WO2018169303A1 - Polyvalent streptococcus pneumoniae vaccine composition - Google Patents

Abstract

The present invention relates to a Streptococcus pneumoniae vaccine composition and, more particularly, provides a vaccine composition comprising: (i) a capsular polysaccharide-protein conjugate; (ii) 2-phenoxyethanol (2-PE); and (iii) formaldehyde (HCHO), and a method for preparing the same.

Description

Multivalent Pneumococcal Vaccine Composition

The present invention relates to a pneumococcal ( Streptococcus pneumoniae ) vaccine composition, and more specifically, (i) capsular polysaccharide-protein conjugate, (ii) 2-phenoxyethanol , 2-PE), and (iii) formaldehyde (HCHO) and a method for preparing the same.

Pneumococcus ( Streptococcus pneumonia ; Pneumococcus) is a gram-positive and hemolytic streptococcus that is a major cause of meningitis, pneumonia, and severe invasive infectious diseases in infants, children, and the elderly worldwide. More than 1.6 million people die each year from pneumococcal disease (2008 International Health Organization), and the incidence of invasive infectious diseases caused by pneumococcal in children under 5 years old and elderly people 65 years or older with low immunity high.

Pneumococci are classified into more than 90 serotypes, depending on the structural and immunological properties of the capsular polysaccharides, the main pathogenic factors surrounding their outer membranes. It is known to be associated with 80-90% pathogenicity. The only host of pneumococci is humans, and they usually exist in colonies in the nasopharynx of healthy normal people (20-40% in infants, 5-10% in adults). In 2005, the US Centers for Disease Control and Prevention (CDC) estimated that approximately 2.1 million children under 5 years of age died from pneumonia, and 1.2 million of them died from developing countries alone annually. In the United States, pneumococcal meningitis and sepsis are reported to be about 3,000 and 50,000 each year (Peters TR, Poehling KA et al. Invasive pneumococcaldisease. JAMA 2007; 297: 1825-6). In addition, pneumoACTION, which is a database of pneumococcal disease, showed that 24,047 cases of pneumococcal infections occurred in Korean children in 2000, and 47 of them died (www.pneumoadip.org). Furthermore, according to a recent study on the analysis of pneumococcal serotypes in children and adolescents published by the Korea Centers for Disease Control and Prevention, pneumococci are the most common cause of invasive infections (43.7%) in infants aged 3 to 59 months. appear. In pneumococci causing invasive infectious diseases all over the world, multidrug resistant bacteria that are resistant to not only penicillin but also three or more drugs are increasing, further increasing the difficulty of treating pneumococcal infectious diseases.

Therefore, the need for pneumococcal vaccination for children and the elderly, which is a high risk group of pneumococcal infectious diseases, has been continuously raised.

To prevent pneumococcal infections, multivalent pneumococcal polysaccharide vaccines have been developed and approved since 1977, and these capsular polysaccharide vaccines have proven useful in preventing pneumococcal disease in elderly and high-risk patients. However, in the case of infants and children, since the maturity of the immune system is lower than that of adults, when only the polysaccharide vaccine is received, the immune system does not recognize the polysaccharide antigen as an external invading factor, so it is difficult to expect a role as a vaccine. In order to solve the problem of lowering immunogenicity of polysaccharide vaccines in infants and children, a 7-valent pneumococcal conjugate vaccine, a capsular polysaccharide-protein conjugated vaccine conjugated with a carrier protein that increases immunogenicity to polysaccharide antigens ( 7vPnC, Prevenar®) has been developed and used and many data have been reported to be effective for the prevention of invasive diseases and otitis media in infants and children. However, the use of the 7-valent vaccine induced a decrease in invasive disease caused by vaccine serotypes used in the vaccine, but also showed a relative increase in pneumococcal disease caused by some non-vaccine serotypes. Synflorix®, a 10-valent capsular polysaccharide-protein conjugate vaccine, and Prevenar13®, a 13-valent pneumococcal conjugate vaccine, in which 6 serotypes were added to the base serotype of Prevena®. Has been developed and is currently commercially available, but the possibility that the efficacy as a vaccine may not be sufficient for some of the serotypes included is reported by Andrews NJ et al, (2014) Lancet Infec Dis (14) 839; EMEA Assessment Report for Prevenar 13 (2009) EMA / 798877/2009], there is a continuing need for the development of new vaccine formulations that show higher and stable potency.

Vaccine dose injections, on the other hand, should use preservatives to prevent microbial contamination. Mixed vaccine products exported to underdeveloped countries through the United Nations have been favored in multi-dose products, including preservatives, due to the country's environment, distribution methods and costs. Preservatives used in vaccine products include chimerosal, phenoxyethanol (2-PE), phenol, and the like, and the amount of preservatives commonly used in the art. For example, chimerosal is used at a concentration of 10 μg / mL, 2-PE is used at a concentration of 5 mg / mL, and the multidose vaccine product including this is EP-B (European Pharmacopoeia B category) or USP (US Pharmacopoeia). The product must be passed the standard antiseptic test.

Chimerosal (Thimerosal, Thiomersal, merthiolate) is an ethyl mercury derivative compound that has been used as a preservative for multidose vaccine injections since the early 1930s. Chimerosal has been used for the purpose of preventing the growth of contaminating microorganisms and maintaining aseptic status when storing or using vaccine products, and a number of pentavalent liquid mixed vaccines (D, T, P, Hib) that have obtained WHO PQ (Prequalified). , HBsAg) contains chimerosal as a preservative.

2-Phenoxyethanol (2-PE) is mainly used as a preservative in cosmetics and transdermal drugs, and also as a preservative in vaccine injections.

However, the amount of these preservative components used to achieve sufficient antiseptic properties of the vaccine can lead to toxicity and / or side effects, and therefore, there is a need for the development of a technique that gives sufficient antiseptic power while reducing their use.

Examples include (i) capsular polysaccharide-protein conjugates of Streptococcus pneumoniae , (ii) 2-phenoxyethanol (2-PE), and (iii) formaldehyde It provides a pneumococcal vaccine composition comprising (Formaldehyde, HCHO). The vaccine composition may be a multiple dose vaccine composition for multiple administrations.

 Another example provides a pharmaceutical composition for the prevention or treatment of pneumococcal infection diseases comprising the pneumococcal vaccine composition described above. The prophylactic or therapeutic pharmaceutical composition may be a multiple dose pharmaceutical composition for multiple administrations.

Other examples include preparing a capsular polysaccharide-protein conjugate of Streptococcus pneumoniae , and the capsular polysaccharide-protein conjugate with 2-phenoxyethanol (2-PE). And a method for preparing stability and / or preservative (or antiseptic) enhanced pneumococcal vaccine composition or the stability and / or preservative (or preservative) of pneumococcal vaccine, comprising mixing formaldehyde (HCHO). Provide a method of promotion. The vaccine composition may be a multiple dose vaccine composition for multiple administrations. Preparing the capsular polysaccharide-protein conjugate may include linking the capsular polysaccharide and the carrier protein through -OC (NH) -NH- by performing a cyanylation method.

Examples include (i) the capsular polysaccharide-protein conjugate of Streptococcus pneumoniae , (ii) 2-Phenoxyethanol (2-PE), and (iii) Formaldehyde (HCHO). It provides a pneumococcal vaccine composition or pneumococcal immunogenic composition comprising.

In the present specification, the pneumococcal immunogenic composition refers to a composition that induces an immune response against pneumococcal, and unless otherwise stated, is used in the same sense as the pneumococcal vaccine composition.

As described herein, "comprising (predetermined components)" may mean comprising essentially or containing components other than the components described. .

The pneumococcal vaccine composition provided herein includes a capsular polysaccharide of Streptococcus pneumoniae and 2-phenoxyethanol and formaldehyde as preservatives, thereby inducing an immune response against pneumococci. It may be an immunogenic composition with enhanced stability and / or preservation (preservation) that (immunogenicity) is maintained for a long time.

The capsular polysaccharide may be a capsular polysaccharide derived from Streptococcus pneumoniae . Specifically, the capsular polysaccharide is derived from two or more of the Streptococcus pneumoniae serotypes, for example, five or more, seven or more, nine or more, eleven or more, 13 or more, or 14 or more serotypes. It may be a capsular polysaccharide. In one embodiment, the capsular polysaccharide is 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 of the Streptococcus pneumoniae serotypes. It may be one containing a capsular polysaccharide derived from 19, 14 to 17, or 14 to 15 serotypes. For example, the capsular polysaccharide is Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A. , 13 to 24 species, 13 to 19 species, 13 to 17 species, 13 to 15 species, 14 to 24 species, 14 to 19 species selected from the group consisting of 19F, 20, 22F, 23F, and 33F. It may include a capsular polysaccharide derived from a species, 14 to 17 species, or 14 to 15 serotypes. In one embodiment, the capsular polysaccharide is 13 capsulomas derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. Polysaccharides; 14 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F; 15 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 12F; 15 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 15B; Or 17 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F. It may be included, but is not limited thereto.

The capsular polysaccharide-protein conjugate may be one in which the capsular polysaccharides derived from two or more serotypes described above are individually conjugated with proteins, respectively, through conventional methods, such as covalent bonds. In one embodiment, the conjugate has a structure in which each capsular polysaccharide (specifically, the hydroxy group of the polysaccharide) and the protein (specifically, the amino group of the protein) are linked via -OC (NH) -NH- using a cyanylation method. ([Polysaccharide] -OC (NH) -NH- [protein]). When the capsular polysaccharide and the protein are conjugated using the cyanylation method as described above, compared to other methods (e.g., reduction amination method, etc.), the degree of structural modification of the capsular polysaccharide is less likely to maintain immunogenicity even after conjugation. It may be more advantageous. For example, when certain serotypes (eg 19F) are conjugated with proteins by reductive amination, depending on the reaction combination, the hexasaccharide ring structure may be cleaved and open, resulting in a decrease in immunogenicity. On the other hand, it is reported that this problem does not occur when the protein is conjugated with cyanylation.

