WO2017048038A1

WO2017048038A1 - Combination vaccine composition for multiple-dosage

Info

Publication number: WO2017048038A1
Application number: PCT/KR2016/010313
Authority: WO
Inventors: Suk Young Choi; Yoon Ae NOH; Eun Ji Kim
Original assignee: LG Life Sciences Ltd
Current assignee: LG Chem Ltd
Priority date: 2015-09-16
Filing date: 2016-09-13
Publication date: 2017-03-23
Anticipated expiration: 2018-03-16
Also published as: BR112018005291A2; KR20170033254A; MA52400B1; PH12018500570A1; TN2018000063A1; MA42317B2; KR101864029B1; EA037283B1; MA52400A1; EA201890502A1

Abstract

The present invention relates to a multiple-dose immunogenic composition for a multivalent combination vaccine, in which (i) the multivalent combination vaccine contains a whole cell pertussis (wP) vaccine or acellular pertussis (aP) vaccine, and a Sabin inactivated polio vaccine (sIPV); and (ii) the composition contains 2-phenoxyethanol (2-PE) and formaldehyde (HCHO) as preservatives; and a method for preparing a multiple-dose immunogenic composition for a multivalent combination vaccine. It was confirmed that the combination of 2-phenoxyethanol and formaldehyde according to the present invention is an excellent preservative capable of maintaining antimicrobial ability and that it can be effectively used for providing a multiple-dose composition maintaining antimicrobial ability of combination vaccine, thus the composition of the present invention can be effectively used in the multiple-dose combination vaccine industry.

Description

COMBINATION VACCINE COMPOSITION FOR MULTIPLE-DOSAGE

The present invention relates to a multiple-dose immunogenic composition for a multivalent combination vaccine, wherein the multivalent combination vaccine contains a whole cell pertussis (wP) vaccine or acellular pertussis (aP) vaccine, and a Sabin inactivated polio vaccine (sIPV); and the composition contains 2-phenoxyethanol (2-PE) and formaldehyde (HCHO) as preservatives; and a method for preparing the multiple-dose immunogenic composition for a multivalent combination vaccine.

Combination vaccines are vaccines containing two or more antigens and are used for preventing at least one infectious disease or for preventing infectious diseases caused by different isoforms or serotypes of the same microorganism. The combination vaccines allowing the simultaneous administration of diphtheria, tetanus, and other antigens have been used since the mid-20^th century.

Although the amount and total range of antigens contained in vaccines vary according to the age range for use (i.e., infants, toddlers, children, adolescents, and adults), recently DTwP-based and DTaP-based combination vaccines are widely used in the world. In particular, the DTwP-based vaccines are exported to less-developed countries via international organizations such as the UN.

For the preparation of a diphtheria (D) vaccine, diphtheria toxin is detoxified with formalin to prepare a diphtheria toxoid, which is used as a vaccine. Symptoms of diphtheria, an upper respiratory tract infection, may include development of an undetachable film on the areas of tonsils, pharynx, and nose for no particular reason, thereby causing a sore throat and mild fever. Sometimes, diphtheria can also cause infections in the skin, conjunctiva, and reproductive organs.

For the preparation of a tetanus (T) vaccine, tetanus toxin is detoxified with formalin to prepare a tetanus toxoid, which is used as a vaccine. The causative pathogen of tetanus is Clostridium tetani, a spore-producing anaerobic gram-positive Bacillus. Tetanus is a neurologic disorder in which Clostridium tetani secretes exotoxin, thereby causing severe spasms of muscle. The pathogen enters through the wounds in the skin or mucous membranes, thereby causing tetanus, and the symptoms occur slowly and progressively develop into more severe muscle contractions.

For the preparation of a whole cell pertussis (wP) vaccine, pertussis is detoxified with heat treatment/formalin, etc., and the resulting pertussis is used as a vaccine, which is called a whole cell pertussis vaccine. An acellular pertussis (aP) vaccine is a vaccine prepared using only the proteins of the pertussis as antigens. The causative pathogen of pertussis is Bordetella pertussis, a gram-positive pleomorphic Bacillus, which is difficult to culture. Pertussis may occur in all ages, but more frequently in children aged 5 or below, and the in-patients are mostly new-born infants aged less than one month, and the ratio of the in-patients decreases along withgetting older. The clinical symptoms of pertussis may continue for 6 to 10 weeks when there is no complication. Examples of the complications may include convulsions, pneumonia, encephalopathy, etc., and may lead to death.

A hepatitis B vaccine (hepatitis B antigen, HBsAg) is a vaccine having the structure of a virus-like particle (VLP) produced by genetic manipulation of the surface antigens of hepatitis B virus (HBsAg). Hepatitis B is a disease caused by the attack of hepatitis B virus on the liver cells, thereby resulting in the loss of liver functions, liver cirrhosis, or liver cancer.

A Haemophilus influenzae type b (Hib) vaccine is a conjugate vaccine prepared by conjugating a carrier protein to polyribose ribitol phosphate (PRP), which is a polysaccharide on the surface membrane of Haemophilus influenzae type b. About 95% of the invasive diseases such as meningitis and sepsis are caused by Haemophilus influenzae type b, and thus Haemophilus influenza is a major causative pathogen of bacterial meningitis in infants, and in particular, the infection rate is highest in young children aged between 6 months and 5 years.

A Sabin inactivated polio virus vaccine for injection is provided in two different types: an inactivated polio virus vaccine of the Sabin strain (attenuated type) (sIPV) and an inactivated polio virus vaccine of the Salk strain (wild type) (wIPV). An attenuated oral polio vaccine was developed by Dr. Albert Sabin in 1961 and the live attenuated species developed by Dr. Sabin is called the Sabin strains polio virus. Recently, the Sabin inactivated polio virus, which was prepared by inactivating the Sabin strains polio virus with formalin, has been developed for injection and also has been available in commercial products. The sIPV vaccine includes 3 different types of strains; type 1, type 2, and type 3. In 1955, Dr. Jonas Salk succeeded in inactivation of the wild type polio virus, thus enabling it in an injection type formulation, and named it as the Salk strain, which includes Mahoney type 1, MEF type 2, and Saukett type 3. Until now, the wIPV vaccine has been widely used. However, according to the wild polio virus eradication policies by the WHO, the sIPV has been studied and developed into commercial products.

