that does not exceed the concentretion 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

2 5   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

30    vaccine is widened by the study on the combining method using whole cell
 

pertussis antigen.  In this case, the combining was perfonned  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 indudes 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 8 antigen, the DTP vaccine was manufactured using the aluminum

hydroxide gel. In this case, the immunity with respect to the Pertussis was

10    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 8 is prepared in the way of that the adsorbent of the same concentration was

previously added before the combination was completed.

15 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

20    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 8 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 resu~ of the group 1, it was evaluated that the

25    combined vaccine showed the equal or beHer 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

30    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

10    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

15    antigen in the mother'sbody, 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
perfonned using a combined vaccine. A proper vaccination with respect to the

2 o infants can be performed by a muscle or hypodermic 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.

25    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 30    ratio of 1:2 in a buffer solution (0.5M NaCI, 10mM EDTA, 0.01% Thimerosal,

0.1M Phosphate, pH 7.0). The diluted solulion flows through a glass bead beater (or Dynomil) for thereby breaking cell walls and so on. The obtained solution is added with a neutral surtactant (Tween group or Triton group) by 0.5% and is uniformly mixed 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'Cfor 5 hours. A diluted hydrochloric acid was added to the resultant solution  for  thereby  obtaining  pH  4.  The  precipitate  was  removed  by  a centr~ugation at  6000rpm  for  15  minutes  using  the  centrifugal  machine (ROTOR: JA-14, Beckman Inc. USA), and the upper solution including the 1o   hepatitis B surtace 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'Cfor

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

3BO(Degussa, USA) which includes fine hydration silica or anhydrous silica

15    having a valid surface area of 100-500mm2/g. In order to remove contaminants from the silica to which the hepatitis B surtace antigen is adsort>ed, 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 cart>onate buffer solution m pH 9.6 for about 2 hours for thereby desorption of

20    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.
2 s Contaminants slightly attached on the column was eluted using the buffer solution including sodium chloride m 0.05-0.1 M. Thereafter, the hepat~is 8 surface antigen was eluted using the said buffer solution including the sodium chloride of 0.2M. The thus eluted solution was concentrated using a ultrafi~ration membrane which is capable of separating the substance of

30    molecular weight above than 100,000, and the precipitate was separated by
 

centr1fugation and removed, and the upper solution was collected, and the gel permeation chromatography (Sepharose CL-48, PHAMACIA, Sweden) was performed 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 8 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~g per 1ml, and then the effect of tween 80 was observed (as shown in Fig.

2).

10    Example 2

Manufacture of diphtheria toxoid

The c01ynebacterium diphtheria PW No. 8 was cultured at 35°C tor 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

15    brain heart infusion culture medium (DIFCO, USA), and 1.2ml of it was inoculated to the modified Muller culture medium (refer to: Stainer, et. a!, Canadian J. Microbial., 14:155, 1968) of 300m! 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.

20    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 1OmM Iris buffer solution pH 8.0 including 25(w/v)% ammonium suWate. The buffer

solution same as the column balance solution and 10mM tris buffer solution(pH

25    8.0) including 15(w/v)% ammonium suWate 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 sal!. The eluted diphtheria toxin solution was collected and was dialyzed to 10mM phosphate buffer solution(pH 7.4) including sodium chloride of 150mM. After the dialyzing process was

30    completed, formalin 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 lor thereby obtaining the final concentration of 0.05M and then, the

resultant solution was detoxified at 37°C for 4 weeks. The_toxoid solution was

dialyzed into 10mM phosphate buffer solution(pH 7.4) with sodium chloride lor thereby fully removing the fonnalin, and thimerosal was added to have 0. 01 (w/v)% of the final conoantration. The resultant solution was used as diphtheria toxoid solution.

Example 3

10    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

15    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 cu~ure 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

20    culture solution, the toxin solution was collected and added with ammonium sulfate at 4•C lor 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

25    dropped into the sephagrill S-100 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 1OmM phosphate buffer solution(pH 7.4) including 150mM sodium chloride. Alter the dialysis, fonnalin

30    was added to have the final conoantration of 0.025(w/v)% and was reacted at


37"C for 1 hour. Thereafter, lysine and sodium hydrogencart>onate were added to have the final concentration of 0.05mM 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.