The protein can be a carrier protein, for example, a protein that is nontoxic and nonreactive and can be obtained in sufficient amount and purity. Threatening polysaccharides from various serotypes can each be conjugated with the same or different proteins, such as the same protein.

In one embodiment, the protein may be CRM197 protein. CRM197 protein is a non-toxic variant of diphtheria toxin isolated from cultures of Corynebacterium diphtheria strain C7 (CRM197; such as grown in casamino acid and yeast extract base medium), ie, toxoid. CRM197 protein is purified from Corynebacterium diphtheria strain C7 culture via ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography, or described in conventional methods such as US Pat. No. 5,614,382 (incorporated herein by reference). It may be obtained recombinantly with reference to the method. In one embodiment, the CRM197 protein is GenBank Accession No. It may comprise an amino acid sequence of 1007216A or an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology with the sequence.

Denatured toxins derived from diphtheria other than Corynebacterium diphtheria strain C7 can also be used as carrier proteins.

Other carrier proteins available

Inactivated bacterial toxins, such as tetanus toxin modified, modified pertussis toxin, cholera toxin-modified (e. G., International Patent Application Publication No. AS as described in WO2004 / 083251 No.), Escherichia coli (E. coli), LT, Escherichia Chitocoli ST, and exotoxin A from Pseudomonas aeruginosa;

Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porins, transferrin binding protein, pneumolysine, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), group A or C5a peptidase from Group B streptococci, Haemophilus influenzae protein D;

Purified protein derivatives (PPD) of ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), tuberculin;

Variants of diphtheria toxins such as CRM197, CRM173, CRM228, CRM45 (modified toxins);

It may be one or more selected from the group consisting of.

The capsular polysaccharide-protein conjugate is derived from two or more of the Streptococcus pneumoniae serotypes, eg, at least 5, at least 7, at least 9, at least 11, at least 13, or at least 14 serotypes. It may be a polyvalent polysaccharide-protein conjugate comprising a conjugate to which each capsular polysaccharide and a carrier protein described above, such as CRM197 protein, are conjugated. In one embodiment, the capsular polysaccharide-protein conjugate is 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24 of the Streptococcus pneumoniae serotypes. , 13 to 24 valent comprising conjugates conjugated to respective capsular polysaccharides and carrier proteins such as CRM197 protein derived from 14 to 19, 14 to 17, or 14 to 15 serotypes, 13-19, 13-17, 13-15, 14-24, 14-19, 14-17, or 14-15 polysaccharide-protein conjugates. For example, the capsular polysaccharide-protein conjugate is a Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 selected from the group consisting of 18C, 19A, 19F, 20, 22F, 23F, and 33F. 13- to 24-valent, 13-valent comprising conjugates conjugated to each of the capsular polysaccharides derived from the species-19 species, 14-17 species, or 14-15 species serotypes and a carrier protein such as CRM197 protein. To 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17, or 14 to 15 polysaccharide-protein conjugates. In one embodiment, the capsular polysaccharide-protein conjugate is 13 derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. 13-valent polysaccharide-protein conjugates comprising 13 conjugates of each species of capsular polysaccharide and a transport protein such as CRM197 protein; 14-valent polysaccharide-protein conjugates derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F; Each of 15 capsular polysaccharides and carrier proteins from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 12F, For example, a 15-valent polysaccharide-protein conjugate comprising 15 conjugates to which CRM197 protein is conjugated; Each of 15 capsular polysaccharides and carrier proteins from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 15B, For example, a 15-valent polysaccharide-protein conjugate comprising 15 conjugates to which CRM197 protein is conjugated; Or 17 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, respectively. And a 17-valent polysaccharide-protein conjugate including but not limited to 17 conjugates to which a carrier protein, such as CRM197 protein, is conjugated.

The pneumococcal vaccine composition provided herein is a Pneumococcal conjugate vaccine (PCV) comprising a capsular polysaccharide-protein conjugate. The pneumococcal vaccine composition is each derived from two or more of the Streptococcus pneumoniae serotypes, eg, at least 5, at least 7, at least 9, at least 11, at least 13, or at least 14 serotypes. It may be a multivalent pneumococcal vaccine composition comprising a polysaccharide-protein conjugate conjugated with a capsular polysaccharide of and a carrier protein described above, such as CRM197 protein. In one embodiment, the pneumococcal vaccine composition is 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, among Streptococcus pneumoniae serotypes, 13 to 24, 13 to 19 species in which each capsular polysaccharide derived from 14 to 19, 14 to 17, or 14 to 15 serotypes and a carrier protein such as CRM197 protein are conjugated. , 13 to 17 species, 13 to 15 species, 14 to 24 species, 14 to 19 species, 14 to 17 species, or 14 to 15 species comprising the polysaccharide-protein conjugate , 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17, or 14 to 15 pneumococcal vaccine composition. . For example, the pneumococcal vaccine composition is Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C. , 13A to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 selected from the group consisting of 19A, 19F, 20, 22F, 23F, and 33F. 13 to 24 valent comprising a polysaccharide-protein conjugate conjugated with a capsular polysaccharide and a carrier protein such as CRM197 protein, each derived from to 19, 14 to 17, or 14 to 15 serotypes, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17, or 14 to 15 pneumococcal vaccine composition.

In one embodiment, the pneumococcal vaccine composition is

(1) each of 13 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F and a transport protein, such as A trivalent pneumococcal vaccine composition comprising 13 polysaccharide-protein conjugates to which CRM197 protein is conjugated;

(2) each of 14 capsular polysaccharides and carrier proteins from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F For example, a 14-valent pneumococcal vaccine composition comprising 14 polysaccharide-protein conjugates to which CRM197 protein is conjugated;

(3) each of 15 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 12F; A 15-valent pneumococcal vaccine composition comprising 15 polysaccharide-protein conjugates to which carrier proteins such as CRM197 protein are conjugated;

(4) each of 15 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 15B; A 15-valent pneumococcal vaccine composition comprising 15 polysaccharide-protein conjugates to which carrier proteins such as CRM197 protein are conjugated; or

(5) 17 different capillaries derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F 17-valent pneumococcal vaccine composition comprising 17 polysaccharide-protein conjugates each conjugated to a polysaccharide and a carrier protein such as CRM197 protein

Can be.

Multivalent pneumococcal vaccine compositions provided herein, such as 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 14 19-, 14-17 or 14- to 15-valent pneumococcal vaccines have significantly superior titers compared to previously developed multivalent vaccines (such as 13-valent vaccines), and can prevent and / or prevent pneumococcal infectious diseases. A very good effect can be expected for treatment.

The pneumococcal vaccine composition may be a formulation formulated in a single dose for multiple administrations or in multiple doses (multi-dose) for multiple administrations. As used herein, a "multidose" is a vaccine dose capable of more than one (eg, two or more) administrations (inoculation) to one administration (inoculation) individual, or one or one of two or more administrations (inoculation) individuals. By formulation unit can be meant comprising a vaccine dose that can be administered (inoculated) more than (eg, two or more times).

The pneumococcal vaccine composition includes a preservative in addition to two or more serotypes of pneumococcal capsular polysaccharide-protein conjugates. Preservatives usable in such vaccine compositions (eg injectable formulations) include 2-phenoxyethanol, formaldehyde, chlorobutanol, m-cresol, methylparaben, propylparaben, benzetonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol It may be one or more selected from the group consisting of phenol, chimerosal, mercury nitrate, and the like.

In one embodiment, the preservative is a multivalent pneumococcal vaccine composition as described above, for example, 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, As a form optimized for the 14-19, 14-17, or 14--15 pneumococcal vaccine composition, it may include 2-phenoxyethanol and formaldehyde. As such, by containing 2-phenoxyethanol as formaldehyde together with formaldehyde, the improved preservative effect is achieved by synergistic action of the two components while reducing the amount of 2-phenoxyethanol to reduce toxicity and / or side effects. can do.

The content of 2-phenoxyethanol contained in the multivalent pneumococcal vaccine composition of the present invention is less than 10 mg / ml, less than 7 mg / ml, 6 mg / ml or less, or 5 mg / ml or less, such as 4 mg / ml or more and less than 10 mg / ml, 4 mg / ml to 7 mg / ml, 4 mg / ml or more but less than 7 mg / ml, 4 to 6 mg / ml, 4.5 mg / ml to 6 mg / ml, 5 mg / ml to 6 mg / ml, 4 to 5 mg / ml, or 4.5 mg / It may be ml to 5 mg / ml (as used herein, “to” as used in the numerical range expression indicates a numerical range below the lower limit or above the upper limit. The same applies hereinafter). The content of 2-phenoxyethanol in the vaccine composition is preferably above the lower limit of the above range in order to exhibit an effective antiseptic effect, and below the upper limit or below the upper limit of the above range in order to have no toxicity and / or side effects or to be acceptable. .

The content of formaldehyde contained in the multivalent pneumococcal vaccine composition of the present invention is 90 to 200 μg / mL, 90 to 190 μg / mL, 90 to 180 μg / mL, 90 to 170 μg / mL, 100 to 200 μg / mL , 100 to 190 μg / mL, 100 to 180 μg / mL, or 100 to 170 μg / mL. The content of formaldehyde in the vaccine composition is preferably higher than the lower limit of the above range in order to exhibit an effective antiseptic effect, and below the upper limit of the above range in order to have no toxicity and / or side effects or be acceptable.

The vaccine composition may be at least about 3 months, at least about 6 months, at least about 1 year, at least about 1.5 years at normal storage conditions, such as at temperatures ranging from 2 ° C. to 8 ° C., 20 ° C. to 25 ° C., or about 37 ° C. , For about 2 years or more, or for about 2.5 years or more. The stability of the vaccine composition herein means that the vaccine composition maintains its original antigenicity (immunogenicity) at an equivalent level, and / or that each component is maintained without degradation or loss, and / or infections such as bacteria / viruses, etc. May mean none.