Meanwhile, a multiple-dose vaccine injection must use a preservative to avoid contamination by microorganisms. For the combination vaccine products exported to less-developed countries by the UN, etc., multiple-dose vaccines containing a preservative are preferred, considering the environments of the countries where the vaccines are to be used, methods of distribution, expenses, etc. Examples of the preservative to be used in the vaccine products may contain thimerosal, 2-PE, phenol, etc., and conventional doses of the preservatives are known in the art. For example, thimerosal is used in a concentration of 100 ㎍/mL and 2-PE is used in a concentration of 5 mg/mL, and multiple-dose vaccine products containing these preservatives can be prepared into commercial products only when they pass the antimicrobial ability tests described in the European Pharmacopeia Category B (EP-B) or the United States Pharmacopeia (USP).

Thimerosal (thimerosal, thiomersal, or merthiolate) is a derivative of ethyl mercury which has been used as a preservative for multiple-dose vaccine injections since the early 1930s. Thimerosal has been used for the purposes of preventing the growth of contaminating microorganisms and maintaining sterile conditions during storage or use of vaccine products, and many pentavalent combination vaccines (including D, T, P, Hib, and HBsAg), which have acquired the WHO Prequalification (PQ), contain thimerosal as a preservative.

2-PE is mostly used as a preservative for cosmetics and dermal pharmaceutical products and also used as a preservative for vaccine injections.

Inactivated polio vaccine (hereinafter, IPV) conventionally uses 2-PE as a preservative. According to a previous report, when the inactivated polio vaccine contains thimerosal, the vaccine potency tends to decrease by 50% or more within a week even when stored in a refrigerator, and thus all IPV vaccine products do not contain thimerosal as a preservative, and instead use 2-PE in a concentration of 5 mg/mL (Vaccine 1994 Volume 12 No. 9 851 - 856. Deleterious effect of thimerosal on the potency of inactivated poliovirus vaccine).

For these reasons, it was reported that hexavalent combination vaccines (including D, T, wP, Hib, HBsAg, and IPV), which are still under development without being developed into commercial products, also use 2-PE in a concentration of 5 mg/mL (US 2011-0195087, WO 2004-110480, and WO 2012-093406).

As such, the present inventors have made efforts to perform antimicrobial ability tests on 2-PE, which has been conventionally used in the IPV vaccines in the art, in a concentration of 5 mg/mL, while developing the hexavalent combination vaccines (including D, T, P, Hib, HBsAg, and IPV vaccines). However, surprisingly, they have failed to obtain satisfactory results to comply with the criteria described in the European Pharmacopeia Category B. Nevertheless, an inconsiderate forced increase of the amount of 2-PE (a preservative) in order to meet the required criteria may cause safety problems in young children, who are the subjects to receive the combination vaccines, and may also affect the manufacture and stability of the final products, and thus the decision must be made carefully.

The present inventors have discovered the combination for each dosage of 2-PE and formaldehyde as preservatives for the hexavalent combination vaccines including D, T, P, and IPV vaccines which satisfied the criteria for antimicrobial ability, while minimizing their contents, thereby completing the present invention.

An object of the present invention is to provide a multiple-dose immunogenic composition for a multivalent combination vaccine, wherein (i) the multivalent combination vaccine contains a diphtheria (D) vaccine, a tetanus (T) vaccine, a whole cell pertussis (wP) vaccine or acellular pertussis (aP) vaccine, and a Sabin inactivated polio vaccine (sIPV); and (ii) the composition contains 2-phenoxyethanol (2-PE) and formaldehyde (HCHO) as preservatives.

Another object of the present invention is to provide a method for preparing the multiple-dose immunogenic composition for a multivalent combination vaccine.

It was confirmed that the combined use of 2-PE and formaldehyde according to the present invention can serve as an excellent preservative capable of maintaining antimicrobial ability of a combination vaccine and can also be effectively used for providing a multiple-dose composition capable of maintaining antimicrobial ability of a multivalent combination vaccine. Accordingly, 2-PE and formaldehyde can be effectively used in the field of multiple-dose combination vaccine preparations.

In order to achieve the above objects, in an aspect, the present invention provides a multiple-dose immunogenic composition for a multivalent combination vaccine, wherein (i) the multivalent combination vaccine contains a diphtheria (D) vaccine, a tetanus (T) vaccine, a whole cell pertussis (wP) vaccine or acellular pertussis (aP) vaccine, and a Sabin inactivated polio vaccine (sIPV); and (ii) the composition contains 2-phenoxyethanol (2-PE) and formaldehyde (HCHO) as preservatives.

In an exemplary embodiment, the present invention provides a multiple-dose immunogenic composition which further contains as a multivalent combination vaccine a hepatitis B vaccine (hepatitis B antigen, HBsAg) and a Haemophilus influenzae type b (Hib) conjugate vaccine.

In another exemplary embodiment, the composition contains 2-PE in an amount of 7 mg/mL to 9.5 mg/mL.

In still another exemplary embodiment, the composition contains formaldehyde in an amount of 120 ㎍/mL to 180 ㎍/mL.

In still another exemplary embodiment, the composition contains 2-PE in an amount of 7 mg/mL to 9.5 mg/mL and formaldehyde in an amount of 120 ㎍/mL to 180 ㎍/mL.

In still another exemplary embodiment, the composition contains a pharmaceutically acceptable adjuvant, wherein the adjuvant is aluminum phosphate, aluminum hydroxide, or a combination thereof.

In still another exemplary embodiment, the composition contains the adjuvant in an amount of 1.25 mg/mL or less.

In still another exemplary embodiment, the composition contains a pharmaceutically acceptable transporter, excipient, binder, carrier, preservative, buffering agent, isotonic agent, emulsifier, or humectant.

In still another exemplary embodiment, the composition has a pH of 5.5 to 8.

In still another exemplary embodiment, the multivalent combination vaccine contains a diphtheria (D) vaccine in an amount of 10 Lf/mL to 50 Lf/mL; a tetanus (T) vaccine in an amount of 5 Lf/mL to 30 Lf/mL; a whole cell pertussis (wP) vaccine or acellular pertussis (aP) vaccine in an amount of 20 OU/mL to 50 OU/mL; a hepatitis B vaccine (hepatitis B antigen, HBsAg) in an amount of 5 ㎍/mL to 30 ㎍/mL; a Haemophilus influenzae type b (Hib) conjugate vaccine in an amount of 5 ㎍/mL to 20 ㎍/mL; and a Sabin inactivated polio vaccine (sIPV), which includes type 1, type 2, and type 3, wherein type 1 is contained in an amount of 5 DU/mL to 100 DU/mL, type 2 is contained in an amount of 10 DU/mL to 100 DU/mL, and type 3 is contained in an amount of 20 DU/mL to 120 DU/mL.

