KR20180105590A - Multivalent vaccine composition against streptococcus - 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. The polyvalent Streptococcus pneumoniae vaccine composition according to the present invention comprises a polyvalent conjugate of a narrow-band polysaccharide-protein having a peculiar junction structure, and contains a combination and a content of 2-phenoxyethanol and formaldehyde optimized as described above as a preservative, and is excellent in stability and/or storage power.

Description

[0001] MULTIVALENT VACCINE COMPOSITION AGAINST STREPTOCOCCUS [0002]

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

Streptococcus pneumonia ( Streptococcus pneumonia ; Pneumococcus) is a gram-positive and haemolytic streptococcus, and is a major cause of meningitis, pneumonia and severe invasive infections in infants, children and the elderly worldwide. More than 1.6 million people die each year from pneumococcal disease (2008, data from the International Health Organization), and the incidence of invasive infectious diseases due to pneumococcal infection in children aged <5 years and> 65 years, high.

Streptococcus pneumoniae is classified into more than 90 serotypes, depending on the structural and immunological characteristics of the capsular polysaccharide, a major virulence factor surrounding the cell membrane (20) It is known to be associated with pathogenicity of 80-90%. The only host of Streptococcus pneumoniae is human, and they are usually populated by healthy nasopharynx (20-40% of infants and 5-10% of adults). In 2005, the Centers for Disease Control and Prevention (CDC), in developing countries, estimated that 2.1 million children under age 5 died of pneumonia per year, of which 1.2 million died of pneumococcal infection In the United States, meningitis and sepsis due to pneumococci have been reported to occur in about 3,000 and 50,000 cases per year, respectively (Peters TR, Poehling KA et al., Invasive pneumococcal disease, JAMA 2007; 297: 1825-6). According to the pneumoACTION database, which is a database of pneumococcal disease, the incidence of pneumococcal infections in children in 2000 was 24,047 cases per year, of which 47 cases were deaths (www.pneumoadip.org). Furthermore, according to a recent study by the Center for Disease Control and Prevention, 'A Study on the Serotype Analysis of Pneumococci in Children and Adolescents in Korea', pneumococci were the most common cause of invasive infections (43.7%) in infants between 3 months and 59 months appear. In addition to penicillin, multidrug-resistant bacteria that are resistant to three or more drugs are increasing in pneumococci that cause invasive infectious diseases globally, thus adding to the difficulty of treating pneumococcal infection diseases.

Therefore, there is a continuing need for pneumococcal vaccination for children and elderly people who are at high risk for pneumococcal disease.

In order to prevent pneumococcal infections, a multivalent pneumococcal polysaccharide vaccine has been developed and approved since 1977, and such a polysaccharide polysaccharide vaccine has proven to be useful in the prevention of pneumococcal disease in elderly and high-risk patients. However, in infants and children, the maturity of the immune system is lower than that of adults. Therefore, if the polysaccharide vaccine alone is met, the vaccine can not be expected to play a role because the immune system does not recognize the polysaccharide antigen as an external invading factor. In order to solve the immunogenicity problem of the polysaccharide vaccine in infants and children, a 7-pneumococcal conjugate vaccine (conjugate polysaccharide-protein conjugate vaccine conjugated with a carrier protein that increases the immunogenicity of the polysaccharide antigen) 7vPnC, Prevenar®) have been developed and used, and many data have been reported to be effective in the prevention of invasive disease and otitis media in infants and children. However, the use of the 7-valent vaccine led to a reduction in invasive disease caused by the vaccine serotypes used in the vaccine, but also showed a relative increase in pneumococcal disease due to some non-vaccinous serotypes. In addition to this, Synflorix®, a 10-bargain polysaccharide-protein conjugate vaccine, and Prevenar 13®, a 13-pneumococcal conjugate vaccine supplemented with 6 serotypes, were added to the primary serotype of Prevenar® ) Has been developed and is currently on the market, but there are reports of possible effects of some serotypes contained in the vaccine as a vaccine [Andrews NJ et al, (2014) Lancet Infec Dis (14) 839; (2009) EMA / 798877/2009], there is a continuing need for the development of new vaccine formulations that demonstrate higher and more stable activity.

On the other hand, preservative should be used to prevent contamination of microorganisms. The combined vaccine products exported to the underdeveloped countries through the United Nations are preferred to multi-dose products including preservatives due to the environment, distribution methods and costs of the countries in use. Preservatives used in vaccine products include thimerosal, 2-phenoxyethanol, 2-PE, phenol and the like, and there are amounts of preservatives commonly used in the art. For example, timerosal is used at a concentration of 10 μg / mL and 2-PE is used at a concentration of 5 mg / mL. Multi-dose vaccine products, including EP-B (European Pharmacopoeia B category) It must pass the standard buoyancy test to be commercialized.