Another example provides a pharmaceutical composition for preventing or treating pneumococcal infection or pneumococcal infection disease, including the multivalent pneumococcal vaccine composition described above. Another example is the prevention of pneumococcal infection or pneumococcal infection disease comprising administering a pharmaceutically effective amount of the multivalent pneumococcal vaccine composition described above to a subject in need of prevention or treatment of pneumococcal infection or pneumococcal infection disease or Provide a method of treatment. The pneumococcal vaccine composition or pneumococcal vaccine composition used in the prophylactic or therapeutic method included in the pharmaceutical composition may be a single dose pharmaceutical composition for single administration or a multi-dose pharmaceutical composition for multiple administration.

The pneumococcal disease refers to all diseases caused by pneumococcal infection, and may be pneumonia, otitis media, sinusitis, bacteremia and the like. "Pneumonia" is a type of acute inflammatory disease of the lung parenchyma. The main infectious agents are Streptococcus pneumoniae and Klebsiella. pneumoniae ). In particular, pneumococcal pneumonia accounts for about 50% of all pneumonia, severe chills, fever, cough and chest pain, sputum is often bloody, complications that can cause pleurisy, meningitis, endocarditis, peritonitis ( Diagn. Microbiol.Infect.Dis., 2001, 39: 181-185).

The term “pneumococcus” in the present invention refers to Streptococcus pneumoniae and is generally a commensal organism that colonizes the mucosal surface of human nasopharynx. If the host's factor allows access to the lower respiratory tract of the organism, then a vigorous inflammatory response follows, which causes dense consolidation when the alveolar space fills the exudate, resulting in pneumonia May cause. The pneumococci can synthesize more than 90 structurally unique capsular polysaccharides, and the serotypes of pneumococci are classified according to the structural and immunological characteristics of these capsular polysaccharides. Thus, when a vaccine preparation is prepared using pneumococcal capsular polysaccharide, the immune response may be different depending on the type of capsular polysaccharide, that is, the serotype of pneumococcal from which capsular polysaccharide is derived.

Since the capsular polysaccharide is recognized as an antigen when administered in the body to produce an antibody against it, a vaccine composition for preventing pneumococcal (or pneumococcal infectious disease) can be prepared using the capsular polysaccharide. As used herein, the term "antigen" refers to a substance capable of specifically inducing an immune response when the substance is invaded into the body. In one embodiment Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C 13 to 24 capsular polysaccharides derived from, 19A, 19F, 20, 22F, 23F, and 33F may act as antigens, respectively.

Another example provides a method of making a pneumococcal vaccine composition having enhanced stability and / or preservative (or antiseptic) or a method of enhancing the stability and / or preservative (or antiseptic) of a pneumococcal vaccine. The method is

(1) preparing a capsular polysaccharide-protein conjugate of Streptococcus pneumoniae , and

(2) mixing the capsular polysaccharide-protein conjugate with 2-phenoxyethanol (2-PE) and formaldehyde (Formaldehyde, HCHO);

It may include.

The capsular polysaccharide, the protein, the conjugate, and the content of 2-phenoxyethanol and formaldehyde are as described above.

In this method, the capsular polysaccharide can be prepared by standard techniques known to those skilled in the art, and are not particularly limited thereto. The capsular polysaccharide can be reduced in size through hydrolysis to reduce viscosity and induce effective immunogenicity.

(1) preparing the capsular polysaccharide-protein conjugate may include linking the capsular polysaccharide and the protein through -O-C (NH) -NH- by performing a cyanylation method.

In one embodiment, the (1) preparing the capsular polysaccharide-protein conjugate,

(i) isolating or isolating and purifying capsular polysaccharides from Streptococcus pneumoniae ;

(ii) dissolving and / or hydrolyzing the separated capsular polysaccharide; And

(iii) performing a cyanylation method to connect the capsular polysaccharide and the protein through -O-C (NH) -NH-.

It may include.

In another example, the preparing of the capsular polysaccharide-protein conjugate (1) may be performed by linking the capsular polysaccharide and the protein through -OC (NH) -NH- by performing the (iii) cyanylation method. Thereafter, one or more steps selected from the ultrafiltration step, the sterile filtration step, and the adsorption step can be further performed. The cyanylation method may be performed using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr.

In one specific embodiment of the present invention, Streptococcus pneumoniae having 13 or 15 different serotypes was lysed using sodium deoxycholate, respectively, to release polysaccharides bound to cells. Then the 21 serotypes 1, 2, 3, 4, 5, 6A, 6B, 9V, 8, 9N, 10A, 11A, 12F, 15B, 17F, 18C, 19A, 19F, 20, 22F, and 23F In the case of CTAB (cetyltrimethylammonium bromide) can be purified by carrying out the CTAB process, and the three serotypes 7F, 14, and 33F that do not react with CTAB was purified using an aluminum phosphate gel (Algel) solution. The reaction with CTAB may comprise, for example, CTAB at a concentration of 1-20% (w / v) or 5-15% (w / v) in the reactant in an amount of 0.5-5% or 1-3% by weight (eg, 2 to 3% by weight for the polysaccharide derived from 23F, 1 to 2% by weight for the serotype-derived polysaccharide other than 23F) may be performed by addition, but is not limited thereto. After the reaction with CTAB, any one or more of pellet recovery after centrifugation, resuspension of the pellet with sodium chloride solution (eg, about 100-500 mM), and removal of CTAB ions with sodium iodide may be further performed. May be, but is not limited thereto. The aluminum phosphate gel reaction may be performed by adding an aluminum phosphate gel solution to the reactants in an amount of 1 to 20 wt% or 5 to 15 wt%, but is not limited thereto.

In the case of using the capsular polysaccharide as a vaccine composition, infants and children whose immune system is lower than adults may not recognize this as an antigen, and thus an immune response may not occur. Thus, in the present invention, the conjugate protein and the capsular polysaccharide are conjugated. It was prepared and used.

The "carrier protein" refers to a protein that can be covalently conjugated with the capsular polysaccharide to increase the immunogenicity of the polysaccharide antigen. Specific types thereof are as described above. In one embodiment CRM197 can be used. The carrier protein may be conjugated with the capsular polysaccharide through a standard conjugation method, and the capsular polysaccharide-carrying protein conjugate formed therefrom may be one or a plurality of capsular polysaccharides conjugated to one carrier protein.

All known methods for preparing conjugates of capsular polysaccharides and carrier proteins can be included within the scope of the present invention, wherein the conjugates are linked to the -OC (NH) -NH- group by using the cyanylation method. Has a structure. The cyanation method may be appropriately performed by those skilled in the art through known methods, for example, may be performed using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr, but is not limited thereto.

As an example for preparing conjugates of capsular polysaccharides and carrier proteins, purified capsular polysaccharides can be chemically activated and each chemically activated capsular polysaccharide can be conjugated to the carrier protein one by one to form a glycoconjugate. Cyanation activity by CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) treatment transforms the hydroxy group of the capsular polysaccharide into a cyanate group, which can be used to form a covalent bond with the amino group of the carrier protein CRM197. Can be. The cyanation reaction by the CDAP may be specifically terminated by adding 3 molar equivalents of glycine (glycine) solution to 1 molar equivalent of CDAP and adjusting the pH to 9.0, but is not limited thereto. The reaction solution and reaction conditions can be adjusted accordingly.

The capsular polysaccharide-carrying protein conjugates obtained can be purified by various methods. Examples of these methods include concentration / dialysis filtration processes, column chromatography and multilayer filtration. Purified polysaccharide-protein conjugates can be mixed and formulated into vaccine compositions of the invention and used respectively. Formulations of vaccine compositions of the invention can be carried out using methods known in the art. For example, 13 individual capsular polysaccharide-carrying protein conjugates can be formulated with a physiologically acceptable vehicle to make a composition. Examples of such vehicles may include, but are not limited to, water, buffered saline, water for injection, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycols) or dextrose solutions.

In a specific embodiment of the present invention, 1) dissolution and hydrolysis of 13 capsular polysaccharides, 2) conjugation reaction process of each capsular polysaccharide and CRM197 using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate), 3) After the conjugation reaction was terminated, 4) ultrafiltration, 5) bactericidal filtration, and 6) adsorption, 13 capsular polysaccharide-carrying protein conjugates were prepared.

As used herein, the term "vaccine" refers to a biological agent containing an antigen that immunizes a living body, and refers to an immunogen or antigenic substance that immunizes the living body by administering it to a human or an animal to prevent infection.

The vaccine composition may further include one or more selected from the group consisting of an adjuvant, a preservative, a buffer, a cryoprotectant, a salt, a divalent cation, a nonionic detergent, and a free radical oxidation inhibitor.