In another aspect, the present invention provides a method for preparing a multiple-dose immunogenic composition for a multivalent combination vaccine, including (a) mixing a diphtheria vaccine (D), a tetanus vaccine (T), and a whole cell pertussis (wP) vaccine or acellular pertussis (aP) vaccine with aluminum phosphate, aluminum hydroxide, or a combination thereof; (b) adding a hepatitis B vaccine (hepatitis B antigen, HBsAg), a Haemophilus influenzae type b (Hib) conjugate vaccine, and a Sabin inactivated polio vaccine (sIPV) to the mixture in step (a) and mixing; and (c) adding 2-PE and formaldehyde to the mixture in step (b), mixing, and adjusting the final pH of the mixture to 5.5 to 8.

Hereinafter, the present invention is explained in detail.

An aspect of the present invention provides a multiple-dose immunogenic composition for a multivalent combination vaccine, wherein:

(i) the multivalent combination vaccine contains a diphtheria (D) vaccine, a tetanus (T) vaccine, a whole cell pertussis (wP) vaccine or acellular pertussis (aP) vaccine, and a Sabin inactivated polio vaccine (sIPV); and

(ii) the composition contains 2-phenoxyethanol (2-PE) and formaldehyde (HCHO) as preservatives.

Specifically, the multivalent combination vaccine may further contain a hepatitis B vaccine (hepatitis B antigen, HBsAg) and a Haemophilus influenzae type b (Hib) conjugate vaccine.

As used herein, the term "multiple-dose" refers to an immunogenic composition containing a vaccine in an amount exceeding a single dose of administration that can be administered to a single subject or subjects exceeding one in different administration steps according to time.

As used herein, the term "a multivalent combination vaccine" refers to a combination of two or more vaccines that can cause immunogenicity against antigens, and may include two or more vaccines that cause immunogenicity with respect to other regions in the same antigen, or may include vaccine(s) for two or more antigens. For the purpose of the present invention, the multivalent combination vaccine of the present invention may contain a diphtheria (D) vaccine, a tetanus (T) vaccine, a whole cell pertussis (wP) vaccine or acellular pertussis (aP) vaccine, and a Sabin inactivated polio vaccine (sIPV), and in particular, a hexavalent combination vaccine further containing a hepatitis B vaccine (hepatitis B antigen, HBsAg) and a Haemophilus influenzae type b (Hib) conjugate vaccine, but is not limited thereto.

The multivalent combination vaccine may be used in a liquid form, but is not limited thereto.

In the present invention, diphtheria toxin is detoxified with formalin to prepare a diphtheria toxoid, which is used as a vaccine. Symptoms of diphtheria, an upper respiratory tract infection, may include development of an undetachable film on the areas of tonsils, pharynx, and nose for no particular reason, thereby causing a sore throat and mild fever. Sometimes, diphtheria can also cause infections in the skin, conjunctiva, and reproductive organs.

In the present invention, tetanus toxin is detoxified with formalin to prepare a tetanus toxoid, which is used as a vaccine. The causative pathogen of tetanus is Clostridium tetani, a spore-producing anaerobic gram-positive Bacillus. Tetanus is a neurologic disorder in which Clostridium tetani secretes exotoxin, thereby causing severe spasms of muscle. The pathogen enters through the wounds in the skin or mucous membranes, thereby causing tetanus, and the symptoms occur slowly and progressively develop into more severe muscle contractions.

In the present invention, the tetanus whole cell pertussis (wP) vaccine is a vaccine in which pertussis is detoxified with heat treatment/formalin, etc., and then the resulting pertussis is used as a vaccine. Meanwhile, the acellular pertussis (aP) vaccine is a vaccine prepared using only the proteins of the pertussis as antigens. The causative pathogen of pertussis is Bordetella pertussis, a gram-positive pleomorphic Bacillus, which is difficult to culture.

In the present invention, an inactivated polio virus vaccine has two different types: an inactivated polio virus vaccine of the Sabin strain (attenuated type) (sIPV) and an inactivated polio virus vaccine of the Salk strain (wild type) (wIPV). An attenuated oral polio vaccine for oral administration was previously developed and the live attenuated species developed by Dr. Sabin is called the Sabin strains polio virus. Recently, a Sabin inactivated polio virus, which was prepared by inactivating the Sabin strains polio virus with formalin, has been developed for injection and has also begun to be developed into commercial products. The sIPV vaccine includes 3 different types of strains; Type 1, Type 2, and Type 3. Until now, the wIPV vaccine has been widely used. Recently, however, according to the wild polio virus eradication policies by the WHO, the sIPV has been studied and developed into commercial products.

In the present invention, a hepatitis B vaccine (hepatitis B antigen, HBsAg) is a vaccine having the structure of a virus-like particle (VLP) produced by genetic manipulation of the surface antigens of hepatitis B virus (HBsAg). Hepatitis B is a disease caused by the attack of hepatitis B virus on the liver cells, thereby resulting in the loss of liver functions, liver cirrhosis, or liver cancer.

In the present invention, a Haemophilus influenzae type b (Hib) vaccine is a conjugate vaccine prepared by conjugating a carrier protein to polyribose ribitol phosphate (PRP), which is a polysaccharide on the surface membrane of Haemophilus influenzae type b. About 95% of the invasive diseases such as meningitis and sepsis are caused by Haemophilus influenzae type b, and thus Haemophilus influenza is a major causative pathogen of bacterial meningitis in infants.

Meanwhile, in the present invention, the multiple-dose vaccine injection must use a preservative to avoid the contamination by microorganisms during the process of multiple administration and storage. For the combination vaccine products exported to less-developed countries by the UN, etc., multiple-dose vaccines containing a preservative are preferred, considering the environments of the countries where the vaccines are to be used, methods of distribution, expenses, etc.

Examples of the preservative to be used in the vaccine products may contain thimerosal, 2-PE, phenol, etc., and conventional doses of the preservatives are known in the art.

The composition of the present invention may contain 2-PE and formaldehyde as preservatives. In particular, the composition of the present invention may contain 2-PE in an amount of 7 mg/mL to 9.5 mg/mL or may contain formaldehyde in an amount of 120 ㎍/mL to 180 ㎍/mL, and specifically, may contain 2-PE in an amount of 7 mg/mL to 9.5 mg/mL and formaldehyde in an amount of 120 ㎍/mL to 180 ㎍/mL.

