EA037283B1 - Combination vaccine composition for multiple-dosage - Google Patents

Abstract

The 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 invention can be effectively used in the multiple-dose combination vaccine industry.

Description

Technology area

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

Prior art

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

Although the amount and full range of antigens contained in vaccines varies according to the age group intended for use (i.e. infants, young children, children, adolescents, and adults), more recently, DTwP (diphtheria-pertussis adsorbed) combination vaccines tetanus vaccine) and DTaP (diphtheria tetanus pertussis toxoid) are widely used throughout the world. In particular, DTwP vaccines are exported to less developed countries through international organizations such as the UN.

To obtain a vaccine against diphtheria (D), diphtheria toxin is detoxified with formalin to obtain diphtheria toxoid, which is used as a vaccine. Symptoms of diphtheria, an upper respiratory tract infection, may include the development of a non-separable film in the tonsils, pharynx, and nose for no specific reason, causing a sore throat and mild fever. Sometimes, diphtheria can also cause infections of the skin, conjunctiva, and reproductive organs.

To obtain a tetanus (T) vaccine, tetanus toxin is detoxified with formalin to produce tetanus toxoid, which is used as a vaccine. Tetanus is caused by Clostridium tetani, a spore-forming anaerobic gram-positive bacterium Bacillus. Tetanus is a neurological disorder in which Clostridium tetani releases an exotoxin, thereby causing severe muscle spasms. The pathogen enters the skin or mucous membranes through wounds, thereby causing tetanus, and symptoms develop slowly and gradually progress to more severe muscle contractions.

To obtain a whole cell pertussis vaccine (wP), the pertussis pathogen is detoxified by heat treatment / formalin, etc. and the resulting pertussis pathogen is used as a vaccine called whole-cell pertussis vaccine. Acellular pertussis vaccine (aP) is a vaccine made using only pertussis proteins as antigens. The causative agent of pertussis is Bordetella pertussis, a gram-positive pleomorphic Bacillus that is difficult to culture. Pertussis can occur in all age groups, but is more common in children aged 5 years or less, and newborn infants less than one month of age are most commonly hospitalized, and the relative proportion of hospitalized patients decreases with increasing age. Clinical symptoms of whooping cough can last for 6-10 weeks when there are no complications. Examples of complications may include seizures, pneumonia, encephalopathy, etc. and can lead to death.

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

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

Sabin Inactivated Poliovirus Vaccine For Injection is available in two different types: Sabin Inactivated Poliovirus Vaccine (Attenuated Type) (sIPV) and Salk Inactivated Poliovirus Vaccine (Wild Type) (wIPV). The attenuated oral polio vaccine was developed in 1961 by Dr. Albert Sabin and the live attenuated species developed by Dr. Sabin are called Sabin poliovirus strains. The recently inactivated Sabin poliovirus, which was obtained by inactivating strains of Sabin poliovirus with formalin, was developed in an injectable form and is also commercially available.

- 1,037283 products. The sIPV vaccine includes 3 different types of strains; type 1, type 2 and type 3. In 1955, Dr. Jonas Salk was able to inactivate wild-type poliovirus, allowing it to be used in an injectable type composition called 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 WHO policy for the elimination of wild-type poliovirus, sIPV has been studied and developed as commercial products.

However, a preservative must be used in multi-dose vaccine injections to avoid microbial contamination. For combination vaccine products exported to the UN, etc. in less developed countries, multi-dose vaccines containing a preservative are preferred, taking into account the climate of the countries in which the vaccines are to be used, distribution methods, costs, etc. Examples of preservatives for use in vaccine products may contain thimerosal, 2-PE, phenol, etc., and typical dosages of preservatives are known in the art. For example, thimerosal is used at a concentration of 100 μg / ml and 2-PE is used at a concentration of 5 mg / ml, and multi-dose vaccine products containing these preservatives can only be prepared commercially after passing the antimicrobial tests described in Part B. European Pharmacopoeia (EP-B) or United States Pharmacopoeia (USP).

Thimerosal (thimerosal, thiomersal, or merthiolate) is a diethylmercury derivative that has been used as a preservative for multi-dose vaccine injections since the early 1930s. Thimerosal has been used to prevent the growth of contaminating microorganisms and maintain sterile conditions during storage or use of vaccine products and many pentavalent combination vaccines (including D, T, P, Hib, and HBsAg) that have received prior WHO approval (PQ) contain thimerosal as a preservative ...

2-PE is mainly used as a preservative for cosmetics and skin pharmaceutical products, and as a preservative for vaccine injections.

In inactivated polio vaccine (below IPV) 2-PE is usually used as a preservative. According to previous information, when inactivated polio vaccine contains thimerosal, the effectiveness of this vaccine tends to decrease by 50% or more within a week, even when stored in the refrigerator, and therefore all IPV vaccine products do not contain thimerosal as a preservative, and instead 2- PE at 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 has been reported that hexavalent combination vaccines (including D, T, wP, Hib, HBsAg and IPV), which are still under development and not used in commercial products, also use 2-PE at a concentration of 5 mg / ml. (US 2011-0195087, WO 2004110480 and WO 2012-093406).