The term "adjuvant" in the present invention refers to a substance used to increase the immunogenicity of the immunogenic composition of the present invention. The adjuvant is often provided to enhance the immune response, which is well known to those skilled in the art. Adjuvants suitable for increasing the effectiveness of the vaccine composition of the present invention may include, but are not limited to, one or more selected from the group consisting of:

(1) aluminum salts (alum) (eg, aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.);

(2) oil-in-water emulsion formulations (with or without muramyl peptide (defined below) or other specific immune stimulating agents, such as bacterial cell wall components), for example (a) MF59 (WO 90/14837) : Contains 5% (w / v) Squalene, 0.5% (w / v) Tween 80 and 0.5% (w / v) Span 85 (optionally varying amounts of MTP-PE And may be formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (microfluidizer, Microfluidics, Newton, Mass.), (B) SAF: 10% (w / v ) Squalene, containing 0.4% (w / v) tween 80, 5% (w / v) pluronic-block polymer L121 and thr-MDP (see below), and microfluidized with submicron emulsion ( microfluidization) or vortex to form a large particle size emulsion, and (c) Ribi ™ Adjuvant System (RAS) (Corixa, Hamilton, MT): 2% (w / v) Allen, 0.2% (w / v) Tween 80 and 3-O-deacylated monophosphoryl lipid A (MPL ™) (Corixa), trehalose dicholate (TDM) and cell wall backbone described in US Pat. No. 4,912,094. One or more bacterial cell wall components from the group consisting of (CWS), preferably MPL + CWS (Detox ™);

(3) Particles using or produced from saponin adjuvant such as Quill A or STIMULON® QS-21 (Antigenics, Framingham, MA, US Pat. No. 5,057,540), such as ISCOM ( Immunostimulatory complexes));

(4) bacterial lipopolysaccharides, synthetic lipid A homologues (eg, aminoalkyl glucoseamine phosphate compounds (AGP)), or derivatives or homologues thereof (commercially available from Corixa and described in US Pat. No. 6,113,918; An example of is 2-[(R) -3-tetradecanoyloxytetradecanoylamino] ethyl 2-deoxy-4-O-phosphono-3-O-[(R) -3-tetradecanoyloxy Tetradecanoyl] -2-[(R) -3-tetradecanoyloxytetradecanoylamino] -bD-glucopyranoside, which is also known as 529 (formerly also known as RC529), which is aqueous or Formulated as a stable emulsion);

(5) synthetic polynucleotides (eg oligonucleotides containing CpG motifs (US Pat. No. 6,207,646));

(6) cytokines, such as interleukins (eg, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL 12, IL-15, IL-18, etc.) Interferon (eg gamma interferon), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (MCSF), tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, and the like;

(7) Wild type cholera toxin (CT) or mutant cholera toxin (Ze WO) wherein, for example, glutamic acid at amino acid position 29 according to WO-2000 / 18434 is replaced with another amino acid, specifically histidine. -2002/098368 and WO-2002 / 098369), pertussis toxin (PT), or E. coli heat-labile toxin (LT), in particular LT-K63, LT-R72, CT-S109, PTK9 / G129 (WO Detoxified mutants of bacterial ADP-ribosylated toxins such as -93/13302 and WO-92 / 19265; And

(8) Complement component C3d, the same complement as the trimer.

The muramyl peptides include N-acetyl-muramil-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2- (1'-2 'dipalmityl) -sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE) and the like, but is not limited thereto.

The aluminum salt adjuvant may be an aluminum-precipitated vaccine or an aluminum-adsorbed vaccine. Aluminum salts include hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, alhydrogel, Superfos, amphogel, aluminum hydroxide, aluminum hydroxyphosphate adjuvant (APA), Amorphous alumina, and the like, but is not limited thereto. APA refers to a suspension of aluminum hydroxyphosphate. When aluminum chloride and sodium phosphate are mixed in a ratio of 1: 1 (by volume), aluminum hydroxyphosphate sulfate is precipitated, and the precipitate is set to 2 to 8 μm using a high shear mixer, and then dialyzed with physiological saline solution. It can be prepared by sterilization. In one embodiment, commercially available Al (OH) 3 (eg alhydrogel or Superfos) is used to adsorb the protein. 50 to 200 g of protein can be adsorbed per mg of aluminum hydroxide, and this ratio is dependent on the protein's pi and the pH of the solvent. Low pI proteins bind more strongly than proteins with high pi. Aluminum salts can form antigen reservoirs that slowly release antigens for two to three weeks to nonspecifically activate macrophages, complement, and innate immune mechanisms.

As used herein, the term "preservative (or preservative)" means an anti-viral and / or antimicrobial agent that inhibits the growth of microorganisms in the vaccine composition, for example, chimerosal, phenoxyethanol ( 2-phenoxyethanol), formaldehyde, or mixtures thereof, but any conventional preservatives used in the art may be used.

In addition, the vaccine composition may comprise one or more physiologically acceptable buffers. For example, when the vaccine composition is an infusion or injectable, the buffer may have buffering capacity at pH 4.0 to 10.0, specifically, pH 5.0 to 9.0, more specifically pH 6.0 to 8.0. The buffer may be at least one selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate, triethanolamine buffer. .

In particular, when the vaccine composition of the present invention is intended for parenteral administration, the buffer may be selected from buffers suitable for USP. For example, buffers include monobasic acids such as acetic acid, benzoic acid, gluconic acid, glyceric acid, lactic acid; Dibasic acids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, tartaric acid; Polybasic acids such as citric acid and phosphoric acid; It may be one or more selected from the group consisting of bases such as ammonia, diethanolamine, glycine, triethanolamine, TRIS.

In addition, the vaccine composition of the present invention may include a nonionic detergent. For example, polysorbate 20 and polysorbate 80 in polyoxyethylene sorbitan esters (commonly called Tweens); Copolymers of ethylene oxide (EO), propylene oxide (PO), butylene oxide (BO) (eg DOWFAX ™); Oxoxynols having a different repeating number of oxy-1,2-ethanediyl groups, especially ostoxynol-9 (Triton-100); Ethylphenoxypolyethoxyethanol (IGEPAL CA-630 / NP-40); Phospholipids such as lecithin; Nonylphenol ethoxylates such as NP series; Polyoxyethylene fatty acid ethers (Brij surfactants) derived from lauryl, cetyl, stearyl, oleyl alcohols, in particular triethyleneglycol monolauryl ether (Brij 30); Sorbitan ethers, also known as SPANs, may include, but are not limited to, surfactants such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Tween 80 may be included in the emulsion and a mixture of nonionic detergents such as Tween 80 / Span 85 may be used. Combinations of polyoxyethylene sorbitan esters such as Tween 80 with octocinols such as Triton X-100 are also suitable, and combinations of Laureth 9 with Tween and or octosinol are also useful. Specifically, polyoxyethylene sorbitan esters such as Tween 80 may be used in an amount ranging from 0.01% (w / v) to 1% (w / v), in particular 0.1% (w / v); Octylphenoxy polyoxyethanol or nonylphenoxy polyoxyethanol (e.g. Triton X-100) may range from 0.001% (w / v) to 0.1% (w / v), in particular from 0.005% (w / v) to 0.02% ( w / v); Polyoxyethylene ethers (e.g. laureth 9) are 0.1% (w / v) to 20% (w / v), preferably 0.1% (w / v) to 10% (w / v), in particular 0.1% (w / v) to 1% (w / v) or about 0.5% (w / v).

The vaccine composition of the present invention may be formulated in the form of a single dose dose vial, multiple dose dose vial or prefilled syringe. Thus, one example provides a vial, or pre-filled syringe, comprising the above-described vaccine composition in a single dose or in multiple doses, such as more than one dose. The vaccine composition may further comprise a physiologically acceptable carrier.

As used herein, a "multidose dose" or "multidose" means a vaccine dose capable of more than one dose (inoculation) to one administration (inoculation) or one or one dose of two or more administrations (inoculation). It can mean a vaccine dose that can be administered more than once (inoculation).

Physiologically acceptable carriers used in liquid formulations include aqueous or non-aqueous solvents, suspensions, emulsions, oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, ethyl oleate. Aqueous carriers include water, alcohol / aqueous solvents, emulsions or suspensions, physiological saline, buffer solutions. Examples of oils are vegetable or animal oils, peanut oils, soybean oils, olive oils, sunflower oils, synthetic oils such as cod liver oil, marine oils, and lipids derived from milk or eggs. Vaccine compositions of the present invention may be isotonic, hypertonic or hypotonic, and pharmaceutical compositions administered by infusion or injection are preferably but is not limited to isotonicity. On the other hand, isotonicity or hypertension may be advantageous for storage of the composition. If the vaccine composition is hypertonic, it can be diluted to isotonic prior to administration. Isotonic agents for dilution can be ionic tonicity agents such as salts or nonionic tonicity agents such as carbohydrates. Ionic isotonic agents include, but are not limited to, sodium chloride, calcium chloride, potassium chloride, magnesium chloride, and the like. Nonionic isotonic agents include, but are not limited to, sorbitol, glycerol, and the like.

The amount of the conjugate at each vaccine dose may be chosen to be an amount that induces an immunoprotective response without significant side effects, which amount may vary depending on the serotype of pneumococcal. Specifically, the vaccine composition, relative to the weight of the capsular polysaccharide derived from serotype 1 (ie, relative to 1 part by weight), serotypes 2, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A Weight ratios of 0.8 to 1.2 or 0.9 to 1.1 (ie 0.8 to 1.2 parts by weight or 0.9 to 1.1, respectively) for capsular polysaccharides derived from, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, respectively. Parts by weight), and in the case of serotype 6B-derived capsular polysaccharide, a weight ratio of 1.6 to 2.4, 1.8 to 2.2, or 1.9 to 2.1 (ie, 1.6 to 2.4 parts by weight, 1.8 to 2.2 parts by weight, or 1.9 to 2.1 parts by weight). It may include, but is not limited to.

As an example for preparing the conjugates, each conjugate may comprise 0.1 to 100 μg, specifically 0.1 to 10 μg, more specifically 1 to 5 μg polysaccharide.

Most specifically, except for the capsular polysaccharide derived from serotype 6B, the remaining polysaccharides, that is, serotypes 1, 2, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B The capsular polysaccharides derived from, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F are each in the amount of 2 to 2.4 μg or 2.1 to 2.3 μg, such as about 2.2 μg, respectively. 4 to 4.8 μg, 4.2 to 4.6 μg, or 4.3 to 4.5 μg, such as about 4.4 μg, but is not limited thereto. In this case, CRM197 protein in the composition may be included in an amount of 0.1 to 100 μg, 1 to 50 μg, 20 to 40 μg, or 28 to 31 μg, such as about 29.3 μg, but is not limited thereto.

Optimal amounts of ingredients for a particular vaccine can be identified by standard studies involving the observation of an appropriate immune response in a subject. For example, the results of animal experiments can be extrapolated to determine the vaccination dose for humans. In addition, one skilled in the art can empirically determine its dose as needed.