Thimerosal (Thimerosal, merthiolate) is an ethyl mercury derivative compound that has been used as a preservative in multi-dose vaccine injections since the early 1930s. TIMEROSAL has been used for the purpose of preventing the growth of contaminated microorganisms and maintaining sterility during storage or use of vaccine products, and has been approved by WHO PQ (Prequalified) for a number of five-liquid mixed vaccines (D, T, P, Hib , HBsAg) contains thiomersal as a preservative.

2-Phenoxyethanol (2-PE) is mainly used as a preservative for cosmetics and percutaneous pharmaceuticals, and is also used as a preservative for vaccine injections.

However, the amount of these preservative ingredients used to achieve the full buoyancy of the vaccine can lead to toxicity and / or side effects, thus requiring the development of techniques that impart sufficient buoyancy while reducing their use.

An example is (i) a capsular polysaccharide-protein conjugate of Streptococcus pneumoniae , (ii) 2-phenoxyethanol, 2-PE, and (iii) formaldehyde (Formaldehyde, &lt; RTI ID = 0.0 &gt; HCHO) &lt; / RTI &gt; The vaccine composition may be a multi-dose vaccine composition for multiple administration.

 Another example provides a pharmaceutical composition for preventing or treating a pneumococcal infection disease comprising the pneumococcal vaccine composition described above. The pharmaceutical composition for preventing or treating may be a multi-dose pharmaceutical composition for multiple administration.

Another example is the preparation of a capsular polysaccharide-protein conjugate of Streptococcus pneumoniae and a step of contacting the capsular polysaccharide-protein conjugate with 2-phenoxyethanol (2-PE) Or preservation (or buoyancy) of the pneumococcal vaccine composition, comprising the step of mixing the pneumococcal vaccine with formaldehyde (HCHO) and formaldehyde (HCHO) ) &Lt; / RTI &gt; The vaccine composition may be a multi-dose vaccine composition for multiple administration. The step of preparing the capsular polysaccharide-protein conjugate may include a step of performing a cyanylation method to link the capsular polysaccharide and the carrier protein through -OC (NH) -NH-.

One example is (i) a dense polysaccharide-protein conjugate of Streptococcus pneumoniae , (ii) 2-phenoxyethanol, 2-PE, and (iii) formaldehyde, HCHO ) Or a pneumococcal immunoconjugate composition.

As used herein, the pneumococcal immunogenic composition means a composition that causes an immune response against pneumococci, and unless otherwise stated, is used with the same meaning as the pneumococcal vaccine composition.

As used herein, "comprising (a given component)" may mean comprising, or consisting essentially of, components other than the recited components .

The pneumococcal vaccine composition provided herein comprises a capsular polysaccharide of Streptococcus pneumoniae and 2-phenoxyethanol and formaldehyde as antiseptic agents to induce an immune response-inducing effect on Streptococcus pneumoniae (Immunogenicity) can be maintained for a long period of time, and / or an immunogenic composition enhanced in retention (repelling) force.

The capsular polysaccharide may be a capsular polysaccharide derived from Streptococcus pneumoniae . Specifically, the capsular polysaccharides are derived from two or more, eg, five or more, seven or more, nine or more, eleven or more, thirteen or more, or fourteen or more serotypes of Streptococcus pneumoniae serotype It may be a capsular polysaccharide. In one example, the capsular polysaccharide comprises 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, and 14 species of Streptococcus pneumoniae serotype To 19 kinds, 14 kinds to 17 kinds, or 14 kinds to 15 kinds of serotypes. For example, the capsular polysaccharide may be a Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19 selected from the group consisting of 19, 20, 22F, 23F and 33F Species, 14 to 17 species, or 14 to 15 species of serotypes. In one embodiment, the capsular polysaccharide is selected from the group consisting of thirteen species of clumps derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, Polysaccharides; 14 clathrin polysaccharides derived from Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F; Fifteen kinds of snack polysaccharides derived from Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2 and 12F; And / or 15 kinds of capsular polysaccharides derived from Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2 and 15B; And 17 strains derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F But are not limited to, polysaccharides.

The above-described snack polysaccharide-protein conjugate may be one in which the above-described two or more serogroup-derived snack polysaccharides are individually bonded to each other through a common method, for example, a covalent bond with the protein. In one example, the conjugate has a structure in which each of the capsular polysaccharides (specifically, the hydroxyl group of the polysaccharide) and the protein (specifically, the amino group of the protein) are linked through -OC (NH) -NH- via a cyanylation method ([Polysaccharide] -OC (NH) -NH- [protein]). When the cyanopolysaccharide and the protein are conjugated using the cyanylation method as described above, the degree of structural modification of the capsular polysaccharide is small compared with other methods (for example, a reductive amination method and the like), and the immunogenicity is maintained even after the conjugation It will be more advantageous. For example, when a specific serotype (19F, etc.) is conjugated with a protein by a reductive amination method, the hexasaccharide ring structure may be cleaved and opened depending on the combination of the reactions, On the other hand, it has been reported that this problem is not likely to occur when a protein is conjugated with a cyanylation method.