Thus, the inventors have made efforts to test the antimicrobial ability of the preservative 2-PE, which is commonly used in IPV vaccines in the art at a concentration of 5 mg / ml, in the development of hexavalent combination vaccines (including vaccines D, T, P, Hib, HBsAg and IPV). However, unexpectedly, they were unable to obtain satisfactory results meeting the criteria described in Part B of the European Pharmacopoeia. At the same time, a reckless forced increase in the amount of 2-PE (preservative) to meet the required criteria can cause safety problems in young children who are subjects of receiving combination vaccines, and can also affect the production and stability of final products, and therefore this decision should be approached carefully. ...

Description of the invention

Technical problem

The present inventors have found a combination for each dose of 2-PE and formaldehyde as preservatives for hexavalent combination vaccines including D, T, P and IPV vaccines that meet the criteria for antimicrobial ability while minimizing their content, and thereby accomplished the present invention.

Technical solution

An object of the present invention is to provide a multi-dose immunogenic composition for a multivalent combination vaccine, wherein (1) the multivalent combination vaccine comprises diphtheria vaccine (D), tetanus vaccine (T), whole cell pertussis vaccine (wP), or acellular pertussis vaccine (aP) and Sabin's inactivated polio vaccine (sIPV); and (2) 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 a multi-dose immunogenic composition for multivalent combination vaccination.

Positive effects of the invention

It has been confirmed that the combined use of 2-PE and formaldehyde in accordance with the present invention can serve as an excellent preservative capable of retaining the antimicrobial activity of a combination vaccine and can also be effectively used to obtain a multi-dose composition capable of retaining the antimicrobial activity of a multivalent

- 2,037283 combination vaccine. Accordingly, 2-PE and formaldehyde can be effectively used in the field of multi-dose combination vaccines.

Best Mode for Carrying Out the Invention

To achieve the above objects, in one aspect of the present invention, there is provided a multi-dose immunogenic composition for a multivalent combination vaccine, wherein (1) the multivalent combination vaccine comprises diphtheria (D) vaccine, tetanus (T) vaccine, whole cell pertussis (wP) vaccine, or acellular vaccine pertussis (aP) and Sabin's inactivated polio vaccine (sIPV); and (2) the composition contains 2-phenoxyethanol (2-PE) and formaldehyde (HCHO) as preservatives.

In a typical embodiment, the present invention provides a multi-dose immunogenic composition that further comprises, as a multivalent combination vaccine, hepatitis B vaccine (hepatitis B antigen, HBsAg) and Haemophilus influenzae type b (Hib) conjugate vaccine.

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

In another typical embodiment, the composition contains formaldehyde in an amount of 120 to 180 μg / ml.

In yet another typical embodiment, the composition comprises 2-PE in an amount of 7 to 9.5 mg / ml and formaldehyde in an amount of 120 to 180 μg / ml.

In yet another typical embodiment, the composition comprises a pharmaceutically acceptable adjuvant, wherein the adjuvant is aluminum phosphate, aluminum hydroxide, or a combination thereof.

In yet another typical embodiment, the composition contains an adjuvant in an amount of 1.25 mg / ml or less.

In yet another typical embodiment, the composition comprises a pharmaceutically acceptable transporter, excipient, binder, carrier, preservative, buffering agent, isotonic agent, emulsifier, or humectant.

In yet another typical embodiment, the composition has a pH of 5.5 to 8.

In yet another typical embodiment, the multivalent combination vaccine comprises diphtheria (D) vaccine in an amount of 10 IU (flocculating units) / ml to 50 IU / ml; tetanus vaccine (T) in an amount of 5 to 30 PU / ml; whole cell pertussis vaccine (wP) or acellular pertussis vaccine (aP) in an amount of 20 IU (turbidity units) / ml to 50 IU / ml; hepatitis B vaccine (hepatitis B antigen, HBsAg) in an amount of 5 to 30 μg / ml; Haemophilus influenzae type b (Hib) conjugate vaccine in an amount of 5 to 20 μg / ml; and Sabin's inactivated polio vaccine (sIPV), which includes type 1, type 2 and type 3, where type 1 is contained in an amount of 5 to 100 IU (D antigenic units) / ml, type 2 is contained in an amount of 10 to 100 IU / ml and type 3 are contained in an amount of 20 to 120 U / ml.

In another aspect, the present invention provides a method for preparing a multi-dose immunogenic composition for a multivalent combination vaccine comprising (a) mixing diphtheria vaccine (D), tetanus vaccine (T) and whole cell pertussis vaccine (wP) or acellular pertussis vaccine (aP) with aluminum phosphate, aluminum hydroxide, or a combination thereof; (b) adding hepatitis B vaccine (hepatitis B antigen, HBsAg), Haemophilus influenzae type b (Hib) conjugate vaccine, and Sabin's inactivated polio vaccine (sIPV) to the mixture from step (a) and mixing; and (c) adding 2-PE and formaldehyde to the mixture from step (b), mixing and adjusting the final pH of the mixture to 5.5-8.