The vaccine composition may further include aluminum element and sodium chloride, but is not limited thereto.

The vaccine composition according to the present invention can be used to protect a subject susceptible to pneumococcal and to prevent pneumococcal disease by administering a pharmaceutically effective amount in a systemic or mucosal route. The term "prevention" of the present invention refers to any action that inhibits or delays infection by the pneumococcal by administration of the vaccine composition of the present invention. As defined herein, a "pharmaceutically effective amount" refers to a dosage required to elicit an antibody that is capable of significantly reducing the probability of infection or the severity of infection. The term "administration" of the present invention refers to the introduction of certain substances into an individual in any suitable way. The vaccine composition of the present invention may be administered by inhalation route through oral, nasal, rectal, transdermal or aerosol, but may be administered by bolus or infused slowly, but is not limited thereto. The administration may be by injection via an intramuscular, intraperitoneal, intradermal or subcutaneous route; Or mucosal administration to the oral / digestive tract, airway or urogenital tract, and the like. In one embodiment, intranasal administration can be used for the treatment of pneumonia or otitis media, in which case more effective prevention of nasopharyngeal carriers of pneumococci can attenuate the infection at an early stage.

The “subject” to which the vaccine composition or pharmaceutical composition of the present invention is administered may mean a living organism to which a pathogen may be infected or a cell, tissue or culture thereof separated from the organism, which organism may be a higher vertebrate. And, more specifically, it may be a mammal such as a human, but is not particularly limited thereto.

In another embodiment of the present invention, the composition of the present invention may be administered in a single inoculation, or two, three, four or more times at appropriate intervals, but is not limited thereto. For example, routine inoculation plans for infants and newborns for invasive diseases caused by Streptococcus pneumoniae can be 2, 4, 6 and 12 to 15 months of age.

In addition, the composition may further comprise one or more proteins from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for inclusion may include all of the proteins identified in WO-2002 / 053855, as well as the proteins described in WO-2002 / 053761, within the scope of the present invention.

In one specific embodiment of the invention, 2.2 μg of each polysaccharide, except that 6B-derived polysaccharide, is 4.4 μg in a total of 0.5 mL of the vaccine composition; About 29.3 μg CRM197 transport protein; 0.5 mg of elemental aluminum (2 mg aluminum phosphate) adjuvant; About 4.25 mg sodium chloride (without preservatives) or about 3.5 mg (with preservatives); About 295 μg succinate buffer; And about 3 mg of 2-phenoxyethanol and about 60 μg of formaldehyde, such as 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24 A pvalent, 14 to 19, 14 to 17, or 14 to 15 pneumococcal vaccine composition is exemplified.

In another specific embodiment of the present invention, serum levels of rabbits inoculated with the vaccine composition confirmed higher serotype specific IgG concentrations than Prevena 13® (Table 1). In addition, it was confirmed that the functional immunogenicity test (Opsonophagocytic assay) showed a superior effect than the prebena 13 ® (Table 2). Thus, it can be seen that the vaccine composition of the present invention has a very excellent effect for the prevention of pneumococcal disease.

Another embodiment of the present invention is capsular polysaccharide-carrying protein conjugate 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19 It is an immunogenic composition for pneumococci, including species, 14 to 17 species, or 14 to 15 species. The conjugate and pneumococcus are as described above.

13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17 species of the capsular polysaccharide-carrying protein conjugate of the present invention Or, the composition comprising 14 to 15 species is 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 To 17, or 14 to 15 different serotypes include streptococcus pneumoniae-derived capsular polysaccharides, and when administered in vivo to recognize the antigen to produce antibodies It causes an immune response and can be used as an immunogenic composition against pneumococci.

Another aspect of the invention is a method of preventing pneumococcal disease by administering the vaccine composition or immunogenic composition to a subject in need thereof.

Another aspect of the invention includes 13 capsular polysaccharide-carrier protein conjugates, wherein the 13 conjugates are Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, Each of 13 capsular polysaccharides derived from 9V, 14, 18C, 19A, 19F and 23F is covalently conjugated to a carrier protein, the carrier protein is CRM197 protein, and the conjugate is a cyanylation method. To provide a use for the preparation of a vaccine composition for the prevention of pneumococcal disease, wherein the composition has a structure in which the capsular polysaccharide and the carrier protein are linked by -OC (NH) -NH- groups.

Vaccine compositions, immunogenic compositions, and prevention of pneumococcal disease are as described above.

Another aspect of the invention provides 13 isolated capillaries from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. Capsular polysaccharide is a method for producing the immunogenic composition comprising the step of conjugating each of the capsular polysaccharide and the carrier protein CRM197 to have a structure linked by -OC (NH) -NH- group using a cyanylation method.

Immunogenic compositions and methods for their preparation are as described above.

The multivalent pneumococcal vaccine composition according to the present invention comprises a multivalent conjugate of capsular polysaccharide-protein of unique conjugation structure, and includes an optimized combination and content of 2-phenoxyethanol and formaldehyde as a preservative, thereby providing excellent immunogenicity. It is characterized by excellent stability and / or preservation while maintaining. Therefore, the vaccine composition and the immunogenic composition according to the present invention can be used more safely and usefully in preventing diseases caused by pneumococci in infants, infants, children, and adults.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Preparation Example 1: Capsular Polysaccharide Preparation

1-1. Preparation of Cell Banks

Streptococcus pneumoniae with 16 different serotypes (1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 12F and 15B) Obtained from the US Center for Disease Control and Prevention (CDC), cell banks were prepared in the following manner.

Streptococcus pneumoniae strains were smeared on blood agar medium to identify pneumococci and the existing media components were removed. After growing a single colony out of 10 or more single colonies, inoculated and cultured in liquid medium (Soytone (Kerry Bio-Science) or Yeast extract (Bio-springer) -derived medium) containing no animal-derived ingredients After the addition, synthetic glycerol was added to prepare a research cell bank (RCB) containing synthetic glycerol.

Master cell banks were prepared by taking out one vial from the cell bank for which the expression of polysaccharides with unique serotypes was confirmed, proliferating the cells in a liquid medium containing no animal-derived components, and adding synthetic glycerol. One vial was taken out of the bank to proliferate cells in a liquid medium containing no animal-derived components, and then synthetic glycerol was added to prepare a cell bank for production.

The prepared cell banks were stored in an ultra-frozen state of -70 deg.

1-2. Fermentation and Polysaccharide Separation

One vial in the cell bank for preparation was thawed and inoculated into a liquid medium containing no animal-derived components. Species were cultured at 37 ± 2 ° C. in an unstirred state until reaching a constant cell density (Optical Density, OD 600). After confirming the contamination of the culture medium after completion of the species culture, inoculated into a fermentor containing a liquid medium (medium from Soytone (Kerry Bio-Science) or Yeast extract (Bio-springer)) containing no animal-derived components (OD600 = 2.5 ± 0.2).

Thereafter, the culture was carried out while maintaining the pH of the medium at 7.2 ± 0.2 using a potassium hydroxide solution sterilized with minimal stirring at 37 ± 2 ° C. Sampling was carried out after 2 hours from the start of the culture to measure the cell concentration in the culture medium and the glucose concentration in the medium. The culture was terminated when glucose in the medium was depleted.

After the incubation, an appropriate amount of sterilized 12% (w / v) sodium deoxycholate was prepared to a final concentration of 0.12% (w / v), and then added to the culture to lyse the cells and The polysaccharide bound to was liberated.

1-3. Purification of Capsular Polysaccharides

After adding phosphoric acid to the sodium deoxycholate-treated sample obtained in Preparation Example 1-3 for a long time, the pH was adjusted to 3.5 ± 0.3 with 85% phosphoric acid and allowed to react for 15 hours ± 3 hours, followed by centrifugation (17,000 xG, room temperature, 1 hour) to recover the supernatant. The recovered supernatant was passed through a depth filter (0.55-9.0 um), concentrated and buffer exchanged with phosphate buffer.

After buffer exchange, the sample was passed through an active carbon filter, and then impurities were removed in two ways:

1) For 14 serotypes of serotypes 1, 2, 3, 4, 5, 6A, 6B, 9V, 12F, 15B, 18C, 19A, 19F and 23F, ionic bonds with CTAB (cetyltrimethylammonium bromide) are possible. CTAB process was carried out, specifically 10% by weight CTAB solution was treated in an amount of 1.5% by weight (except 23F) or 2.5% (in the case of 23F) relative to the weight of the process solution and reacted for 1 hour ± 10 minutes. Thereafter, the pellets were recovered by centrifugation at 17,000 xG for 1 hour. Then, to treat sodium chloride (NaCl) and sodium iodide (NaI), 200-300 mM sodium chloride (NaCl) solution was used to resuspension the pellet recovered through the CTAB treatment, and iodide corresponding to 50% by weight of CTAB usage. Sodium (NaI) was used to remove CTAB ions. For serotype 3, 350 mM NaCl solution was used. After treatment with sodium chloride and sodium iodide, the supernatant was recovered by centrifugation and used in subsequent processes. 2) For the two serotypes of serotypes 7F and 14 that do not react with CTAB, the reaction was performed by adding an aluminum phosphate gel (Algel) solution, followed by treating 10% by weight of an aluminum phosphate gel solution with respect to the total process solution 1 The reaction was time. The supernatant obtained through centrifugation (1 l centrifugation at 17,000 × G conditions) was recovered and used for the subsequent process.

After completing the two types of impurities removal process, the sample was subjected to a depth filter and ultrafiltration (UF / DF), and then the amount of ethanol and sodium chloride was adjusted and stored in a raw form.