Example4

1o    Manufacture of purilied Pertussis antigen protein •

Borrietella pertussis, Tohama phase I was cu~ured in BodeH3engo agar culture medium(DIFCO, USA) including 15% rabbit blood at 35"C for 72 hours

and was passaged two times.  A few colonies were inoculated into 50ml of

stanorwsholte  culture  medium  and  was cultured  at  35°C  for  48  hours.  The

15    thusly cultured solution were rewinoculated in 500ml of changed stanorwsholte cullure medium(refer to lmazumi et al., INFECT.-IMMUN., 1983, val 41, pp. 1138) and was cultured at 35"C for 36 hours. 10% thimerosal aqueous solution was added to the cultured solution lo have the final concentration of 0.01% for thereby preventing the growth of the Pertussis bacteria, and then the cells were

20    removed by centr~ugation. Three times volume of distilled water was added to the resultant solution, and was mixed well. The current "PH was decreased to pH 6.0 using 1 N su~uric acid. The resultant solution was dropped into CM-sapharose column which was previous~ balanced using 10mM sodium

phosphate buffer of pH 6.0. The buffer solution same as the column balance

2 5   solution was flown to the column for thereby removing the substances which

were not coupled to the column, and the impunties which were slightly coupled

to the column were removed using the buffer solution same as the column

balance solution including 100mM sodium chloride. V\lhen the impurities were

not eluted anymore through the column, the bound materials were eluted wtth

30    linear gradient formed by the column balance solution including 100mM 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 1OmM 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 1OmM sodium phosphate buffer solution of pH 8.0. The buffer solution same as the column balance solution including 1OOmM sodium chloride was flown to the column for thereby removing the

substances which were not coupled to the column. 80mM sodium phosphate

10    buffer solution of pH 8.0 including 100mM 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 1OOmM sodium chloride was flown at the same rate
as the dropping rate for thereby separating FHA(filamentous hemagglutinin)

15    fraction. Separated Pertussis toxin and FHA(filamentous hemagglutinin) were dialyzed in 10mM sodium phosphate buffer solution(pH 7.4) including 0.15% sodium 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'Cfor 4 hours. Sodium aspartate was

20    added to have the final concentration of 0.025M for thereby completing the detoxifying process. Glycerol and formalin were added into the dialyzed FHA (filamentous hemagglutinin) sample solution to have 50(w/v)% and 0.025(w/V)% and were detoxified at 37'Cfor 24 hours. Lysine was added to

have the final concentration of 0.025M, so that the detoxifying process was

25    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

30    a Pertussis antigen protein.

Example 5

Manufacture of a whole cell Pertussis antigen

Pertussis bacteria, Bon1etella pertussis, Tohama phase I was cultured in 8odet-Gengo agar culture medium(OIFCO, USA) including 15% rabbit blood at 35"C for 72 hours and was passaged two times. Some of the colonies were inoculated into 50ml of Stanor-sholte culture medium and were cuRured at 35"C for 48 hours. The resultant solution was re-inoculated in the changed stanor-sholte culture medium(refer to lmazumi et al., INFECT.-IMMUN., 1983,

10    vel 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 1Om M sodium phosphate buffer sotution(pH

15    7.4) including 0.15% sodium chloride at 4"C. Formalin 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 detox~ying process. The sample solution was dialyzed into 1OmM sodium phosphate

20    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

2 5 Manufacture of combined vaccine including purified Pertussis antigen Step 1. Manufacture of hepatitis 8 vaccine

The hepatitis 8 vaccine was manufactured using the hepatitis 8 surface antigen  manufactured  in  Example  1.  aluminum  hydroxide  solution  was manufactured  by  adding  aluminum  hydroxide  get to the  phosphate  buffer 30   solution.  The  resultant  solution was slowly mixed,  and  the above  antigen


solution was dropped and was mixed by stining 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~g/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).

10    Step 2. Manufacture of DTP combined vaccine

The  DIP  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

15    was slowly mixed, eadl antigen solution was dropped thereto and was mixed by stining slowly for thereby implementing a un~orm adsorption, so that the

DIP 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 DIP vaccine. The amount of the aluminum ion

20    in the aluminum hydroxide gel was 0.3mg/ml.

Step 3. Manufacture of DIP-hepatitis 8 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, 2 5   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

3 o   and  was  continuously  mixed  by  stining  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 flrst and second samples were 0. 7mg/ml. For the test of antibody tner, the composnion of the combined vaccine was 20~ of the hepatitis B surface antigen, 25Lf of diphtheria toxoid, 3Lf of tetanus toxoid, 2.5~gPN of Pertussis toxoid, and

10~PN of Pertussis FHA antigen(Pertussis FHA(filamenlous hemaggh.Jtinin) antigen based on 1ml(as shown in Fig. 2).

Example 7

10    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

15    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'Cfor 15 hours for thereby manufacturing the

hepatitis B vaccine. At this time, the amount of the hepatitis 8 surface antigen

was 60~g/ml which was concentrated three times high compared to the amount

20    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).

2 5    Steo 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

30    slowly mixed, the above antigen solution was dropped and was fully mixed. A

un~orm 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 0.3mglml in the aluminum hydroxide gel.