In an exemplary embodiment, compositions containing a hexavalent combination vaccine, which contains a (D) vaccine, a tetanus (T) vaccine, a whole cell pertussis (wP) vaccine, a hepatitis B vaccine (hepatitis B antigen, HBsAg), and a Haemophilus influenzae type b (Hib) conjugate vaccine, and a Sabin inactivated polio vaccine (sIPV) and aluminum phosphate; and 2-PE and formaldehyde as preservatives according to concentrations were prepared, and the antimicrobial ability of the compositions was measured. As a result, when compositions contained 2-PE in an amount of 7.2 mg/mL or 8.5 mg/mL and formaldehyde in an amount of 135 ㎍/mL or 150 ㎍/mL, they were confirmed to have the antimicrobial ability satisfying the requirement by the European Pharmacopeia Category B (EP-B).

From the above, it was confirmed that when the hexavalent combination vaccine contained only 2-PE in a concentration described above or contained only formaldehyde, the hexavalent combination vaccine of the present invention could not have the antimicrobial ability satisfying the requirement by the European Pharmacopeia Category B, whereas the combined use of 2-PE and formaldehyde had a synergistic effect in antimicrobial ability and when 2-PE and formaldehyde were used in concentrations described above, the hexavalent combination vaccine of the present invention could have the antimicrobial ability satisfying the requirement by the European Pharmacopeia Category B.

Meanwhile, the composition of the present invention may further contain a pharmaceutically acceptable adjuvant, and the adjuvant may be aluminum phosphate, aluminum hydroxide, or a combination thereof. In particular, the composition of the present invention may contain the adjuvant in an amount of 1.25 mg/mL or less, and specifically, in an amount of 0.5 mg/mL to 1.25 mg/mL.

As used herein, the term "adjuvant" refers to a material that improves the immunogenicity of the immunogenic composition of the present invention. Accordingly, adjuvants are often provided for the purpose of boosting immune responses and are well known to one of ordinary skill in the art.

The immunogenic composition of the present invention can easily be achieved using the methods and techniques well known in the art, and the composition may be administered in various methods, preferably, parenterally or intranasally. The composition of the present invention may further contain a pharmaceutically acceptable transporter, excipient, binder, carrier, preservative, buffering agent, isotonic agent, emulsifier, or humectant. Examples of the formulations that are suitable for parenteral or intranasal administration may include an aqueous and non-aqueous sterile injection solution which can contain a solute that makes the formulation for an antioxidant, a buffer, a bacteriostatic agent, and the body fluid of the subject of issue isotonic; and an aqueous and non-aqueous sterile suspension which can contain a suspending agent or thickening agent. Active immunogenic ingredients are often mixed with a pharmaceutically acceptable excipient, e.g., water, saline, dextrose, glycerol, ethanol, etc. Additionally, the immunogenic composition may contain a small amount of a supplementary material, e.g., a humectant, emulsifier, a pH buffering agent, a binder, a carrier, or a preservative.

The hexavalent combination vaccine composition may contain a buffering agent. The buffer in the solution composition of the present invention plays the role of maintaining the pH of the solution to prevent a rapid pH change, and preferred examples of the buffer may include a phosphate buffer, a histidine buffer, a citrate buffer, a Tris buffer, etc.

The composition of the present invention contains an isotonic agent. Examples of the isotonic agent may include amino acids, multivalent alcohols, sodium chloride, etc., and the isotonic agent plays the role of maintaining a suitable osmotic pressure in vivo when a vaccine is administered in a liquid form into the body.

The composition of the present invention may contain a pharmaceutically acceptable excipient. Examples of the excipient may include sugars and polyols, surfactants, polymers, etc. Examples of the sugars and polyols to be used may include sucrose, trehalose, lactose, maltose, galactose, mannitol, sorbitol, glycerol, etc. Examples of the surfactants may include non-ionic surfactants such as polysorbate 20, polysorbate 80, etc. Examples of the polymers may include dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin, etc.

These materials may be used alone or in a combination of two or more, but one of ordinary skill in the art may appropriately select them for use.

The composition of the present invention may have a pH of 5.5 to 8.0, but is not limited thereto.

In an exemplary embodiment, the present inventors prepared an immunogenic composition using the hexavalent combination vaccine, which contained a (D) vaccine, a tetanus (T) vaccine, a whole cell pertussis (wP) vaccine, a hepatitis B vaccine (hepatitis B antigen, HBsAg), and a Haemophilus influenzae type b (Hib) conjugate vaccine, and a Sabin inactivated polio vaccine (sIPV), and adjusted the pH of the composition to 6.7.

The composition of the present invention may include a multivalent combination vaccine, which contains a (D) vaccine, a tetanus (T) vaccine or acellular pertussis (aP) vaccine, a hepatitis B vaccine (hepatitis B antigen, HBsAg), and a Haemophilus influenzae type b (Hib) conjugate vaccine, and a Sabin inactivated polio vaccine (sIPV), and in particular, the Sabin inactivated polio vaccine (sIPV) may include type 1, type 2, and type 3. The multivalent combination vaccine may contain the diphtheria vaccine (D) in an amount of 10 Lf/mL to 50 Lf/mL; the tetanus vaccine (T) in an amount of 5 Lf/mL to 30 Lf/mL; the whole cell pertussis vaccine (wP) or acellular pertussis vaccine (aP) in an amount of 20 OU/mL to 50 OU/mL; the hepatitis B vaccine (HBsAg) in an amount of 5 ㎍/mL to 30 ㎍/mL; the Haemophilus influenzae type b conjugate vaccine (Hib) in an amount of 5 ㎍/mL to 20 ㎍/mL; and the Sabin inactivated polio vaccine (sIPV), which includes type 1, type 2, and type 3, wherein type 1 may be contained in an amount of 5 DU/mL to 100 DU/mL, type 2 in an amount of 10 DU/mL to 100 DU/mL, and type 3 in an amount of 20 DU/mL to 120 DU/mL.

In an exemplary embodiment, the present inventors prepared an immunogenic composition using the hexavalent combination vaccine, which contained the diphtheria vaccine (D) in an amount of 30 Lf/mL; the tetanus vaccine (T) in an amount of 20 Lf/mL; the whole cell pertussis vaccine (wP) in an amount of 36 OU/mL; the hepatitis B vaccine (HBsAg) in an amount of 20 ㎍/mL; the Haemophilus influenzae type b conjugate vaccine (Hib) in an amount of 10 ㎍/mL; and the Sabin inactivated polio vaccine (sIPV), wherein type 1 was contained in an amount of 10 DU/mL, type 2 in an amount of 16 DU/mL, and type 3 in an amount of 32 DU/mL; or an immunogenic composition, which contained the diphtheria vaccine (D) in an amount of 30 Lf/mL; the tetanus vaccine (T) in an amount of 20 Lf/mL; the whole cell pertussis vaccine (wP) in an amount of 36 OU/mL; the hepatitis B vaccine (HBsAg) in an amount of 20 ㎍/mL; the Haemophilus influenzae type b conjugate vaccine (Hib) in an amount of 10 ㎍/mL; and the Sabin inactivated polio vaccine (sIPV), wherein type 1 was contained in an amount of 60 DU/mL, type 2 in an amount of 64 DU/mL, and type 3 in an amount of 90 DU/mL.