The present invention is explained in more detail below.

In one aspect, the present invention provides a multi-dose immunogenic composition for a multivalent combination vaccine, wherein:

(1) the multivalent combination vaccine contains diphtheria (D) vaccine, tetanus (T) vaccine, whole cell pertussis vaccine (wP) or acellular pertussis vaccine (aP), and Sabin's inactivated polio vaccine (sIPV);

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

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

As used herein, the term multi-dose refers to an immunogenic composition containing a vaccine in an amount in excess of a single administration dose, which can be administered to one subject or more than one subject at different stages of administration, depending on time.

As used herein, the term multivalent combination vaccine refers to a combination of two or more vaccines that can induce immunogenicity against antigens and may include two or more vaccines that induce immunogenicity against other regions in the same antigen, or may include vaccine (s) against two or more antigens. For the purposes of the present invention, the multivalent combination vaccine of the present invention may comprise diphtheria (D) vaccine, tetanus (T) vaccine, whole cell pertussis vaccine

- 3 037283 (wP) or acellular pertussis vaccine (aP) and Sabin's inactivated polio vaccine (sIPV), and in particular a hexavalent combination vaccine additionally containing hepatitis B vaccine (hepatitis B antigen, HBsAg) and Haemophilus influenzae conjugate vaccine, (Hib), but not limited to them.

The multivalent combination vaccine can be used in liquid form, but is not limited to it.

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

In the present invention, tetanus toxin is detoxified with formalin to obtain tetanus toxoid, which is used as a vaccine. The causative agent of tetanus is Clostridium tetani, a spore-forming anaerobic gram-positive Bacillus. Tetanus is a neurological disorder in which Clostridium tetani releases an exotoxin, thereby causing severe muscle spasms. The pathogen enters the skin or mucous membranes through wounds, thereby causing tetanus, and symptoms develop slowly and gradually progress to more severe muscle contractions.

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

In the present invention, the Sabin inactivated poliovirus vaccine is of two different types: the Sabin strain (attenuated type) inactivated poliovirus vaccine (sIPV) and the Salk strain inactivated poliovirus vaccine (wild type) (wIPV). The oral attenuated oral polio vaccine was previously developed and the live attenuated species developed by Dr. Sabin are called Sabin poliovirus strains. The recently inactivated Sabin poliovirus, which was obtained by inactivating strains of Sabin poliovirus with formalin, was developed in an injectable form and has also begun to be developed in commercial products. The sIPV vaccine includes 3 different types of strains; type 1, type 2 and type 3. The wIPV vaccine is still widely used. However, according to the WHO policy for the elimination of wild-type poliovirus, sIPV has been studied and developed as commercial products.

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

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

However, in the present invention, it is necessary to use a preservative in the multi-dose vaccine injection in order to avoid contamination by microorganisms during the multiple administration process and during storage. For combination vaccine products exported to less developed countries through the UN, etc., multi-dose vaccines containing a preservative are preferred, taking into account the climate of the countries in which these vaccines are used, distribution methods, costs, etc.

Examples of a preservative for use in vaccine products may contain thimerosal, 2-PE, phenol, etc., and typical dosages of preservatives are known in the art.

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

In an exemplary embodiment, compositions were prepared comprising a hexavalent combination vaccine comprising diphtheria (D) vaccine, tetanus (T) vaccine, whole cell pertussis vaccine (wP), hepatitis B vaccine (hepatitis B antigen, HBsAg), and Haemophilus conjugate vaccine influenzae type b (Hib) and Sabin's inactivated polio vaccine (sIPV) and aluminum phosphate; and 2-PE and formaldehyde as preservatives in accordance with the concentrations and the antimicrobial activity of these compositions was measured. As a result, when the compositions contained

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2-PE at 7.2 or 8.5 mg / ml and formaldehyde at 135 or 150 μg / ml were confirmed to have antimicrobial activity meeting the requirement of EP-B Part B.

Based on the above, it was confirmed that when the hexavalent combination vaccine contained only 2-PE at the concentration described above, or contained only formaldehyde, the hexavalent combination vaccine of the present invention could not have antimicrobial activity meeting the requirements of Part B of the European Pharmacopoeia, whereas the combination the use of 2-PE and formaldehyde has a synergistic effect of antimicrobial activity, and when 2-PE and formaldehyde are used at the above concentrations, the hexavalent combination vaccine of the present invention can have antimicrobial activity meeting the requirements of Part B of the European Pharmacopoeia.

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

As used herein, the term adjuvant refers to a substance that improves the immunogenicity of the immunogenic composition of the present invention. Accordingly, adjuvants are often present for the purpose of enhancing immune responses and are well known to the person skilled in the art.