Step 3. Manufacture of DTP-hepat[is 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 firsl sample, a

10    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

15    gel was not added, and the solution was continuously mixed at 25'Ctor 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~g of the  hepatitis  B surface  antigen,  50Lf of

20    diphlheria toxoid, 1Olf of tetanus toxoid, and 200E 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 25   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 adsorbed to the aluminum san was removed by the cenlrifugation for measuring lhe adsorption ratio of each antigen, and the
30    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

antigenictty of each antigen.

Example 9. Test of antibody formation by combined vaccine

Test of antibody formation by hepatitis 8 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

10    8 surface antigen was obtained based on the unit of IU/ml using the EIA(Enzyme Immune Assay) and calculated as in geomean antibody titer. The ttter of the combined vaccine was computed with respect to the hepatitis 8

vaccine.

Fig. 2c and 2d shows relative antibody titer of combined vaccine wtth

15    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 Example's6

and 7 were inoculated to the ICR mice in which 16 mice are grouped as the

20    group 1. The blood was collected from the CIR mice attar 28 days. Serum was separated from the blood. The antibody level against the diphtheria antigen of the serum was measured using the EIA(Enzyme Immune Assay) based on the

unit of IU/ml and calculated as in geomean antibody titer. Thereafter, the titer of

the combined vaccine was measured compared to the DTP vaccine.

2 5 Fig. 2c and 2d shows relative antibody titer of combined vaccine wtth 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 30 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 bbod. The antibody level against the telanus antigen of the serum was measured using the EIA(Enzyme Immune Assay) based on the unit of IU/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 vaccne with repect to each antigen, and Table 1 shows the values of each titer.

Test of antibody formation by Pertussis antigen

10    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 Immune Assay) based on the unH
15    of IU/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 combined vaccine with repect to each antigen, and Table 1 shows the values of each titer.


Table 1

        DTwP vaccine    OTwP    OTwP-HepB        DTwP-HepB    Creterion    of
        (Aluminium    vaccine    combined        combined    rrter   
        Phosphate   ..    (Aluminium    vaccine        vaccine               
        an adsorbent)    Hydroxide  as    (Sample A)        (Sample B)               
            an adsorbent                           
                                   
    Oiph1heria    >800    249.7    601.1        128.1    Over           
    Potency(IUJml}                        301U/ml   
                               
    Tetanus    176.5    196.0    216.9        230.7    Over   
    Potency(IU/mO                        40 IU/ml   
                               
    Pertussis    20.2    3.2    25.4        5.5    Over   
    Potency(IPU/mO                        BIPU/ml   
                               
    Hepatitis 8            196.8        103.9    Equal    or
    Relative                        over   Value
    Potency(%)                        of  Standard
                            sample   
                                       
    Example10.                               
    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 eal:h
    component vaccine was vaccinated to 6 monkeys(group 1) fanned of the same
1o    numbers of male end 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•, 291",  sa'", and
    86'"days from the in~ial vaccination date, and the antibody titer against each
    disease was measured based on the ELISA method proper to the serum of the
15    monkey.                                   
    Fig. 3 shows the geomean antibody titer against each antigen of each

INDUSTRIAL APPLICABILITY

According  to  the  present  invention,  ft  is  directed  to  a  method  for

manufacturing a combined vaccine for concurrently preventing the diseases

such as diphtheria, tetanus, pertussis, and hepatitis 8, 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 hepatftis 8

which should be prevented in an Infant using the combined vaccine according

to the present invention.

10    As  the  present invention  may  be  embodied  in  several  forms  without

departing from the spirit or essential characteristics thereof, ft 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 construed broadly within its spirit and scope as defined in the

15    appended daims, 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, wtlerein 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 0.5-1.25mg/ml.

5.    The method of either claim 3 or 4, further comprising a step of:

adding the adsorbent in which the final alumlnuril 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 8  surface

antigen as the hepatitis 8 antigen is combined by the amount of 5-1 O~g 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 eo and

triton X-100.

B. The method of claim 6, wherein said hepatitis 8 antigen is mixed by stilling w~h an adsorbent at 2-a•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 dosa.

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 200E 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 daimed in Claims 1 to 12.
 

group 1. The blood was collected from the ICR mice alter 28 days. Serum was separated tram the blood. The antibody level against the tetanus antigen of the serum was measured using the EIA(Enzyme Immune Assay) based on the unit of IU/ml and calculated as in geomean antibody titer. Thereafter, the titer of
the combined vaccine was measured compared tp the DTP vaccine.

Fig. 2c and 2d shows relative antibody titer of combined vaccine wllh repect to each antigen, and Table 1 shows the values of eacih titer.

Test of antibody formation by Pertussis antigen

1 o 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 Immune Assay) based on the uM

15    of IU/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 cOmbined vaccine with repect to each antigen, and Table 1 shtlws the values of each titer.
 

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