The protein may be a carrier protein, for example, a protein that exhibits non-toxicity and non-reactivity and can be obtained in sufficient amount and purity. The intensifying polysaccharides from various serotypes can each be conjugated to the same or different proteins, such as the same protein.

In one embodiment, the protein may be a CRM197 protein. The CRM197 protein is a non-toxic variant of the diphtheria toxin isolated from cultures of Corynebacterium diphtheriae strain C7 (CRM197; for example, grown in casamino acid and yeast extract-based media), a metamorphic toxoid. The CRM197 protein can be purified from Corynebacterium diphtheria strains C7 cultures via ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography, or purified by conventional methods, such as those described in U.S. Patent No. 5,614,382 (herein incorporated by reference) May be obtained recombinantly with reference to the method. In one example, the CRM197 protein is the GenBank Accession No. &lt; RTI ID = 0.0 &gt; 1007216A or an amino acid sequence having 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with the amino acid sequence of the above sequence.

In addition, a modified toxin derived from diphtheria other than Corynebacterium diphtheriae strain C7 can also be used as a carrier protein.

Other available transport proteins include

Inactivated bacterial toxins such as tetanus toxoid, pertussis toxin, cholera degenerative toxins (e.g., as described in International Patent Application Publication No. WO 2004/083251), E. coli LT, Chia coli ST, and Pseudomonas aeruginosa exotoxin A;

(OMPC), porins, transferrin binding protein, neomolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), group A or C5a peptidase derived from Group B streptococci, Haemophilus influenzae protein D;

(KLH), bovine serum albumin (BSA), purified protein derivatives (PPD) of tuberculin;

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

And the like.

The clathrin polysaccharide-protein conjugate is derived from two or more, eg, five or more, seven or more, nine or more, eleven or more, thirteen or more, or fourteen or more serotypes of Streptococcus pneumoniae serotype May be a polyvalent polysaccharide-protein conjugate comprising each conjugate polysaccharide and a conjugate conjugated with a carrier protein, e. G. CRM197 protein, as described above. In one example, the clathrin polysaccharide-protein conjugate is selected from the group consisting of 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24 of the Streptococcus pneumoniae serotype 13 to 24 comprising a conjugate in which the respective narrow-spectrum polysaccharides derived from 14 to 19, 14 to 17, or 14 to 15 different serotypes and a transport protein such as CRM197 protein are conjugated, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17, or 14 to 15 may be polysaccharide-protein conjugates. For example, the clathrin polysaccharide-protein conjugate may be a polypeptide of 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 13, 17 to 13, 15 to 14, and 24 to 14 selected from the group consisting of 18C, 19A, 19F, 20, 22F, 23F, 13 to 24, and 13 to 24, including a conjugate in which a contractile polysaccharide derived from a serotype of 19 to 14 species, 14 to 17 species, or 14 to 15 species and a transport protein such as CRM197 protein is conjugated, To 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17, or 14 to 15 may be polysaccharide-protein conjugates. In one embodiment, the capsular polysaccharide-protein conjugate is selected from the group consisting of Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, A 13-polysaccharide-protein conjugate comprising 13 kinds of conjugates in which each of the species of the capsular polysaccharide is conjugated with a transport protein, for example, CRM197 protein; 14 polysaccharide-protein conjugates derived from Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F; 15 kinds of the capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, For example, a 15 polysaccharide-protein conjugate comprising 15 conjugates with CRM197 protein conjugated; 15 kinds of the capsular polysaccharides derived from Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, For example, a 15 polysaccharide-protein conjugate comprising 15 conjugates with CRM197 protein conjugated; Or 17 kinds of 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 But not limited to, a 17 polysaccharide-protein conjugate comprising 17 conjugates in which the 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 may comprise at least two, more preferably at least seven, at least nine, at least eleven, at least thirteen, or at least fourteen serotypes of Streptococcus pneumoniae serotype And a polysaccharide-protein conjugate to which the above-described transport protein, for example, CRM197 protein, is conjugated, may be a polyvalent pneumococcal vaccine composition. In one example, the pneumococcal vaccine composition comprises 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, and 12 to 24 of the Streptococcus pneumoniae serotype, 14 to 19, 14 to 17, or 14 to 15 different serotypes, and 13 to 24, 13 to 19, 13 to 24, including 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17, or 14 to 15 polysaccharide- , 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17, or 14 to 15 may be a pneumococcal vaccine composition . For example, the pneumococcal vaccine composition can be used for the treatment of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C 13 to 24, 13 to 17, 13 to 15, 14 to 24, and 14 species selected from the group consisting of 19A, 19F, 20, 22F, 23F and 33F 13 to 24, including polysaccharide-protein conjugates in which the respective contractile polysaccharides derived from the serotypes 14 to 19, 14 to 17, or 14 to 15 species and the transport protein such as CRM197 protein are conjugated, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17, or 14 to 15 may be a pneumococcal vaccine composition.