The immunogenic composition of the present invention can be easily prepared using methods and techniques well known in the art, and the composition can be administered in a variety of ways, preferably parenterally or intranasally. The composition of the present invention may further comprise a pharmaceutically acceptable transporter, excipient, binder, carrier, preservative, buffering agent, isotonic agent, emulsifier, or humectant. Examples of compositions suitable for parenteral or intranasal administration may include an aqueous and non-aqueous sterile injection solution, which may contain a solute that renders the composition of the antioxidant, buffer, bacteriostatic agent, and body fluid of the subject of interest isotonic; and an aqueous and non-aqueous sterile suspension, which may contain a suspending agent or thickening agent. The active immunogenic ingredients are often mixed with a pharmaceutically acceptable excipient such as water, saline, dextrose, glycerol, ethanol, etc. In addition, the immunogenic composition may contain a small amount of an additional substance, such as a humectant, emulsifier, pH buffering agent, binding agent, carrier, or preservative.

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

The composition of the present invention contains an isotonic agent. Examples of the isotonic agent may include amino acids, polyvalent alcohols, sodium chloride, etc., and the isotonic agent plays a role in maintaining a suitable osmotic pressure in vivo when the vaccine is administered in liquid form to 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 sugars and polyols used may include sucrose, trehalose, lactose, maltose, galactose, mannitol, sorbitol, glycerin, etc. Examples of surfactants may include nonionic surfactants such as polysorbate 20, polysorbate 80, etc. Examples of polymers may include dextran, carboxymethyl cellulose, hyaluronic acid, cyclodextrin, etc.

These substances can be used alone or in combination of two or more, but the person skilled in the art can suitably select them for use.

The composition of the present invention can have a pH of 5.5 to 8.0, but is not limited to these values.

In an exemplary embodiment, the present inventors have prepared an immunogenic composition using a hexavalent combination vaccine that contains diphtheria vaccine (D), tetanus vaccine (T), whole cell pertussis vaccine (wP), hepatitis B vaccine (hepatitis B antigen, HBsAg) and Haemophilus influenzae type b (Hib) conjugate vaccine and Sabin's inactivated polio vaccine (sIPV) and the pH of the composition was adjusted to 6.7.

The composition of the present invention may include a multivalent combination vaccine that contains diphtheria (D) vaccine, tetanus (T) vaccine or acellular pertussis vaccine (aP), hepatitis B vaccine (hepatitis B antigen, HBsAg), and Haemophilus conjugate vaccine influenzae type b (Hib) and Sabin inactivated polio vaccine (sIPV), and in particular Sabin inactivated polio vaccine (sIPV), may include type 1, type 2 and type 3.

The multivalent combination vaccine may contain the diphtheria (D) vaccine in the amount of 10 to 50 PU / ml; tetanus vaccine (T) in an amount of 5 to 30 PU / ml; whole cell pertussis vaccine (wP) or acellular pertussis vaccine (aP) in an amount of 20 to 50 IU / ml; hepatitis B vaccine (HBsAg) in an amount of 5 to 30 μg / ml; Haemophilus influenzae type b (Hib) conjugate vaccine in an amount of 5 to 20 μg / ml; Sabin's inactivated polio vaccine (sIPV), which includes type 1, type 2 and type 3, where type 1 can be present in an amount of 5 to 100 U / ml, type 2 in an amount of 10 to 100 U / ml and type 3 in an amount of 20 to 120 CU / ml.

In a typical embodiment, the present inventors have prepared an immunogenic composition using a hexavalent combination vaccine that contains the diphtheria (D) vaccine at 30 PU / ml; tetanus vaccine (T) in the amount of 20 PU / ml; whole cell pertussis vaccine (wP) at 36 IU / ml; hepatitis B vaccine (HBsAg) at 20 μg / ml; conjugate vaccine Haemophilus influenzae, type b (Hib), in an amount of 10 μg / ml; and Sabin's inactivated polio vaccine (sIPV), where type 1 was contained in an amount of 10 U / ml, type 2 in an amount of 16 U / ml and type 3 in an amount of 32 U / ml; or an immunogenic composition that contains a vaccine against diphtheria (D) in an amount of 30 PU / ml; tetanus vaccine (T) in the amount of 20 PU / ml; whole cell pertussis vaccine (wP) at 36 IU / ml; hepatitis B vaccine (HBsAg) at 20 μg / ml; conjugate vaccine Haemophilus influenzae type, b (Hib), in an amount of 10 μg / ml; and Sabin's inactivated polio vaccine (sIPV), where type 1 is present at 60 U / ml, type 2 at 64 U / ml and type 3 at 90 U / ml.

In another aspect, the present invention provides a method for preparing a multi-dose immunogenic composition for a multivalent combination vaccine, comprising:

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

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

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

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

In a typical embodiment, the inventors added diphtheria vaccine (D), tetanus vaccine (T) and whole cell pertussis vaccine (wP) and aluminum phosphate (Brenntag) at a concentration of 1 mg / ml and mixed thoroughly. Hepatitis B vaccine (hepatitis B antigen, HBsAg), Haemophilus influenzae type b (Hib) conjugate vaccine, and Sabin's inactivated polio vaccine (sIPV) were then added to the mixture in that order and mixed. Finally, 2-PE and formaldehyde were added in accordance with 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 of preventing infection in a subject, which comprises administering to the subject an immunogenic composition of the present invention.

The immunogenic composition of the present invention can be administered by any effective and convenient route, including topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, and intradermal administration.