Another aspect of the present invention provides a method for preparing a multiple-dose immunogenic composition for a multivalent combination vaccine, including:

(a) mixing a diphtheria vaccine (D), a tetanus vaccine (T), and a whole cell pertussis (wP) vaccine or acellular pertussis (aP) vaccine with aluminum phosphate, aluminum hydroxide or a combination thereof;

(b) adding a hepatitis B vaccine (hepatitis B antigen, HBsAg), a Haemophilus influenzae type b (Hib) conjugate vaccine, and a Sabin inactivated polio vaccine (sIPV) to the mixture in step (a) and mixing; and

(c) adding 2-PE and formaldehyde to the mixture in step (b), mixing, and adjusting the final pH of the mixture to 5.5 to 8.

In the present invention, the multivalent combination vaccine, multiple-dose, diphtheria vaccine, tetanus vaccine, whole cell pertussis vaccine or acellular pertussis vaccine, hepatitis B vaccine (hepatitis B antigen, HBsAg), Haemophilus influenzae type b (Hib) conjugate vaccine, Sabin inactivated polio vaccine, etc., are the same as explained above.

In an exemplary embodiment, the present inventors added a diphtheria vaccine (D), a tetanus vaccine (T), and a whole cell pertussis (wP) vaccine, and aluminum phosphate (Brenntag) in a concentration of 1 mg/mL were added and stirred to mix them well. The mixture was then treated with a hepatitis B vaccine (hepatitis B antigen, HBsAg), a Haemophilus influenzae type b (Hib) conjugate vaccine, and a Sabin inactivated polio vaccine (sIPV) in this order and mixed. Finally, 2-PE and formaldehyde were added according to the concentration, mixed well, and the final pH of the mixture was adjusted to 6.7.

In another exemplary embodiment, the present invention provides a method for preventing the infection of an individual which includes administering the immunogenic composition of the present invention to a subject.

The immunogenic composition of the present invention may be administered by any effective and convenient method including local, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, and intradermal administrations.

As used herein, the term "subject" refers to a multicellular eukaryotic animal which includes, without limitation, metazoa, mammals, ovids, bovine animals, birds, primates, and humans.

In the present invention, the above infection may refer to infections caused by pertussis, polio virus, diphtheria, tetanus, hepatitis B virus, and Haemophilus influenzae type b virus.

Hereinbleow, the present invention will be described in detail with accompanying exemplary embodiments. However, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present invention.

Example 1: Protocols and criteria for antimicrobial ability test of vaccines

In the present invention, the antimicrobial ability test was performed according to the European Pharmacopeia Category B (EP-B) criteria requested by the WHO on vaccine products, between the European Pharmacopeia and the United States Pharmacopeia (USP).

In specific details of the test, a total of four different kinds of microorganisms including two different kinds of bacteria (Pseudomonas aeruginosa (ATCC NO. 9027, PA) and Staphylococcus aureus (ATCC NO. 6538, SA)), one yeast (Candida albicans (ATCC NO. 10231, CA)), and one fungus (Aspergillus niger (ATCC NO. 16404, AN)) were inoculated in an amount of 10⁵ to 10⁶ colony forming units (CFU)/mL into vaccine preparations at 0 hours, respectively. Then, samples were collected at 24 hours, day 7, day 14, and day 28, cultured in solid media, and the number of colonies was counted between day 3 and day 5.

The criteria for evaluation of antimicrobial activity by the European Pharmacopeia Category B and Pharmacopeia from other countries with respect to the results according to the above method are shown in Table 1 below.

	Pharmacopoeia Criteria	Log Reduction (log CFU/mL reduction)
		6 Hours	24 Hours	7 Days	14 Days	28 Days
Bacteria	EP-A	2	3			NR*
	EP-B	-	1	3	-	NI**
	USP	-	-	1	3	NI
	JP	-	-	-	3	NI
Fungi and Yeasts	EP-A	-	-	2	-	NI
	EP-B	-	-	-	1	NI
	USP	-	-	NI	NI	NI
	JP	-	-	-	NI	NI

*NR: No Recovery

**NI: No Increase

As can be confirmed in Table 1 above, the criteria requested by the European Pharmacopeia are stricter than the US Pharmacopeia or the Japanese Pharmacopeia, and in particular, the European Pharmacopeia is divided into categories A and B according to the preparations. The criteria requested by the WHO are the European Pharmacopeia category B (EP-B) (EP 5.1.3. Efficacy of antimicrobial preservation, USP 37-51, Antimicrobial effectiveness testing).

The present inventors, while developing hexavalent combination vaccines containing D, T, wP, Hib, HBsAg, and IPV vaccines, have performed tests on their antimicrobialantimicrobial abilities by adding 2-PE, which has been conventionally used as a preservative in IPV vaccines in the art, in a concentration of 5 mg/mL to the combination vaccines. Unexpectedly, however, the result was not satisfactory, failing to meet the acceptance criteria on antimicrobialantimicrobial ability.

Nevertheless, an inconsiderate forced increase of the amount of 2-PE (a preservative) to meet the required criteria may raise safety issues in young children, who are the subjects to receive the combination vaccines and also affect the manufacture and stability of the final products, and thus the decision should be made carefully. The amount of preservative(s) to be contained in the vaccines should be selected so as to meet the requirements defined in the US Pharmacopeia, the European Pharmacopeia, the WHO Pharmacopeia, or a combination thereof with respect to the safety of vaccines.

In this regard, the present inventors have performed experiments in an effort to develop a novel composition which can satisfy the requirements on antimicrobial ability by containing a minimal amount of 2-PE as a preservative of a hexavalent combination vaccine, which contains wP and IPV vaccines, and as a result, obtained the following results.