In one embodiment, the pneumococcal vaccine composition comprises

(1) Thirteen kinds of barnyard polysaccharides derived from Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, , A 13-pneumococcal vaccine composition comprising 13 polysaccharide-protein conjugates conjugated with CRM197 protein;

(2) Each of 14 kinds of the capsular polysaccharides derived from Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, For example, a 14-pneumococcal vaccine composition comprising 14 polysaccharide-protein conjugates conjugated with CRM197 protein;

(3) 15 kinds of the capsular polysaccharides derived from Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, A 15-pneumococcal vaccine composition comprising fifteen polysaccharide-protein conjugates conjugated with a carrier protein, e.

(4) 15 kinds of the capsular polysaccharides derived from Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, A 15-pneumococcal vaccine composition comprising fifteen polysaccharide-protein conjugates conjugated with a carrier protein, e. or

(5) Seventeen clusters derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F A 17-pneumococcal vaccine composition comprising 17 polysaccharide-protein conjugates with each of the polysaccharides and a transport protein, e.

Lt; / RTI &gt;

The polyvalent pneumococcal vaccine composition provided in the present invention, for example, the above-mentioned composition 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 19, 14 to 17, or 14 to 15, the pneumococcal vaccine has remarkably excellent potency as compared with the previously developed polyvalent vaccine (for example, the 13-mer vaccine), and the prevention of pneumococcal infectious diseases and / A very excellent effect for treatment can be expected.

The pneumococcal vaccine composition may be a composition formulated in a single dose (multiple doses) for single dose or multiple doses for single dose administration. As used herein, the term "multi-dose" refers to a dose of a vaccine capable of being administered more than once (eg, two or more times) to a single dose (inoculation) May include a dosage unit containing a dose of vaccine capable of being administered (over two times) (inoculated).

The pneumococcal vaccine composition includes a preservative in addition to two or more serotype pneumococcal capsular polysaccharide-protein conjugates. Preservatives that can be used in the vaccine composition (such as injectable formulations) include, but are not limited to, 2-phenoxyethanol, formaldehyde, chlorobutanol, m-cresol, methylparaben, propylparaben, benzethonium chloride, benzalkonium chloride, benzoic acid, , Phenol, thimerosal, phenylmercuric nitrate, and the like.

In one example, the antiseptic is the antibiotic pneumococcal vaccine composition described above, for example, the above-mentioned 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 may be in the form optimized for the pneumococcal vaccine composition, including 2-phenoxyethanol and formaldehyde. Thus, the inclusion of 2-phenoxyethanol as a preservative with formaldehyde reduces the amount of 2-phenoxyethanol used to reduce toxicity and / or side effects while achieving improved preservative effect by the synergistic action of the two components can do.

The content of 2-phenoxyethanol contained in the polyvalent pneumococcal vaccine composition of the present invention is less than 10 mg / ml, less than 7 mg / ml, less than 6 mg / ml, or less than 5 mg / ml, such as less than 4 mg / 4 mg / ml to 7 mg / ml, 4 mg / ml to 7 mg / ml, 4 to 6 mg / ml, 4.5 to 6 mg / ml, 5 to 6 mg / ml to 5 mg / ml. (The term "to" used in the expression of the numerical range as used herein denotes a numerical value range lower than the upper limit value and lower than the upper limit value. The content of 2-phenoxyethanol in the vaccine composition is preferably lower than or equal to the lower limit of the above range in order to exhibit an effective preservative effect and less than or equal to the upper limit of the above range so as to be toxic and / .

The content of formaldehyde contained in the polyvalent pneumococcal vaccine composition of the present invention is 90 to 200 / / mL, 90 to 190 / / mL, 90 to 180 / / mL, 90 to 170 / / mL, 100 to 200 / / mL , 100 to 190 / / mL, 100 to 180 / / mL, or 100 to 170 / / mL. The content of formaldehyde in the vaccine composition is preferably at least the lower limit of the above range in order to exhibit an effective preservative effect and less than or equal to the upper limit of the above range so that there is no toxicity and / or side effects.

The vaccine composition may be stored for at least about 3 months, at least about 6 months, at least about 1 year, at least about 1.5 years at a temperature of about 2 ° C to about 8 ° C, about 20 ° C to about 25 ° C, , About 2 years or more, or 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 infection such as bacteria / Can mean none.