As used herein, the term “subject” refers to a multicellular eukaryotic animal, which includes, but is not limited to, metazoans, mammals, ovids, cattle, birds, primates, and humans.

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

Implementation of the invention

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

Example 1. Protocols and criteria for testing antimicrobial activity of vaccines.

In the present invention, testing for antimicrobial activity was performed in accordance with the European Pharmacopoeia (EP-B) criteria for vaccine products, part B, between the European Pharmacopoeia and the United States Pharmacopoeia (USP).

In the specific details of this test, all four different types of microorganisms, including two different types of bacteria (Pseudomonas aeruginosa (ATCC NO. 9027, PA) and Staphylococcus aureus (ATCC NO.

- 6 037283

6538, SA)), one type of yeast (Candida albicans (ATCC NO. 10231, CA)) and one type of fungi (Aspergillus niger (ATCC NO. 16404, AN)) were inoculated at 105-10 ⁶ colony forming units (CFU) / ml in vaccine preparations at 0 h, respectively. Then the samples were collected after 24 hours, on the 7th day, on the 14th day and on the 28th day, cultured in a solid medium, and the number of colonies was counted between 3 and 5 days.

The criteria for evaluating antimicrobial activity according to part B of the European Pharmacopoeia and Pharmacopoeia of other countries in comparison with the results in accordance with the above method are shown in table. 1 below.

Table 1

Pharmacopoeia criteria Log - decrease (decrease in log CFU / ml) 6 o'clock 24 hours 7 days 14 days 28 days Bacteria EP-A 2 3 NR * EP-B - one 3 - NJ ** USP - - one 3 NI JP - - - 3 NI Mushrooms and yeast EP-A - - 2 - NI EP-B - - - one NI USP - - NI NI NI JP - - - NI NI

* NR: not highlighted ** NI: no magnification

As you can see from the table. 1 above, the criteria of the European Pharmacopoeia are more stringent than those of the USP or the Japanese Pharmacopoeia, and in particular the European Pharmacopoeia is divided into parts A and B according to the formulation. The WHO criteria refer to part B of the European Pharmacopoeia (EP-B) (EP 5.1.3. Efficacy of antimicrobial preservation, USP 37-51, Antimicrobial effectiveness testing).

The inventors of the present invention, while developing hexavalent combination vaccines containing D, T, wP, Hib, HBsAg and IPV vaccines, tested their antimicrobial activity by adding 2-PE to combination vaccines, which has traditionally been used as a preservative in IPV vaccines in the art. at a concentration of 5 mg / ml. However, unexpectedly, the result was unsatisfactory, not meeting the criteria for assessing antimicrobial activity.

At the same time, a reckless forced increase in the amount of 2-PE (preservative) to meet the required criteria can cause safety problems in young children who are subjects of receiving combination vaccines, and can also affect the production and stability of final products, and therefore this decision should be approached carefully. The amount of preservative (s) to be contained in vaccines should be selected to meet the safety requirements of the United States Pharmacopoeia, European Pharmacopoeia, WHO Pharmacopoeia, or a combination thereof.

In this regard, the inventors of the present invention conducted experiments in an attempt to develop a new composition that can meet the requirements for antimicrobial activity, containing a minimum amount of 2-PE as a preservative of a hexavalent combination vaccine that contains the wP and IPV vaccines, and thereby obtained the following results.

Example 2. Obtaining a hexavalent combination vaccine containing 2-PE as a preservative.

Diphtheria vaccine (D), tetanus vaccine (T) and whole cell pertussis vaccine (wP) were treated with aluminum phosphate (Brenntag) at a concentration of 1 mg / ml and stirred to mix well. Hepatitis B vaccine (hepatitis B antigen, HBsAg), Haemophilus influenzae type b (Hib) conjugate vaccine, and Sabin's inactivated polio vaccine (sIPV) were then added sequentially and mixed. Finally, 2-PE was added, mixed and the final pH of the resulting product was adjusted to 6.7.

In particular, the concentration of each ingredient of the combined vaccine preparation was as follows: D = 30 PU / ml; T - 20 PU / ml; wP - 36 U / ml; hepatitis B vaccine (hepatitis B antigen, HBsAg) - 20 μg / ml; Hib - 10 μg / ml; Sabin's IPV (type 1 - 60 U / ml; type 2 - 64 U / ml; type 3 - 90 U / ml).

Combination vaccines were prepared in comparative examples 1-3 according to the concentration of 2-PE, the content of 2-PE was measured using HPLC and the results are shown in table. 2 below. The formaldehyde content was measured by analysis using a colorimetric reaction. Formaldehyde below detoxifies or inactivates diphtheria (D), tetanus (T) vaccines, and whole cell pertussis (wP) vaccines among the six antigens

- 7,037283 tape combination vaccines, and thus the measured values are formaldehyde originating from the antigen stock solution remaining in the hexavalent combination vaccines.

table 2

Target PE concentration Experimental value for PE Experimental value for formaldehyde Comparative example 1 5.2 mg / ml 5.2 mg / ml 37.0 μg / ml Comparative example 2 6.5 mg / ml 6.7 mg / ml 37.5 μg / ml Comparative example 3 7.8 mg / ml 8.0 mg / ml 37.3 μg / ml

As you can see from the table. 2 above, 2-PE as a preservative was contained at the target concentration level and formaldehyde was confirmed to be present at a concentration of about 37 μg / ml (Table 2).