Example 2: Preparation of a hexavalent combination vaccine containing 2-PE as a preservative

Diphtheria (D), tetanus (T), and whole cell pertussis (wP) vaccines were treated with aluminum phosphate (Brenntag) in a concentration of 1 mg/mL and stirred to be mixed well. Then, a hepatitis B vaccine (hepatitis B antigen, HBsAg), a Haemophilus influenzae type b (Hib) conjugate vaccine, and a Sabin inactivated polio vaccine (sIPV) were sequentially added thereto and mixed. Finally, 2-PE was added thereto, mixed, and the final pH of the resultant was adjusted to 6.7.

In particular, the concentration of each of the ingredients of the combination vaccine preparation was as follows: D: 30 Lf/mL; T: 20 Lf/mL; wP: 36 OU/mL; hepatitis B vaccine (hepatitis B antigen, HBsAg): 20 ㎍/mL; Hib: 10 ㎍/mL; and sabin IPV (type 1: 60 DU/mL; type 2: 64 DU/mL; and type 3: 90 DU/mL).

The combination vaccines were prepared in Comparative Examples 1 to 3 according to the concentration of 2-PE, and the content of 2-PE was measured using HPLC and the results are shown in Table 2 below. For formaldehyde, the content was measured by analyzing using a color reaction method. The formaldehyde below detoxifies or inactivates diphtheria (D), tetanus (T), and whole cell pertussis (wP) vaccines among the antigens constituting the hexavalent combination vaccines, and thus the measured values represent the formaldehyde derived from the antigen stock solution remained in the hexavalent combination vaccines.

	Target PE Conc.	Experimental Value of PE	Experimental Value of Formaldehyde
Comparative Example 1	5.2 mg/mL	5.2 mg/mL	37.0 ㎍/mL
Comparative Example 2	6.5 mg/mL	6.7 mg/mL	37.5 ㎍/mL
Comparative Example 3	7.8 mg/mL	8.0 mg/mL	37.3 ㎍/mL

As can be confirmed in Table 2 above, 2-PE as a preservative was contained at the level of the target concentration and it was confirmed that formaldehyde was contained in a concentration of about 37 ㎍/mL (Table 2).

Example 3: Antimicrobial ability test

Antimicrobial ability test was performed according to the test method described in the EP-B. The hexavalent combination vaccine preparations of Comparative Examples 1 to 3 were inoculated with a total of four microorganisms including two different kinds of bacteria (Pseudomonas aeruginosa (ATCC NO. 9027, PA) and Staphylococcus aureus (ATCC NO. 6538, SA)), one yeast (Candida albicans (ATCC NO. 10231, CA)), and one fungus (Aspergillus niger (ATCC NO. 16404, AN)) in an amount of 10⁵ to 10⁶ CFU/mL into vaccine preparations at 0 hours, respectively. Then, bacteria samples were collected at 0 hours, 24 hours, day 7, and day 28, cultured in solid media, and the number of colonies was counted between day 3 and day 5 and the log reduction of the colonies was calculated. Yeast and fungi samples were collected at 0 hours, day 14, and day 28, cultured in solid media, and the number of colonies was counted between day 3 and day 5 and the log reduction of the colonies was calculated. The results are shown in Table 2 below.

	Sampling Time	Log Reduction (log CFU/mL reduction)
		Bacteria			Fungi and Yeasts
		P.A.	S.A.	EP-BCriteria	C.A.	A.N.	EP-BCriteria
Comparative Example 1(2-PE: 5.0 mg/mL)	24 hours	*NR	0.5	1 or higher	-	-	-
	7 days	NR	2.1	3 or higher	-	-	-
	14 days	-	-	-	3.4	0.7	1 or higher
	28 days	NR	NR	**NI	NR	NR	NI
Comparative Example 2(2-PE: 6.5 mg/mL)	24 hours	NR	0.6	1 or higher	-	-	-
	7 days	NR	2.0	3 or higher	-	-	-
	14 days	-	-	-	4.6	1.1	1 or higher
	28 days	NR	NR	NI	NR	4.0	NI
Comparative Example 3(2-PE: 8.0 mg/mL)	24 hours	NR	0.7	1 or higher	-	-	-
	7 days	NR	2.2	3 or higher	-	-	-
	14 days	-	-	-	NR	1.0	1 or higher
	28 days	NR	NR	NI	NR	NR	NI

*NR: No Recovery

**NI: No Increase

Referring to Table 3, the hexavalent combination vaccines of Comparative Examples 1 to 3 containing 2-PE in an amount of 5 mg/mL to 8 mg/mL failed to pass the antimicrobial ability test criteria of the EP-B with respect to Staphylococcus aureus (ATCC NO. 6538, SA). The hexavalent combination vaccines of Comparative Examples 1 to 3 met the test criteria of the EP-B with respect to Pseudomonas aeruginosa (ATCC NO. 9027, PA) and Candida albicans (ATCC NO. 10231, CA), and the hexavalent combination vaccines of Comparative Examples 2 and 3 met the test criteria of the EP-B with respect to Aspergillus niger (ATCC NO. 16404, AN). As a result, it was confirmed that when the combination vaccines contained 2-PE in an amount of 5 mg/mL to 8 mg/mL, the combination vaccines did not meet the antimicrobial ability of the EP-B. The IPV-containing vaccines in the art conventionally contain 2-PE as a preservative in an amount of 5 mg/mL. However, contrary to the expectation, the hexavalent combination vaccines of the present invention failed to meet the required antimicrobial ability of the EP-B, even when the 2-PE content was increased to 8.0 mg/mL.

For the screening of preservatives, a screening method for preservatives that can minimize the 2-PE content within the range of the preservatives used in the existing vaccine products using 2-PE with certified stability of the IPV antigen in IPV-containing vaccines was designed. From the result shown in Table 3, the hexavalent combination vaccines containing 2-PE were shown to have poor antimicrobial ability with respect to Staphylococcus aureus (ATCC NO. 6538, SA), and thus the screening of preservatives was performed directed toward the selection of the preservative(s) which meet the criteria of the EP-B at 24 hours with respect to SA.

Example 4: Preparation of a hexavalent combination vaccine containing 2-PE and formaldehyde as preservatives

Diphtheria (D), tetanus (T), and whole cell pertussis (wP) vaccines were treated with aluminum phosphate (Brenntag) in a concentration of 1 mg/mL and stirred to be mixed well. Then, a hepatitis B vaccine (hepatitis B antigen, HBsAg), a Haemophilus influenzae type b (Hib) conjugate vaccine, and an inactivated polio vaccine were sequentially added thereto and mixed. Finally, 2-PE and formaldehyde were added thereto, mixed, and the final pH of the resultant was adjusted to 6.7.