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

The pneumococcal infection disease means 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. Infection sources are mainly Streptococcus pneumoniae and Klebsiella pneumoniae ). In particular, pneumococcal pneumonia accounts for about 50% of all pneumonia, and severe chills, fever, cough, and chest pain are present, and sputum is often a hemorrhagic complication and can cause pleurisy, meningitis, endocarditis, and peritonitis Diagn. Microbiol. Infect. Dis., 2001, 39: 181-185).

As used herein, the term "pneumococcus " refers to Streptococcus pneumoniae and is a commensal organism that generally colonizes the mucosal surface of human noninvasive (nasopharynx). If the host's factor allows access to the lower respiratory tract, a vigorous inflammatory response will follow, thereby causing dense consolidation when the alveolar space fills the exudate, Lt; / RTI &gt; The pneumococcus can synthesize more than 90 structurally unique capsular polysaccharides, and the serotype of pneumococcus is classified according to the structural and immunological characteristics of these capsular polysaccharides. Therefore, when a vaccine preparation is prepared using the capsular polysaccharide of Streptococcus pneumoniae, the immune response may be different depending on the type of the capsular polysaccharide, that is, the serotype of Streptococcus pneumoniae derived from the capsular polysaccharide.

Since the capsular polysaccharide is recognized as an antigen when administered into the body and can produce an antibody against the same, a vaccine composition for the prevention of Streptococcus pneumonia (or Streptococcus pneumoniae infection) can be prepared. The term "antigen" in the present invention refers to a substance capable of specifically inducing an immune response when a substance enters 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 , And 13 to 24 kinds of capsular polysaccharides derived from 19A, 19F, 20, 22F, 23F and 33F, respectively, can act as antigens.

Another example provides a method for preparing a pneumococcal vaccine composition with enhanced stability and / or retention (or buoyancy) or a method for enhancing the stability and / or retention (or buoyancy) of a pneumococcal vaccine. The method

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

(2) mixing the above-mentioned snack polysaccharide-protein conjugate with 2-phenoxyethanol (2-PE) and formaldehyde (HCHO)

. &Lt; / RTI &gt;

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

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

The step (1) of preparing the clathrin polysaccharide-protein conjugate may include a step of carrying out a cyanylation method to connect the protein and the capsaicin polysaccharide through -O-C (NH) -NH-.

In one embodiment, the step of preparing the (1) snack polysaccharide-protein conjugate comprises:

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

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

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

. &Lt; / RTI &gt;

In another embodiment, the step (1) of preparing the clathrin polysaccharide-protein conjugate comprises: (iii) performing a cyanylation method to link the capsular polysaccharide and the protein through -OC (NH) -NH- Thereafter, at least one selected from the ultrafiltration step, the bacteria elimination filtration step, and the adsorption step may be further performed. The cyanylation method may be carried out using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) or CNBr.

In one specific example of the present invention, Streptococcus pneumoniae having 13 or 15 different serotypes were dissolved using sodium deoxycholate to liberate the polysaccharide bound to the cells. The following polysaccharides originated from 21 serotypes 1, 2, 3, 4, 5, 6A, 6B, 9V, 8, 9N, 10A, 11A, 12F, 15B, 17F, 18C, 19A, 19F, 20, 22F, (CTAB), which was able to ionic bond with CTAB (cetyltrimethylammonium bromide), and purified three serotypes 7F, 14, and 33F, which did not react with CTAB, using aluminum phosphate gel (Algel) solution. The reaction with the CTAB can be effected, for example, by adding 0.5 to 5% by weight or 1 to 3% by weight of the CTAB to the reaction in concentrations of 1 to 20% (w / v) or 5 to 15% (w / v) 2 to 3% by weight in the case of the polysaccharide derived from 23F, and 1 to 2% by weight in the case of the polysaccharides derived from serotype other than 23F), but the present invention is not limited thereto. After the reaction with the CTAB, one or more of pellet recovery after centrifugation, resuspension of the pellet using a sodium chloride solution (e.g., about 100 to 500 mM), and removal of CTAB ion using sodium iodide are further performed But is not limited thereto. The aluminum phosphate gel reaction may be performed by adding an aluminum phosphate gel solution to the reaction product in an amount of 1 to 20 wt% or 5 to 15 wt%, but is not limited thereto.

In case of using as a vaccine composition using only the capsular polysaccharide, infants and children, whose immune system is lower than adults, may not recognize it as an antigen and may not develop an immune reaction. Therefore, in the present invention, a conjugate form of a carrier protein and a capsular polysaccharide And used.

The "carrier protein" refers to a protein that can be covalently conjugated to a capsular polysaccharide to increase the immunogenicity of a polysaccharide antigen, and the specific type thereof is as described above. CRM197 may be used in one embodiment. The transport protein can be conjugated to the capsular polysaccharide through a standard conjugation method, and the resulting capsular polysaccharide-carrier protein conjugate formed may be one in which one or more capsular polysaccharides are conjugated to one transport protein.