Example 3. Analysis of antimicrobial activity.

The analysis of antimicrobial activity was performed in accordance with the analysis method described in EP-B. The hexavalent combination vaccine formulations of Comparative Examples 1-3 were inoculated with a total of four microorganisms including two different types of bacteria (Pseudomonas aeruginosa (ATCC NO. 9027, PA) and Staphylococcus aureus (ATCC NO. 6538, SA)), one type of yeast ( Candida albicans (ATCC NO. 10231, CA)) and one type of fungi (Aspergillus niger (ATCC NO. 16404, AN)) in an amount of 10 ⁵ to 10 ⁶ CFU / ml in vaccine preparations at 0 h, respectively. Then, bacterial samples were collected at 0 o'clock, at 24 o'clock, on day 7 and on day 28, cultured in solid medium, and the number of colonies was counted between 3 and 5 days, and the logarithmic contraction of the colonies was calculated. Samples of yeast and fungi were collected at 0 h, on day 14, and on day 28, cultured in solid medium, and the number of colonies was counted between 3 and 5 days, then the logarithmic contraction of the colonies was calculated. The results are presented in table. 3 below.

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Table 3

Sampling time Log reduction (reduction of log CFU / ml) Bacteria Mushrooms and yeast R.A. S.A. Criteria EP-B S.A. A.N. Criteria EP-B 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: not highlighted ** NI: no magnification

With reference to table. 3, the hexavalent combination vaccines of Comparative Examples 13 containing 2-PE in an amount of 5 to 8 mg / ml did not meet the criteria for antimicrobial activity assay according to EP-B against Staphylococcus aureus (ATCC NO. 6538, SA). The hexavalent combination vaccines of Comparative Examples 1-3 met the EP-B assay criteria for 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 criteria for analysis according to EP-B for 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 to 8 mg / ml, these combination vaccines did not correspond to the antimicrobial activity according to the EPB. Vaccines containing IPV in the art usually contain 2-PE as a preservative at 5 mg / ml. However, contrary to expectations, the hexavalent combination vaccines of the present invention do not meet the required antimicrobial activity according to EP-B even when the 2-PE content is increased to 8.0 mg / ml.

For preservative screening, a preservative screening method has been developed that can minimize the 2-PE content in the range of preservatives used in existing vaccine products using 2-PE certified IPV antigen stability in IPV-containing vaccines. From the results presented in table. 3, it can be seen that the hexavalent combination vaccines containing 2-PE have weak antimicrobial activity against Staphylococcus aureus (ATCC NO. 6538, SA), and thus preservative screening was performed regarding the choice of preservative (s) that meet the criteria EP-B at 24 h for SA.

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Example 4. Obtaining a hexavalent combination vaccine containing 2-PE and formaldehyde as preservatives.

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

In particular, the concentration of each of the ingredients of the combined vaccine preparation was as follows: D - 30 PU / ml; T - 20 PU / ml; wP - 36 U / ml; hepatitis B vaccine (hepatitis B antigen, HBsAg) - 20 μg / ml; Hib - 10 μg / ml; and Sabin's IPV (type 1 10 U / ml; type 2 16 U / ml; and type 3 32 U / ml).

The different concentrations of 2-PE and formaldehyde in the experimental examples are summarized in table. 4 below. In examples 1, 4, 7, and 10, no additional formaldehyde was added. The concentration of 2-PE was measured by HPLC analysis, and the concentration of formaldehyde was measured by a colorimetric reaction method. The results are presented in table. 4 below.

Table 4

Target concentration of 2-PE (mg / ml) Experimental value for 2-PE (mg / ml) Target formaldehyde concentration (μg / ml) Experimental value for formaldehyde (μg / 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 you can see from the table. 4 above, it was confirmed that the concentrations of 2-PE and formaldehyde as preservatives were contained in the hexavalent combination vaccines in accordance with the target values. In examples 1, 4, 7 and 10 diphtheria vaccine (D), tetanus vaccine (T)

- 10 037283 and inactivated polio vaccine (IPV), along with the antigens constituting the hexavalent combination vaccines, additionally not containing 2-PE, were detoxified or inactivated with formaldehyde, and thus the formaldehyde originating from the original antigen solution remained in the hexavalent combination vaccines, and it was confirmed that the concentration of formaldehyde was about 37 μg / ml. After confirming the concentration of preservatives, an antimicrobial activity assay was performed.

Example 5. Screening for antimicrobial activity.

To screen for antimicrobial activity, Staphylococcus aureus (ATCC NO. 6538, SA), which is difficult to die in the presence of 2-PE, was selected as a bacteria for screening based on the result confirmed in Example 3, and the experiment was carried out in the direction of choosing a combination of each dose 2-PE and formaldehyde, which meet the criteria of EP-B, by assessing the antimicrobial activity using samples collected at 24 hours. Accordingly, the analyzes were carried out in accordance with the analysis method according to EP-B.