Preparation Example 2 Preparation of Streptococcus pneumoniae capsular polysaccharide-protein conjugate

2-1: Comparison of Conjugation Methods between Reductive Amination and Cyanylation

Purification of serotype 9V polysaccharide and CRM197 carrier protein (GenBank Accession No. 1007216A; SEQ ID NO: 1; mgaddvvdssk sfvmenfssy hgtkpgyvds iqkgiqkpks gtqgnydddw kefystdnky daagysvdne nplsgkaggv vkvtypgltk vlalkvdnae tikkelglsl teplmeqvgt eefikrfgdg asrvvlslpf aegsssveyi nnweqakals veleinfetr gkrgqdamye ymaqacagnr vrrsvgssls cinldwdvir dktktkiesl kehgpiknkm sespnktvse ekakqyleef hqtalehpel selktvtgtn pvfaganyaa wavnvaqvid setadnlekt taalsilpgi gsvmgiadga vhhnteeiva qsialsslmv aqaiplvgel vdigfaaynf vesiinlfqv vhnsynrpay spghktqpfl hdgyavswnt vedsiirtgf qgesghdiki taentplpia gvllptipgk ldvnkskthi svngrkirmr craidgdvtf crpkspvyvg ngvhanlhva fhrsssekih sneissdsig vlgyqktvdh using tkvnsklslf feiks), attempts to laminating method of reductive amino speech (reductive amination), and cyano carbonyl speech (cyanylation) The bonding yields of the two bonding methods were compared. Specific bonding process is as follows.

2-1-1. Reductive amination

11.7 mg of sodium periodate was added to the serotype 9V polysaccharide stock solution, and the polysaccharide was activated while stirring at 21 to 25 ° C. The oxidized polysaccharide was concentrated and diafiltered using a 100 KDa ultrafiltration filter and WFI (water for injection), and the remaining residual polysaccharide was lyophilized by mixing CRM197 protein with saccharide / CRM197 = 0.5 (weight ratio). Lyophilized complexes were thawed and stabilized (equilibrated) at 21-25 ° C. The equilibrated complex was dissolved by incubation (37 ± 2 ° C.) in sodium phosphate (Na ₃ PO ₄ ) buffer solution at a rate of 0.1 M per 20 g of saccharides, and then cyanoborohydride (100 mg / mL) was added to the protein and The conjugation reaction between sugars was initiated. After incubation at 37 ± 2 ° C. for about 44-52 hours, the temperature was lowered to 23 ± 2 ° C. and 1 mL of 0.9% (w / v) NaCl solution was added to the reactor. Sodium borohydride solution (100 mg / mL) was added so as to have 1.8 to 2.2 molar equivalent of sodium borohydride per mole of saccharide, and the reaction mixture was incubated with stirring at 23 ± 2 ° C. to be present in saccharide. Any aldehydes that did not react were reduced. 5 mL of 0.9% (w / v) aqueous sodium chloride solution was added to the obtained saccharide-protein conjugate mixture, and the diluted conjugate mixture was dialyzed and filtered using a 100 kDa MWCO membrane.

2-1-2. Cyanylation

A 2M NaCl polysaccharide solution was prepared by adding sodium chloride powder to a serotype 9V polysaccharide stock solution prepared without hydrolysis. In order to activate the polysaccharides, a CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) solution was added to the serotype 9V polysaccharide solution to a concentration of 0.5% (w / w) relative to the polysaccharides, followed by stirring for 15 minutes to perform the polysaccharide activation reaction. Induced. Sodium hydroxide solution was added to the reaction mixture to raise the pH until it became 9.5 ± 0.1 ° C., and then stirred for 3 minutes so that the hydroxyl group of the polysaccharide could be sufficiently activated by CDAP. CRM197 was added to the polysaccharide solution after the polysaccharide activation process so that the ratio of CRM197 to polysaccharide was 1.0% (w / w) (CRM197 weight / polysaccharide weight), and the conjugation reaction was performed at room temperature for 1 hour.

The conjugation reaction was terminated by adding 2M glycine solution in 3 molar equivalents to 1 molar equivalent of CDAP and incubating overnight at room temperature with pH adjusted to 9.0. The terminated conjugate was concentrated and diafiltered into an ultrafiltration filter through a buffer containing 0.9% (w / w) sodium chloride.

As a result, it was confirmed that the conjugate produced by the cyanation method was 4 times or more in yield compared to the conjugate produced by the reductive amination method. Thus, we prepared conjugates using the capsular polysaccharide and the cyanylation method with CRM197.

2-2. Conjugation and Purification of Conjugates

Preparation of the conjugate of the capsular polysaccharide and CRM197 of Streptococcus pneumoniae was carried out through the following steps.

Step 1. Dissolution and Hydrolysis of Capsular Polysaccharides

The original capsular polysaccharide derived from each serotype was dissolved in water for injection, respectively, so that the final concentration range was within the range described below and filtered through a 0.45 μm filter:

1) for serotypes 1, 2 and 4, in the range of 0.8 to 2.0 mg / ml,

2) for serotypes 5, 6B, 9V, 18C and 19F, in the range of 4-8 mg / ml,

3) serotypes 6A, 12F and 19A range from 8 to 12 mg / ml, and

4) for serotypes 3, 7F, 14, 15B and 23F, in the range of 2-4 mg / ml.

Each serotype was subjected to an incubation of the solution in the pH and temperature ranges described below:

1) for serotypes 1, 2, 4, 5, 6B, 7F, 14 and 23F, overnight at 70-80 ° C.,

2) for serotypes 6A and 19F, 70 to 80 ° C. for 0.5 to 4 hours,

3) For serotypes 3, 9V, 12F and 18C, incubate at pH 2.0 and 65 to 80 ° C. for 0.5 to 4 hours with phosphoric acid solution.

4) For serotypes 15B and 19A, no hydrolysis was performed.

The hydrolysis was then stopped by cooling to 21 ° C.-24 ° C. and adding sodium hydroxide to a target pH of 6.0 ± 1.0.

Step 2. Conjugation Reaction Process of Capsular Polysaccharide and CRM197

Sodium chloride powder was added to all serotypes to prepare a 2M NaCl polysaccharide solution. A solution of the appropriate CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) dissolved at a ratio of 100 mg of CDAP per mL of 1/1 acetonitrile / water for injection (v / v) in each serotype in the amounts described below. Added. In detail

1) for serotypes 6A, 9V and 14, CDAP 1 to 1.5 (w / w) relative to polysaccharides,

2) CDAP 2 (w / w) versus polysaccharide for serotypes 2 and 4,

3) CDAP 3 (w / w) versus polysaccharide for serotypes 1, 3, 7F, 15B, 19F and 19A, 4) CDAP 4 (w / w) versus polysaccharide for serotypes 5, 6B, 18C, and 23F;

5) Serotype 12F was dissolved at a CDAP 5 (w / w) to polysaccharide ratio and added to each polysaccharide solution.

Then, sodium hydroxide solution was added to raise the pH to 9.5, followed by stirring for 3 to 7 minutes so that the hydroxyl group of the polysaccharide could be sufficiently activated by CDAP. CRM197 was added to each serotype polysaccharide solution in an amount of CRM197 0.75 (w / w) relative to the polysaccharide, followed by conjugation reaction for 2 hours. Thereafter, the reaction conversion was measured using SE-HPLC, and additional CDAP was added as necessary.

Step 3. Termination of Conjugation Reaction

For all serotypes, 3-6 molar equivalents of glycine solution relative to 1 molar equivalent of added CDAP were added and the reaction was terminated by adjusting the pH to 9.0. The conjugation solution was stirred at 21 ° C.-24 ° C. for one hour and then stored overnight at 2-8 ° C. low temperature.

Step 4. Ultrafiltration

The diluted conjugation mixture was concentrated and diafiltered in an ultrafiltration filter using a minimum of 20 volumes of buffer (5 mM Succinate buffer, pH 5.8 with 150 mM NaCl). A buffer containing 0.9% (w / v) sodium chloride was used while maintaining a pH range of 5.5 to 6.5 as a buffer. Fractional molecular weight of the ultrafiltration filter was carried out using 300 kDa in all serotypes, and the permeate was discarded.

Step 5. Sterile Filtration

The residue after diafiltration was diluted to less than 0.4 g / L based on polysaccharide content concentration using a buffer (5 mM Succinate buffer (pH 5.8) with 150 mM NaCl) and filtered through a 0.22 μm filter. The filtered product was subjected to in-process control (sugar content, residual DMAP). In-process controls were performed on the filtered residue to determine if further concentration, diafiltration and / or dilution were needed.

Step 6. Adsorption

Aluminum salt (aluminum phosphate) was added to the sterile filtrate so that the final concentration was 1 mg / mL based on aluminum ions, and an additional salt was added to maintain a pH range of 5.5 to 6.5. After the adsorption, the stock solution was subjected to quality inspection to confirm quality suitability, and refrigerated at 2 to 8 ° C. until use.

Example 1 Formulation of Multivalent Pneumococcal Conjugate Vaccine

The capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 12F and 15B, respectively prepared in Preparation Example 2 Polyvalent pneumococcal polysaccharide-protein conjugates were prepared from the capsular polysaccharide-protein conjugates combined with and CRM197 protein as follows:

(1) trivalent conjugates: serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F;

(2) 15-valent A conjugates: serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 12F;

(3) 15-valent B conjugates: serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 15B.

The required amount of conjugate bulk solution was calculated based on the batch volume and the polysaccharide concentration of the conjugate stock solution. As in Step 6 of Preparation Example 2, to the conjugate bulk solution of each serotype and to the addition of aluminum phosphate, 0.85% (w / v) saline and 5 mM succinate buffer (pH 5.8) were added, Bulk solutions of each conjugate were prepared containing 0.85% sodium chloride, 5 mM succinate buffer (pH 5.8) and 1 mg / mL elemental aluminum (concentration of elemental aluminum in aluminum phosphate). Finally, as a preservative, chimerosal (85 or 100 ug / ml) or 2-phenoxyethanol (2-PE) (0, 5.0, or 10.0 mg / ml) and formaldehyde (0, 10, 25, 40, 50, 80 , 100, 120, or 170 ug / ml) was added and slowly mixed into the serotype combinations above to prepare a formulated multivalent pneumococcal conjugate vaccine composition. At this time, the concentration of each serotype capsular polysaccharide of the multivalent vaccine composition is 4.4 μg / mL (where Type 6B is 8.8 μg / mL). pH was checked and adjusted to pH 5.8 if necessary. The formulated final vaccine composition stock prepared above was filled into Type 1 borosilicate glass vials. The filled vaccine composition was stored at 2-8 ° C.