In particular, the concentration of each of the ingredients of the combination vaccine preparation was as follows: D: 30 Lf/mL; T: 20 Lf/mL; wP: 36 OU/mL; hepatitis B vaccine (hepatitis B antigen, HBsAg): 20 ㎍/mL; Hib: 10 ㎍/mL; and Sabin IPV (type 1: 10 DU/mL; type 2: 16 DU/mL; and type 3: 32 DU/mL).

Various concentrations of 2-PE and formaldehyde in Experimental Examples are summarized in Table 4 below. In Examples 1, 4, 7, and 10, formaldehyde was not additionally added. The concentration of 2-PE was measured by HPLC analysis and the concentration of formaldehyde was measured by a color reaction method. The results are shown in Table 4 below.

	Target 2-PE Conc.(mg/mL)	Experimental Value of 2-PE(mg/mL)	Target Formaldehyde Conc. (㎍/mL)	Experimental Value of Formaldehyde (㎍/mL)
Experimental Example 1	5.0	5.1	-	37.3
Experimental Example 2	5.0	5.0	100	91.0
Experimental Example 3	5.0	5.0	150	140.9
Experimental Example 4	6.0	5.8	-	37.5
Experimental Example 5	6.0	5.6	100	91.5
Experimental Example 6	6.0	6.0	150	142.3
Experimental Example 7	7.2	6.9	-	37.2
Experimental Example 8	7.2	7.1	100	91.8
Experimental Example 9	7.2	7.2	150	142.9
Experimental Example 10	8.5	8.2	-	37.3
Experimental Example 11	8.5	8.3	100	93.7
Experimental Example 12	8.5	8.5	130	127.1
Experimental Example 13	8.5	8.5	150	142.8

As can be seen in Table 4 above, it was confirmed that the concentrations of 2-PE and formaldehyde as preservatives were contained in the hexavalent combination vaccines to be suitable for the target values. In Examples 1, 4, 7, and 10, a diphtheria (D) vaccine, a tetanus (T) vaccine, and an inactivated polio vaccine (IPV), among the antigens constituting the hexavalent combination vaccines without additionally containing 2-PE, were detoxified or inactivated with formaldehyde, and thus the formaldehyde derived from the antigen stock solution remained in the hexavalent combination vaccines and the formaldehyde concentration was confirmed to be about 37 ㎍/mL. After confirming the concentration of the preservatives, the antimicrobial ability test was performed.

Example 5: Screening of antimicrobial ability

For antimicrobial ability screening, Staphylococcus aureus (ATCC NO. 6538, SA), which is not readily killed by 2-PE, was selected as the bacteria for screening based on the result confirmed in Example 3, and the experiment was performed directed toward the selection of a combination of each dosage of 2-PE and formaldehyde that meets the criteria of the EP-B by evaluating the antimicrobial ability using the samples collected at 24 hours. Accordingly, the tests were performed according to the test method of the EP-B.

Specifically, the hexavalent combination vaccine preparations of Experimental Examples 1 to 13, prepared in Example 4, were inoculated with Staphylococcus aureus (ATCC NO. 6538, SA) in an amount of 10⁵ to 10⁶ CFU/mL into vaccine preparations at 0 hours. The samples were collected at 0 hours and 24 hours, cultured in solid media, and the number of colonies was counted between day 3 and day 5 and the log reduction of the colonies was calculated. The results are shown in Table 5 below.

	Target 2-PE Conc.(mg/mL)	Target Formaldehyde Conc. (㎍/mL)	SA Antimicrobial Ability (24 Hours) EP-B Criteria
Experimental Example 1	5.0	-	Fail
Experimental Example 2	5.0	100	Fail
Experimental Example 3	5.0	150	Fail
Experimental Example 4	6.0	-	Fail
Experimental Example 5	6.0	100	Fail
Experimental Example 6	6.0	150	Fail
Experimental Example 7	7.2	-	Fail
Experimental Example 8	7.2	100	Fail
Experimental Example 9	7.2	150	Pass
Experimental Example 10	8.5	-	Fail
Experimental Example 11	8.5	100	Fail
Experimental Example 12	8.5	130	Pass
Experimental Example 13	8.5	150	Pass

As can be seen in Table 5 above, the antimicrobial ability of the SA passed the criteria of the EP-B, starting from the combination of each dosage of 2-PE (7.2 mg/mL) and formaldehyde (150 ㎍/mL). Surprisingly, the present inventors have discovered that the combination of 2-PE and formaldehyde, as preservatives, showed a synergistic effect in antimicrobial ability and the combination of each dosage of 2-PE (7.2 mg/mL) and formaldehyde (150 ㎍/mL) or higher was suitable as preservatives for the multiple-dose hexavalent combination vaccines.

Based on the above results, the present inventors continued to develop multiple-dose hexavalent combination vaccines containing 2-PE (5 mg/mL), which was conventionally used in the existing IPV-containing vaccines, in order to solve the issue of lacking antimicrobial ability.

Example 6: Preparation of a hexavalent combination vaccine containing 2-PE (8.5 mg/mL) and formaldehyde ( 150 ㎍ /mL)

A hexavalent combination vaccine with a novel composition, i.e., that of Experimental Example 14, was prepared based on the result of Example 5. Specifically, the hexavalent combination vaccine of Experimental Example 14 was prepared by the method described below. Diphtheria (D), tetanus (T), and whole cell pertussis (wP) vaccines were treated with aluminum phosphate (Brenntag) in a concentration of 1 mg/mL and stirred to be mixed well. Then, a hepatitis B vaccine (hepatitis B antigen, HBsAg), a Haemophilus influenzae type b (Hib) conjugate vaccine, and an inactivated polio vaccine were sequentially added thereto and mixed. Finally, 2-PE (8.5 mg/mL) and formaldehyde (115 ㎍/mL) were added thereto to a final concentration of 150 ㎍/mL, mixed, and the final pH of the resultant was adjusted to 6.7. In particular, the concentration of each of the ingredients of the combination vaccine preparation was as follows: D: 30 Lf/mL; T: 20 Lf/mL; wP: 36 OU/mL; hepatitis B vaccine (hepatitis B antigen, HBsAg): 20 ㎍/mL; Hib: 10 ㎍/mL; and Sabin IPV (type 1: 60 DU/mL; type 2: 64 DU/mL; and type 3: 90 DU/mL).

As a result of performing an antimicrobial ability test on the hexavalent combination vaccine of Experimental Example 14 according to the method of Example 3, the combination vaccine was confirmed to meet the criteria of the EP-B. The content of the preservatives was tested after storing the combination vaccine at 37°C for two weeks and the recovery rate of 2-PE and formaldehyde contents was revealed to be in the range of 90% to 110%, thus confirming that the combination vaccine was stably maintained without the loss of the preservatives.