In particular, the hexavalent combination vaccine preparations from Experimental Examples 1-13 obtained 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 h. Samples were collected at 0 h and 24 h, cultivated in a solid medium, counted the number of colonies in the interval between 3 and 5 days, and calculated the logarithm of the contraction of colonies. The results are shown in table. 5 below.

Table 5

Target concentration of 2-PE (mg / ml) Target formaldehyde concentration (μg / ml) Antimicrobial activity against SA (24 hours) EP-B criteria Experimental Example 1 5.0 - Does not match Experimental Example 2 5.0 100 Does not match Experimental Example 3 5.0 150 Does not match Experimental Example 4 6.0 - Does not match Experimental Example 5 6.0 100 Does not match Experimental Example 6 6.0 150 Does not match Experimental Example 7 7.2 - Does not match Experimental Example 8 7.2 100 Does not match Experimental Example 9 7.2 150 Compliant Experimental Example 10 8.5 - Does not match Experimental example AND 8.5 100 Does not match Experimental Example 12 8.5 130 Compliant Experimental Example 13 8.5 150 Compliant

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As you can see from the table. 5 above, SA antimicrobial activity meets the EP-B criteria starting with a combination of each dose of 2-PE (7.2 mg / ml) and formaldehyde (150 μg / ml). Surprisingly, the present inventors found that the combination of 2-PE and formaldehyde as preservatives showed a synergistic effect of antimicrobial activity, and the combination of each dose of 2-PE (7.2 mg / ml) and formaldehyde (150 μg / ml) or higher was suitable as a preservative for multi-dose hexavalent combination vaccines.

Based on the above results, the present inventors continued to develop multi-dose hexavalent combination vaccines containing 2-PE (5 mg / ml), which is commonly used in existing IPV-containing vaccines, to solve the problem of lack of antimicrobial activity.

Example 6. Obtaining a hexavalent combination vaccine containing 2-PE (8.5 mg / ml) and formaldehyde (150 μg / ml).

The hexavalent combination vaccine with a new composition, that is, the composition from Experimental Example 14, was prepared based on the result of Example 5. In particular, the hexavalent combination vaccine from Experimental Example 14 was prepared by the method described below. Diphtheria (D), tetanus (T) vaccine and whole cell pertussis vaccine (wP) were treated with aluminum phosphate (Brenntag) at a concentration of 1 mg / ml and stirred to mix well. Then hepatitis B vaccine (hepatitis B antigen, HBsAg), Haemophilus influenzae type b (Hib) conjugate vaccine, and inactivated polio vaccine were sequentially added and mixed. Finally, 2-PE (8.5 mg / ml) and formaldehyde (115 μg / ml) were added to a final concentration of 150 μg / ml, mixed and the final pH of the resulting product was adjusted to 6.7. In particular, the concentration of each of the ingredients of the combination vaccine preparation was as follows: D - 30 PU / ml; T - 20 PU / ml; wP - 36 U / ml; hepatitis B vaccine (hepatitis B antigen, HBsAg) - 20 μg / ml; Hib - 10 μg / ml; Sabin's IPV (type 1 - 60 U / ml; type 2 - 64 U / ml; and type 3 - 90 U / ml).

As a result of the analysis of the antimicrobial activity of the hexavalent combination vaccine from Experimental Example 14 in accordance with the method of Example 3, the combination vaccine was confirmed to meet the EP-B criteria. The content of preservatives was analyzed after storing the combination vaccine at 37 ° C for two weeks and it was found that the recovery of 2-PE and formaldehyde was from 90 to 110%, thus confirming that the combination vaccine was stably maintained without loss of preservatives.

Example 7. Obtaining a hexavalent combination vaccine and analysis of antimicrobial activity.

The final product of the hexavalent combination vaccine was obtained by varying the composition of the inactivated polio vaccine, which is a component of the hexavalent combination vaccine, and the product was subjected to antimicrobial analysis by the method of Example 3. When preparing the vaccine product, diphtheria vaccine (D), tetanus vaccine (T) and whole cell pertussis vaccine (wP) was treated with aluminum phosphate at a concentration of 1 mg / ml and stirred for good mixing. Then hepatitis B vaccine (hepatitis B antigen, HBsAg), Haemophilus influenzae type b (Hib) conjugate vaccine, and inactivated polio vaccine were sequentially added and mixed. Finally, 2-PE at a concentration of 8.5 mg / ml and formaldehyde were added to a final concentration of 130 μg / ml, stirred and the final pH of the resulting product was adjusted to 6.7. In particular, the concentration of each of the ingredients of the combination vaccine preparation was as follows: D - 30 PU / ml; T - 20 PU / ml; wP - 36 U / ml; hepatitis B vaccine (hepatitis B antigen, HBsAg) - 20 μg / ml; Hib - 10 μg / ml; and IPV (the composition of the inactivated polio vaccine is shown in Table 6 below).