Any of the known methods for preparing conjugates of capsular polysaccharides and carrier proteins may be included within the scope of the present invention, wherein the conjugates are conjugated using a cyanylation method such that the capsular polysaccharide and the transport protein are linked by-OC (NH) -NH- groups Structure. The cyanylation can be performed by a person skilled in the art through a known method. For example, the cyanylation can be carried out using, for example, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate or CNBr.

One example for preparing a conjugate of a capsular polysaccharide and a carrier protein is to chemically activate the purified capsular polysaccharide and form glycoconjugates by joining each chemically activated capsular polysaccharide to the carrier protein one by one. The activity of cyanation by treatment with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) changes the hydroxyl group of the capsular polysaccharide into a cyanate group, which is used to form a covalent bond with the amino group of the carrier protein CRM197 . The cyanation reaction by CDAP may be performed by adding 3 molar equivalents of a glycine solution to 1 equivalent of CDAP and adjusting the pH to 9.0, but the present invention is not limited thereto. Accordingly, the reaction solution and the reaction conditions can be appropriately controlled.

The resulting capsular polysaccharide-carrier protein conjugate can be purified by various methods. Examples of these methods include concentration / dialysis filtration processes, column chromatography and multi-layer filtration. The purified polysaccharide-protein conjugates may be formulated into a vaccine composition of the present invention by mixing them, and they may be used. The formulation of the vaccine composition of the present invention may be carried out using methods known in the art. For example, a composition can be prepared by formulating each of the thirteen individual capsular polysaccharide-carrier protein conjugates together with a physiologically acceptable vehicle. Examples of such vehicles include, but are not limited to, water, buffered saline, water for injection, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol) or dextrose solution.

In one specific embodiment of the present invention, 1) the dissolution and hydrolysis of 13 kinds of capsular polysaccharides, 2) the reaction process of CRM197 with the respective capsular polysaccharides using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) End complexing, 4) ultrafiltration, 5) bactericidal filtration, and 6) adsorption step to produce 13 kinds of capsular polysaccharide - transport protein conjugates.

The term "vaccine " in the present invention refers to a biological agent containing an antigen that immunizes a living body. The term &quot; vaccine &quot; refers to an immunogenic agent or an antigenic substance that causes immunity to a living body by administration to a human or animal for prevention of infection.

The vaccine composition may further comprise at least one member 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" as used herein refers to a substance used to increase the immunogenicity of an immunogenic composition of the 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) (e.g., aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.);

(2) a water-in-oil type emulsion formulation (with or without other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), such as (a) MF59 (WO90 / 14837) : 5% (w / v) squalene, 0.5% (w / v) Tween 80 and 0.5% (w / v) Span 85 (optionally supplemented with various amounts of MTP- (B) a SAF of 10% (w / v), which may be further contained, such as a microfluidizer, Microfluidizer, Microfluidics, Newton, Mass. ) Squalene, 0.4% (w / v) Tween 80, 5% (w / v) pluronic-block polymer L121 and thr-MDP (see below) (R) (Corixa, Hamilton, MT): 2% (w / v) &lt; tb &gt; ______________________________________ &lt; tb &gt; ______________________________________ &lt; Allene, 0.2% (w / v) tween 80 and 3-O-deacylated monophosphoryl lipid A (MPL ™) (Corixa), trehalose dimeric cholate (TDM) and cell wall skeleton described in US Pat. No. 4,912,094 (CWS), preferably MPL + CWS (Detox ™);

(3) Quil A or STIMULON? Particles (e.g., ISCOMs (immunostimulatory complexes)) in which saponin adjuvants such as QS-21 (Antigenics, Framingham, MA, U.S. Patent No. 5,057,540) are used or produced;

(4) Bacteriostatic lipopolysaccharides, synthetic lipid A analogs, such as aminoalkylglucosamine amine phosphate compounds (AGP), or derivatives or homologues thereof, which are available from Corixa and are described in U.S. Patent No. 6,113,918; One example of an amino acid is 2 - [(R) -3-tetradecanoyloxytetradecanoylamino] ethyl 2-deoxy-4-O-phosphono-3-0- [ Which is also known as 529 (formerly known as RC529), which can be either male-shaped or &lt; RTI ID = 0.0 &gt; Formulated as a stable emulsion);

(5) synthetic polynucleotides such as oligonucleotides containing CpG motifs (US Patent No. 6,207,646);

IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.) , Interferons (e.g., gamma interferon), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (MCSF), tumor necrosis factor (TNF), co-stimulating molecules B7-1 and B7-2 and the like;

(7) a wild-type cholera toxin (CT) or a mutant cholera toxin in which glutamic acid at position 29 of amino acid according to WO-2000/18434 is substituted with another amino acid, specifically histidine (PT), or E. coli heat-labile toxin (LT), particularly LT-K63, LT-R72, CT-S109, PTK9 / G129 (see WO02 / 0998368 and WO-2002/098369), pertussis toxin -93 / 13302 and WO-92/19265); a mutant of a bacterial ADP-ribosylated toxin; And

(8) Complement component Complement such as trimer of C3d.