Example 7: Preparation of a hexavalent combination vaccine and antimicrobial ability test

A hexavalent combination vaccine final product was prepared by varying the composition of the inactivated polio vaccine, which is a component of the hexavalent combination vaccine, and the product was subjected to an antimicrobial ability test by the method of Example 3. In preparing the vaccine product, diphtheria (D), tetanus (T), and whole cell pertussis (wP) vaccines were treated with aluminum phosphate in a concentration of 1 mg/mL and stirred to be mixed well. Then, a hepatitis B vaccine (hepatitis B antigen, HBsAg), a Haemophilus influenzae type b (Hib) conjugate vaccine, and an inactivated polio vaccine were sequentially added thereto and mixed. Finally, 2-PE was added in a concentration of 8.5 mg/mL and formaldehyde was added to a final concentration of 130 ㎍/mL, mixed, and the final pH of the resultant was adjusted to 6.7. In particular, the concentration of each of the ingredients of the combination vaccine preparation was as follows: D: 30 Lf/mL; T: 20 Lf/mL; wP: 36 OU/mL; hepatitis B vaccine (hepatitis B antigen, HBsAg): 20 ㎍/mL; Hib: 10 ㎍/mL; and IPV (the composition of the inactivated polio vaccine is shown in Table 6 below).

	Inactivated Sabin Polio Vaccine (DU/mL)			Other Antigens	Preservative
	Type 1	Typ 2	Type 3	D: 30 Lf/mL;T: 20 Lf/mL;wP: 36 OU/mL;hepatitis B vaccine (hepatitis B antigen, HBsAg): 20 ㎍/mL;Hib: 10 ㎍/mL	2-PE: 8.5 mg/mLFormaldehyde: 130 ㎍/mL
Experimental Example 15	10	64	32
Experimental Example 16	10	32	32
Experimental Example 17	10	16	32

As a result of the antimicrobial ability test of the combination vaccine of Experimental Examples 15 to 17 were subjected to the antimicrobial ability test according to the method of Example 3, and they were all confirmed to meet the requirement of EP-B criteria.

As such, the present inventors have discovered the optimized combination for each dosage of 2-PE and formaldehyde as preservatives, for the multiple-dose hexavalent combination vaccine containing a diphtheria (D) vaccine, a tetanus (T) vaccine, a whole cell pertussis (wP) vaccine, a hepatitis B vaccine (hepatitis B antigen, HBsAg), and an improved inactivated polio vaccine (IPV), thus enabling provision of a multiple-dose composition capable of maintaining the antimicrobial ability of the hexavalent combination vaccine based on the whole cell pertussis (wP) vaccine.

Summarizing the above results, it was confirmed that the combination of 2-PE and formaldehyde according to the present invention is an excellent preservative that can maintain the antimicrobial ability of the combination vaccine and also that it can be effectively used for providing a multiple-dose composition capable of maintaining the antimicrobial ability of the multivalent combination vaccine.

From the foregoing, a skilled person in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present invention. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present invention. On the contrary, the present invention is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the present invention as defined by the appended claims.

Claims

A multiple-dose immunogenic composition for a multivalent combination vaccine, wherein:

(i) the multivalent combination vaccine comprises a diphtheria (D) vaccine, a tetanus (T) vaccine, a whole cell pertussis (wP) vaccine or acellular pertussis (aP) vaccine, and a Sabin inactivated polio vaccine (sIPV); and

(ii) the composition comprises 2-phenoxyethanol (2-PE) and formaldehyde (HCHO) as preservatives.
The composition of claim 1, wherein the multivalent combination vaccine further comprises a hepatitis B vaccine (hepatitis B antigen, HBsAg) and a Haemophilus influenzae type b (Hib) conjugate vaccine.
The composition of claim 1, wherein the composition comprises 2-PE in an amount of 7 mg/mL to 9.5 mg/mL.
The composition of claim 1, wherein the composition comprises formaldehyde in an amount of 120 ㎍/mL to 180 ㎍/mL.
The composition of claim 1, wherein the composition comprises 2-PE in an amount of 7 mg/mL to 9.5 mg/mL and formaldehyde in an amount of 120 ㎍/mL to 180 ㎍/mL.
The composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable adjuvant, wherein the adjuvant is aluminum phosphate, aluminum hydroxide, or a combination thereof.
The composition of claim 6, wherein the composition comprises the adjuvant in an amount of 1.25 mg/mL or less.
The composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable transporter, excipient, binder, carrier, preservative, buffering agent, isotonic agent, emulsifier, or humectant.
The composition of claim 1, wherein the composition has a pH of 5.5 to 8.
The composition of claim 2, wherein the multivalent combination vaccine comprises a diphtheria (D) vaccine in an amount of 10 Lf/mL to 50 Lf/mL; a tetanus (T) vaccine in an amount of 5 Lf/mL to 30 Lf/mL; a whole cell pertussis (wP) vaccine or acellular pertussis (aP) vaccine in an amount of 20 OU/mL to 50 OU/mL; a hepatitis B vaccine (hepatitis B antigen, HBsAg) in an amount of 5 ㎍/mL to 30 ㎍/mL; a Haemophilus influenzae type b (Hib) conjugate vaccine in an amount of 5 ㎍/mL to 20 ㎍/mL; and a Sabin inactivated polio vaccine (sIPV), which comprises type 1, type 2, and type 3, wherein type 1 is comprised in an amount of 5 DU/mL to 100 DU/mL, type 2 is comprised in an amount of 10 DU/mL to 100 DU/mL, and type 3 is comprised in an amount of 20 DU/mL to 120 DU/mL.
A method for preparing a multiple-dose immunogenic composition for a multivalent combination vaccine, comprising:

(a) mixing a diphtheria vaccine (D), a tetanus vaccine (T), and a whole cell pertussis (wP) vaccine or acellular pertussis (aP) vaccine with aluminum phosphate, aluminum hydroxide or a combination thereof;

(b) adding a hepatitis B vaccine (hepatitis B antigen, HBsAg), a Haemophilus influenzae type b (Hib) conjugate vaccine, and a Sabin inactivated polio vaccine (sIPV) to the mixture in step (a) and mixing; and

(c) adding 2-phenoxyethanol (2-PE) and formaldehyde to the mixture in step (b), mixing, and adjusting the final pH of the mixture to 5.5 to 8.