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Table 6

Sabin's inactivated polio vaccine (DE / ml) Other antigens Preservative Type 1 Type 2 Type 3 D: 30 PU / ml; T: 20 PU / ml; wP: 36 U / ml; vaccine against hepatitis B (hepatitis B antigen, HBsAg): 20 μg / ml; Hib: 10 μg / ml 2-PE: 8.5 mg / ml Formaldehyde: 130 μg / ml Experimental Example 15 10 64 32 Experimental 10 32 32 example 16 Experimental Example 17 10 sixteen 32

As a result of the analysis of the antimicrobial activity of the combination vaccines of Experimental Examples 15-17, they were tested for antimicrobial activity in accordance with the method of Example 3, and all of them were confirmed to meet the criteria of EP-B.

Thus, the present inventors have found an optimized combination for each dose of 2-PE and formaldehyde as preservatives for a multi-dose hexavalent combination vaccine containing diphtheria (D) vaccine, tetanus (T) vaccine, whole cell pertussis (wP) vaccine, hepatitis B vaccine (hepatitis B antigen, HBsAg) and improved inactivated polio vaccine (IPV), which allows for a multi-dose composition capable of supporting the antimicrobial activity of a hexavalent combination vaccine based on the whole cell pertussis vaccine (wP).

Combining the above results, it was confirmed that the combination of 2-PE and formaldehyde of the present invention is an excellent preservative that can maintain the antimicrobial activity of a combination vaccine, and which can also be effectively used to prepare a multi-dose composition capable of supporting the antimicrobial activity of a multivalent combination vaccine.

From the foregoing, a person skilled in the art to which the present invention relates will be able to understand that the present invention may be embodied in other specific forms without changing the technical principles or essential characteristics of the present invention. In this regard, the embodiments given herein as examples are for illustrative purposes only 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 typical 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 (10)

CLAIM 1. Immunogenic multivalent combined vaccine composition for repeated dosing, containing: (1) a multivalent liquid combination vaccine that contains diphtheria (D) vaccine, tetanus (T) vaccine, whole cell pertussis vaccine (wP) or acellular pertussis vaccine (aP), and Sabin's inactivated polio vaccine (sIPV); and (2) 2-phenoxyethanol (2-PE) and formaldehyde (HCHO) as preservatives, wherein the composition contains 2-PE in an amount of 7 to 9.5 mg / ml and formaldehyde in an amount of 120 to 180 μg / ml. - 13 037283 2. The composition according to claim 1, containing 2-phenoxyethanol in an amount of 7.2 to 8.5 mg / ml and formaldehyde in an amount of 130 to 150 μg / ml. 3. 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. 4. The composition of claim 1, further comprising a pharmaceutically acceptable adjuvant of aluminum phosphate, aluminum hydroxide, or a combination thereof. 5. A composition according to claim 4, which contains the adjuvant in an amount of 1.25 mg / ml or less. 6. The composition of claim 1, further comprising a pharmaceutically acceptable binder, carrier, buffering agent, isotonic agent, emulsifier, or humectant. 7. A composition according to claim 1, which has a pH of 5.5-8. 8. The composition according to claim 3, in which the multivalent combination vaccine contains a vaccine against diphtheria (D) in an amount from 10 IU (flocculating units) / ml to 50 IU / ml; tetanus vaccine (T) in an amount of 5 to 30 PU / ml; whole cell pertussis vaccine (wP) or acellular pertussis vaccine (aP) in an amount of 20 IU (turbidity units) / ml to 50 IU / ml; hepatitis B vaccine (hepatitis B antigen, HBsAg) in an amount of 5 to 30 μg / ml; Haemophilus influenzae type b (Hib) conjugate vaccine in an amount of 5 to 20 μg / ml; Sabin's inactivated polio vaccine (sIPV), which contains type 1, type 2 and type 3, where type 1 is contained in an amount of 5 U (D antigenic unit) / ml to 100 U / ml, type 2 is contained in an amount of 10 to 100 CU / ml and type 3 is contained in an amount of 20 to 120 CU / ml. 9. A method of obtaining a multiple dose immunogenic multivalent combined vaccine composition, including: (a) mixing diphtheria (D) vaccine, tetanus (T) vaccine and whole cell pertussis vaccine (wP) or acellular pertussis vaccine (aP) with aluminum phosphate, aluminum hydroxide, or a combination thereof; (b) adding hepatitis B vaccine (hepatitis B antigen, HBsAg), Haemophilus influenzae type b (Hib) conjugate vaccine, and Sabin's inactivated polio vaccine (sIPV) to the mixture from step (a) and mixing; (c) adding 2-phenoxyethanol (2-PE) to a final concentration of 7 to 9.5 mg / ml and formaldehyde to a final concentration of 120 to 180 μg / ml to the mixture from step (b), mixing and adjusting the final pH value of the mixture to 5.5-8.0. 10. The method according to claim 9, wherein the composition contains 2-phenoxyethanol in an amount of 7.2 to 8.5 mg / ml and formaldehyde in an amount of 130 to 150 μg / ml.