The muramyl peptides include N-acetyl-muramyl-L-tryonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L- -sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE), and the like.

The aluminum salt adjuvant may be an aluminum-precipitated vaccine or an alum-adsorbed vaccine. Aluminum salts include hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, Alhydrogel, Superfos, Ampogel, Aluminum Hydrophosphate Adjuvant (APA) Amorphous alumina, and the like, but are not limited thereto. APA refers to a suspension of aluminum hydroxyphosphate. When aluminum chloride and sodium phosphate are mixed at a ratio of 1: 1 (by volume), aluminum hydroxide phosphate sulfate is precipitated. The precipitate is sized to 2 ~ 8 ㎛ by using a high shear mixer and dialyzed with physiological saline And sterilized. In one embodiment, the protein is adsorbed using commercially available Al (OH) 3 (e.g., Alhydrogel or Superfos). 50 to 200 g of protein can be adsorbed per 1 mg of aluminum hydroxide, which is dependent on the pH of the protein and the pH of the solvent. Low pI proteins bind strongly to high pI proteins. Aluminum salts can activate the macrophage, complement, and innate immune mechanisms nonspecifically by forming an antigen reservoir that slowly releases the antigen for 2 to 3 weeks.

The term "preservative (or preservative) " in the present invention means an anti-viral and / or antimicrobial substance which inhibits the growth of microorganisms in the vaccine composition. Examples thereof include thimerosal, phenoxyethanol 2-phenoxyethanol, formaldehyde, or mixtures thereof, but any conventional preservative used in the art may be used.

In addition, the vaccine composition may comprise one or more, physiologically acceptable buffering agents. For example, when the vaccine composition is an infusion or injecting agent, the buffer may have a buffering capacity at a pH of 4.0 to 10.0, specifically, a pH of 5.0 to 9.0, more specifically a pH of 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, triethanol amine 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, the buffer may be a monobasic acid 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; Ammonia, diethanolamine, glycine, triethanolamine, TRIS, and the like.

In addition, the vaccine composition of the present invention may comprise a nonionic detergent. For example, polyoxyethylene sorbitan esters (commonly called Tweens), especially polysorbate 20 and polysorbate 80; Copolymers of ethylene oxide (EO), propylene oxide (PO), butylene oxide (BO) (e.g. DOWFAX TM); Oxytoxynols, in particular the octyloxynol-9 (Triton-100), in which the number of repeats of the oxy-1,2-ethanediyl group is different; Ethylphenoxypolyethoxyethanol (IGEPAL CA-630 / NP-40); Phospholipids such as lecithin; Nonylphenol ethoxylate such as NP series; Polyoxyethylene fatty acid ethers (Brij surfactants) derived from lauryl, cetyl, stearyl, oleyl alcohol, especially triethylene glycol monolauryl ether (Brij 30); But are not limited to, sorbitan ethers known as SPANs, especially surfactants such as sorbitan triolate (Span 85) and sorbitan monolaurate. Tween 80 may be included in the emulsion, and mixtures of nonionic detergents such as Tween 80 / Span 85 may also be used. A combination of a polyoxyethylene sorbitan ester such as Tween 80 and an octocinol such as Triton X-100 is also suitable, and a combination of Laureth 9, Tween, and or Octocinol is also useful. Specifically, 0.01% (w / v) to 1% (w / v), especially 0.1% (w / v) of polyoxyethylene sorbitan ester such as Tween 80; (W / v) to 0.1% (w / v), especially 0.005% (w / v) to 0.02% (w / v) of octylphenoxy polyoxyethanol or nonylphenoxy polyoxyethanol w / v); (W / v) to 20% (w / v), preferably 0.1% (w / v) to 10% (w / v), especially 0.1% (w / v), of polyoxyethylene ethers 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 vial, a multi-dose vial or a prefilled syringe. Thus, one example provides a vial, or pre-field syringe, containing the vaccine composition described above in a single dose or in multiple, e.g., one more dose. The vaccine composition may further comprise a physiologically acceptable carrier.

As used herein, a "multi-dose dose" or "multi-dose dose" refers to a dose of a vaccine that can be overdosed once (inoculated) to a single dose (inoculation) May mean the dose of vaccine that can be overdosed (inoculated).