Example 2: Immunogenicity Evaluation of Multivalent Pneumococcal Conjugate Vaccines

Among the vaccine compositions prepared in Example 1 above, 13 vaccine (serum type 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) vaccines, In 0.5 mL, 2.2 μg of each capsular polysaccharide (4.4 μg of serotype 6B capsular polysaccharide); About 29.3 μg CRM197 transport protein; 0.5 mg of elemental aluminum (2 mg aluminum phosphate) adjuvant; About 4.25 mg sodium chloride; About 295 μg succinate buffer; A multivalent pneumococcal vaccine composition (named LBVE013) containing about 3 mg of 2-phenoxyethanol, and about 60 μg of formaldehyde (selected LBVE013) was selected to assess the ability of the rabbit to induce an immune response. This immunogenicity was confirmed by antigen-specific ELISA for serum IgG concentration and by Opsonophagocytic assay (OPA) for antibody function.

Prevenar 13®, used as the vaccine composition LBVE013 or positive control, was administered in New Zealand White at parking 0, parking 2 and parking 4 at planned human clinical dose (2.2 μg of each polysaccharide, exception: 4.4 μg of 6B). ) Rabbits were immunized into muscles and serum was taken at 2 week intervals after inoculation. Table 1 shows the results of IgG measurement using ELISA on the collected serum. This will be described in detail as follows.

2-1. Serotype Specific IgG Concentration Measurement

Capsular polysaccharides for each of the 13 serotypes were treated in 96-well plates at 5 μg / well and coated for 16 hours at room temperature. In order to minimize the nonspecific antigen-antibody response of the serum, serum of each individual was taken in the same amount and pooled between the same groups. Serum pool was adsorbed with 333.3 ㎍ / mL of C-PS (Cell Wall Polysaccharide; State Serum Institute) and 333.3 ㎍ / mL of capsular polysaccharide (PnPs22F) of serotype 22F at room temperature for 30 minutes. , Diluted with an appropriate dilution multiple (1: 100 ~ 1: 40000) in the antibody dilution buffer containing Tween 20. The coated plate was washed four times with washing buffer, 50 μl of previously adsorbed and diluted serum was added to the coated well-plate and allowed to react at room temperature for 1 hour. The reacted well-plates were washed four times in the same manner, and each well was added with goat anti-Rabbit IgG-HRP conjugates (1: 20000), followed by reaction at room temperature for 30 minutes. I was. Wash the plate 4 times in the same manner as above and add 100 μl of TMB substrate solution (3,3 ', 5,5'-Tetramethylbenzidine; Sigma, St. Louis, MO, USA) stabilized at room temperature in each well and then The reaction was carried out for 15 minutes at. 100 μl of 1 N sulfuric acid solution was added to stop the reaction, and the absorbance at 450 nm was measured using 650 nm as a control wavelength. For objective immunogenicity evaluation, blood samples obtained by immunizing Prevena 13® as a control were analyzed in the same manner.

The results are shown in Table 1 below.

LBVE013		Prevenar13®
Serotype	IgG (μg / mL)	Serotype	IgG (μg / mL)
19F	82.4	6A	46.5
6A	64.7	6B	22.4
14	61.1	23F	18.3
6B	59.5	19F	17.6
19A	26.7	19A	17.2
23F	21.8	14	10.1
7F	13.4	5	8.4
5	11.4	4	6.7
4	9.1	7F	6.3
One	5.3	18C	4.4
18C	4.7	3	2.7
3	3.0	One	1.6
19F	82.4	6A	46.5

As shown in Table 1, it can be seen that the serotype-specific immunogenic response pattern of the multivalent (13-valent) pneumococcal vaccine composition (LBVE013) is significantly different from that of Prevena 13®. A higher level of titer than prevena 13® was observed in all serotypes compared to prevena 13® in rabbits inoculated with the trivalent vaccine composition (LBVE013) according to this example, in particular, serotypes 1, 6B, 7F, and 9V. At 14 and 19F, they were two to six times better than Prevena 13®.

2-2. Functional immunogenicity test Opsonophagocytic  assay, OPA )

OPA analysis was performed on sera from rabbits to evaluate the function of antibodies induced by serotypes.

Specifically, the same amount of serum was taken for each individual and the same groups were pooled. Streptococcus Pneumoniae was incubated in THY medium (Todd-Hewitt Broth w / 2% Yeast Extract) for each serotype and diluted with Opsonization buffer to 200 to 300 CFU / 10 μl. 20 μl of diluted serum and 10 μl of diluted Streptococcus pneumoniae were mixed and reacted at room temperature for 30 minutes. Then 50 μl of a mixture of pre-differentiated HL-60 cells and complement (cell: complement = 4: 1) was added and reacted for 45 minutes in a CO 2 incubator (37 ° C.).

Phagocytosis was stopped by lowering the temperature, and 10 μl of the reaction solution was plated in dried agar medium for 30 to 60 minutes in advance. Next, the cells were incubated for 12 to 18 hours in a CO ₂ incubator (37 ° C.) and the number of colonies was counted. OPA titers were expressed as dilution multiples where 50% killing was observed.

The results obtained are shown in Table 2 below:

Serotype	OPA titer		LBVE013 / Prevenar-13
	LBVE013	Prevenar13
One	208	44	4.7
3	218	190	1.1
4	1775	836	2.1
5	493	228	2.2
6A	1606	1375	1.2
6B	1751	784	2.2
7F	2187	2187	1.0
9 V	1032	512	2.0
14	2187	1761	1.2
18C	1222	1035	1.2
19A	2187	1468	1.5
19F	2187	847	2.6
23F	752	320	2.4

In Table 2, for example, the OPA titer indicated as 2187 means that the titer is very high even when the most diluted section does not reach the 50% level compared to the negative control. Through the test results, it was confirmed that the multivalent pneumococcal vaccine composition according to the present example had a significantly superior serum IgG titer compared to Prevena 13®. Therefore, the multivalent pneumococcal vaccine composition according to the present embodiment can be very useful for preventing diseases caused by pneumococcal.

Reference Example : vaccine Antiseptic  Test protocols and criteria

In this example, the antiseptic test of the vaccine was conducted according to the EP-B standard, which is a standard required for vaccine products by the World Health Organization (WHO) among the US Pharmacopoeia (USP) and the European Pharmacopoeia (EP).

Specifically, the two bacteria bacteria Pseudomonas aeruginosa Four species of (ATCC No. 9027, PA), Staphylococcus aureus (ATCC No. 6538, SA), Yeast Candida albicans (ATCC No. 10231, CA), and Fungal Aspergillus niger (ATCC No. 16404, AN) were added to the vaccine composition. Hours were inoculated with 10 ⁵ to 10 ⁶ CFU / mL (CFU; Colony forming units). Thereafter, the samples were sampled at 24 hours, 7 days, 14 days and 28 days, cultured in a solid medium, and the number of colonies was counted at 3 to 5 days.

Table 3 shows the antiseptic properties of EP-B and the pharmacopoeia of the countries according to the method.

	Pharmacopoeia	Log Reduction (log CFU / mL reduction)
		6 hours	24 hours	7 days	14 days	28 days
Germ	EP-A	2	3			NR *
	EP-B	-	One	3	-	NI **
	USP	-	-	One	3	NI
	JP	-	-	-	3	NI
Yeast and Fungi	EP-A	-	-	2	-	NI
	EP-B	-	-	-	One	NI
	USP	-	-	NI	NI	NI
	JP	-	-	-	NI	NI

* NR: No Recovery ** NI: No Increase

As can be seen in Table 3 above, the EP requirements are more stringent than the USP or USP, and are classified into categories A and B according to the formulation, and the level required by the WHO for vaccine products is EP-B (EP 5.1.3.Efficacy of antimicrobial perserbation, USP 37-51, Antimicrobial effectiveness testing).

 In the present embodiment, in developing a multivalent pneumococcal polysaccharide-protein conjugated vaccine, chimerosal and 2-PE, which are vaccine preservatives commonly used in the art, were added and subjected to an antiseptic test, and chimerosal or a low concentration of 2-PE. When used alone it was confirmed that does not satisfy the antiseptic force criteria.

In addition, when using a high concentration of 2-PE (7mg / mL or more), the company has already applied for a patent and could not be used in the company.

Accordingly, the inventors of the present invention experiment to develop a new composition that can increase the antiseptic effect while minimizing the content of 2-PE in order to satisfy the antiseptic standard without infringing patents of other companies as a preservative of the multivalent pneumococcal protein conjugate vaccine. As a result, the following results were obtained.

Example 3: Antiseptic Screening of Multivalent Pneumococcal Polysaccharide-Protein Conjugate Vaccine

Two kinds of bacteria Pseudomonas aeruginosa (ATCC No. 9027, PA), Staphylococcus aureus (ATCC No. 6538, SA), Yeast Candida albicans (ATCC No. 10231, CA), among the four fungal strains of the fungus Aspergillus niger (ATCC No. 16404, AN), Staphylococcus aureus (ATCC No. 6538, SA), which is known to not die well in 2-PE, is used for antiseptic screening. The fungus was selected. The preservation force was evaluated by the sample sampled at 24 hours, and the objective was to select the 2-PE and formaldehyde combination which satisfy | fills EP-B. Accordingly, the test was conducted according to the test method of EP-B.

Specifically, referring to the multivalent pneumococcal polysaccharide-protein conjugated vaccine preparation method of Example 1, the multivalent pneumococcal protein conjugate of vaccine compositions 1 to 12 was added by adding chimerosal, or 2-PE and / or formaldehyde, as shown in Table 2 below. Vaccine compositions were prepared and these agents were inoculated with Staphylococcus aureus (ATCC No. 6538, SA) at 10 ⁵ to 10 ⁶ CFU / mL (CFU = colony forming units) at 0 hours. Thereafter, the samples were sampled at 0 hours and 24 hours, cultured in a solid medium, and the number of colonies was counted at 3 to 5 days to calculate log reduction of colonies. The results are shown in Table 4 below.

	2-PE concentration (mg / mL)	Formaldehyde concentration (µg / mL)	Chimerosal concentration (㎍ / mL)	SA antiseptic force (24 hours) EP-B standard
Vaccine Composition 1	-	-	85	Fail
Vaccine Composition 2			100	Fail
Vaccine Composition 3	5.0	-	-	Fail
Vaccine Composition 4	5.0	10	-	Fail
Vaccine Composition 5	5.0	25	-	Fail
Vaccine Composition 6	5.0	40	-	Fail
Vaccine Composition 7	5.0	50	-	Fail
Vaccine Composition 8	5.0	80	-	Fail
Vaccine Composition 9	5.0	100	-	Pass
Vaccine Composition 10	5.0	120	-	Pass
Vaccine Composition 11	5.0	170	-	Pass

As can be seen in Table 4, from the combination of 2-PE 5.0 mg / mL and formaldehyde 100 μg / mL SA preservation force passed the EP-B standard. Based on this result, it can be confirmed that a combination of about 5.0 mg / mL of 2-PE and 100 μg / mL or more of 2-PE is suitable as a preservative of the multivalent pneumococcal polysaccharide-protein conjugated vaccine.

Example  4: Multivalent Pneumococcal Polysaccharide-protein Conjugate Vaccine Antiseptic  Test 1

Antiseptic test was carried out according to the test method of EP-B. Two Bacterial Pseudomonas in Multivalent Pneumococcal Protein Conjugate Vaccine Formulations of Vaccine Compositions 13-15 Four species of aeruginosa (ATCC No. 9027, PA), Staphylococcus aureus (ATCC No. 6538, SA), yeast Candida albicans (ATCC No. 10231, CA), and fungi Aspergillus niger (ATCC No. 16404, AN) Were inoculated with 10 ⁵ to 10 ⁶ CFU / mL (CFU = colony forming units). Since bacteria were sampled at 0 hours, 24 hours, 7 days, 28 days and cultured in a solid medium to count the number of colonies on the 3 to 5 days to calculate the log reduction of colonies. Fungi and yeasts were sampled at 0 hours, 14 days, 28 days and cultured in solid medium and counted colonies on day 3 to 5 to calculate the log reduction of colonies. The results are shown in Table 5.

	Sampling time	Log Reduction (log CFU / mL reduction)
		Germ			Fungus and Yeast
		P.A.	S.A.	EP-B standard	C.A.	A.N.	EP-B standard
Vaccine Composition 13: 13-valent (2-PE 5.1 mg / mL + Formaldehyde 102 μg / mL)	24 hours	1.7	2.2	1 or more	-	-	-
	7 days	NR	NR	More than 3	-	-	-
	14 days	-	-	-	NR	1.2	1 or more
	28 days	NR	NR	NI	NR	NR	NI
Vaccine Composition 14: 15-valent A (2-PE 5.1 mg / mL + Formaldehyde 102 μg / mL)	24 hours	NR	2.1	1 or more	-	-	-
	7 days	NR	NR	More than 3	-	-	-
	14 days	-	-	-	NR	NR	1 or more
	28 days	NR	NR	NI	NR	NR	NI
Vaccine Composition 15: 15-valent B (2-PE 5.1 mg / mL + Formaldehyde 102 μg / mL)	24 hours	NR	1.9	1 or more	-	-	-
	7 days	NR	NR	More than 3	-	-	-
	14 days	-	-	-	NR	NR	1 or more
	28 days	NR	NR	NI	NR	NR	NI

* NR: No Recovery ** NI: No Increase

As shown in Table 5, two kinds of bacteria Pseudomonas aeruginosa (ATCC No. 9027, PA) in the multivalent pneumococcal polysaccharide-protein conjugated vaccine compositions 13 to 15, including the serotypes of 13-, 15-A and 15-B, respectively. , Staphylococcus aureus (ATCC No. 6538, SA), yeast Candida Antibacterial activity of four species of albicans (ATCC No. 10231, CA) and fungus Aspergillus niger (ATCC No. 16404, AN) was determined. The antiseptic force against the species of bacteria met the EP-B standard. As a result, when the preservative of the multivalent pneumococcal polysaccharide-protein conjugated vaccine contains 2-PE in the amount of about 5 mg / mL and formaldehyde in the amount of about 100 µg / mL, it was confirmed that the antiseptic capacity of EP-B standard was satisfied. .

In the art, the pneumococcal protein conjugate vaccine typically uses 2-PE 7 mg / mL or more as a preservative, and together with the result of Example 3, the result shows that when formaldehyde is added in an amount of 100 μg / mL or more, Lowering the -PE content to 5 mg / mL shows that the antiseptic power of EP-B can be achieved. Thus, the present specification provides a strong and effective antiseptic force by applying an optimized combination of 2-phenoxyethanol (2-PE) and formaldehyde (HCHO) as a preservative of the multivalent pneumococcal polysaccharide-protein conjugate vaccine. It has become possible to provide a multidose composition to maintain.

Example 5: Stability of Multivalent Pneumococcal Polysaccharide-Protein Conjugate Vaccine Compositions

Based on the results of Example 4, a multivalent pneumococcal polysaccharide-protein conjugated vaccine composition 16 was prepared. Specifically, vaccine composition 16 was prepared by the following method: 14 polysaccharide-protein conjugates (Type 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F) ) And 1 mg / mL of aluminum phosphate (based on aluminum concentration) were stirred to mix well. 5 mM succinic acid and 0.85% (w / v) sodium chloride were dissolved in distilled water for injection, followed by mixing with a sterile filtered buffer. Finally, 2-PE was added at a concentration of 6.0 mg / mL to formaldehyde at a concentration of 120 μg / mL, and then homogenized to adjust the final pH to 5.8. The concentration of each serotype in the vaccine composition thus prepared is 4.4 μg / mL (where 6B is 8.8 μg / mL).

After the vaccine composition 16 was stored at 2 to 8 ° C. for 6 months, the content of the preservative was tested. The recovery rate of 2-PE and formaldehyde was maintained at 90 to 110% without loss of the preservative. Confirmed.

Summarizing the above results, the combination of 2-PE and formaldehyde as exemplified in the examples of the present invention is an excellent composition capable of effectively maintaining the antiseptic power of the multivalent pneumococcal polysaccharide-protein conjugated vaccine for a long time, and the polyvalent polysaccharide-protein conjugate It has been found that the vaccine can be usefully used to provide multidose compositions with improved antiseptic or extended antiseptic retention periods.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (12)

1. (i) capsular polysaccharide-carrying protein conjugates,

   (ii) at least 4 mg / mL and less than 7 mg / mL 2-phenoxyethanol (2-PE), and

   (iii) formaldehyde (HCHO) 90-200 μg / mL,

   The capsular polysaccharide is a multivalent pneumococcal vaccine composition comprising 13 to 24 Streptococcus pneumoniae serotype-derived capsular polysaccharides.
2. The method of claim 1, wherein the capsular polysaccharide is Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F A polyvalent pneumococcal vaccine composition comprising 13 to 24 selected from capsular polysaccharides derived from, 18C, 19A, 19F, 20, 22F, 23F, and 33F.
3. The method of claim 2, wherein the capsular polysaccharide is

   13 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F;

   14 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F;

   15 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 12F;

   15 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 15B; Or 17 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

   That comprises, multivalent pneumococcal vaccine composition.
4. The multivalent pneumococcal vaccine composition of claim 1, wherein the carrier protein is CRM197 protein.
5. The multivalent pneumococcal vaccine composition according to claim 1, wherein the capsular polysaccharide-carrying protein conjugate has a structure in which the capsular polysaccharide and the carrier protein are connected by an -O-C (NH) -NH- group by using a cyanation method.
6. The multivalent pneumococcal vaccine composition according to claim 5, wherein the cyanation method is performed using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr.
7. According to claim 1, Per 1 part by weight of capsular polysaccharide derived from serotype 1,

   Capsular polysaccharides derived from serotypes 2, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 23F, 12F and 15B are each 0.9 to 1.1 parts by weight, and

   1.8-2.2 parts by weight of capsular polysaccharides derived from serotype 6B

   Comprising, multivalent pneumococcal vaccine composition.
8. The multi-dose multivalent pneumococcal vaccine composition according to any one of claims 1 to 7.
9. A pharmaceutical composition for preventing or treating pneumococcal infection or pneumococcal infectious disease comprising the multivalent pneumococcal vaccine composition of any one of claims 1 to 7.
10. The pharmaceutical composition of claim 9, wherein the pneumococcal disease is pneumonia.
11. (1) preparing a capsular polysaccharide-protein conjugate of Streptococcus pneumoniae , and

    (2) mixing the capsular polysaccharide-protein conjugate with 2-phenoxyethanol (2-PE) and formaldehyde (Formaldehyde, HCHO);

    Including,

    The capsular polysaccharide comprises 13 to 24 Streptococcus pneumoniae serotype derived capsular polysaccharides,

    The amount of 2-phenoxyethanol used is 4 mg / mL or more and less than 7 mg / mL, and the amount of formaldehyde is 90-200 µg / mL,

    Method for producing a multivalent pneumococcal vaccine composition.
12. 12. The method of claim 11, wherein (1) preparing the capsular polysaccharide-protein conjugate comprises performing a cyanation method to link the capsular polysaccharide and the protein through -OC (NH) -NH-. That is, a method for producing a multivalent pneumococcal vaccine composition.