Physiologically acceptable carriers for use in liquid preparations include aqueous or nonaqueous solvents, suspensions, emulsions, and oils. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, and 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 oil, soybean oil, olive oil, sunflower oil, synthetic oil such as liver oil, marine oil, and lipids obtained from milk or eggs. The vaccine composition of the present invention may be isotonic, hypertensive, or shelf stable, and pharmaceutical compositions administered by infusion or injection are preferably but not limited to isotonicity. On the other hand, isotacticity and toughness may be advantageous for the storage of the composition. If the vaccine composition is malfunctioning, it may be diluted to become isotonic before administration. An isotonic agent for dilution may be an ionic isotonic agent such as a salt or a non-ionic isotonic agent such as a carbohydrate. Ionic isotonic agents include, but are not limited to, sodium chloride, calcium chloride, potassium chloride, magnesium chloride, and the like. Non-ionic isotonic agents include, but are not limited to, sorbitol, glycerol, and the like.

The amount of the conjugate in each vaccine dose can be selected to an amount that induces an immunoprotective response without significant side effects, and this amount can vary depending on the serotype of S. pneumoniae. Specifically, the vaccine composition was prepared by mixing the serotypes 1, 2, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A , 0.8 to 1.2 parts by weight or 0.8 to 1.2 parts by weight or 0.9 to 1.1 parts by weight, respectively, in the case of the capsular polysaccharides derived from 12F, 14F, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 1.8 to 2.2 parts by weight, or 1.9 to 2.1 parts by weight of the serotype 6B-derived capsular polysaccharide in the range of 1.6 to 2.4, 1.8 to 2.2, or 1.9 to 2.1 (i.e., 1.6 to 2.4 parts by weight, But are not limited thereto.

As an example for producing a conjugate, each conjugate may contain 0.1 to 100 μg, specifically 0.1 to 10 μg, more particularly 1 to 5 μg of polysaccharide.

The most specific polysaccharide except for the serotyped 6B-derived polysaccharide, namely serotypes 1, 2, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B , And the barley polysaccharides from 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F are in amounts of 2 to 2.4 μg or 2.1 to 2.3 μg, 4 to 4.8 [mu] g, 4.2 to 4.6 [mu] g, or 4.3 to 4.5 [mu] g, such as about 4.4 [mu] g. In this case, the CRM197 protein may be contained in the composition in an amount of 0.1 to 100 μg, 1 to 50 μg, 20 to 40 μg, or 28 to 31 μg, for example, about 29.3 μg, but is not limited thereto.

The optimal amount of a component for a particular vaccine can be ascertained by standard studies involving observation of a suitable immune response in a subject. For example, an animal study can be extrapolated to determine the dose of a vaccine in humans. In addition, a person skilled in the art can empirically determine the capacity, if necessary.

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

The vaccine composition according to the present invention can be used to protect individuals susceptible to pneumococci and prevent pneumococcal disease by administering a pharmacologically effective amount in a systemic or mucosal route. The term "prophylactic" of the present invention refers to any act that inhibits or delays infection by the pneumococcus by administration of the vaccine composition of the present invention. As used herein, the term "pharmaceutically effective amount" as used herein refers to the dosage required to induce antibodies that are capable of significantly reducing the probability of infection or the severity of the infection with S. pneumoniae. The term "administering" of the present invention refers to the introduction of a predetermined substance into an individual by any suitable method. The vaccine composition of the present invention may be administered via the inhalation route via oral, nasal, rectal, transdermal or aerosol administration, but may be administered by bolus or by slow infusion, but is not limited thereto. Such administration may be by intramuscular, intraperitoneal, intradermal or subcutaneous injection; Or mucosal administration to the oral / digestive tract, airway or genitourinary tract, and the like. In one embodiment, intranasal administration can be used for the treatment of pneumonia or otitis media, which can more effectively prevent non-invasive infections of S. pneumoniae and attenuate infection at an early stage.

The "subject" of the vaccine composition or pharmaceutical composition of the present invention may be a live organism from which the pathogens can be infected or cells, tissues, or cultures thereof isolated therefrom, and the organism may be a high vertebrate animal And more specifically 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 dose, or may be administered two times, three times, four times or more at appropriate intervals, but is not limited thereto. For example, for an invasive disease caused by Streptococcus pneumoniae, the routine vaccination schedule for infants and newborns may be 2, 4, 6 and 12 to 15 months of age.

In addition, the composition may further comprise one or more proteins derived from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for inclusion include proteins identified in WO-2002/053761, as well as proteins identified in WO-2002/083855, all within the scope of the present invention.

In one specific embodiment of the present invention, 2.2 μg of each polysaccharide, 4.4 μg of polysaccharide derived from 6B in the total 0.5 mL of the vaccine composition; About 29.3 [mu] g CRM197 transport protein; 0.5 mg aluminum element (2 mg aluminum phosphate) adjuvant; Approximately 4.25 mg of sodium chloride (without preservatives) or about 3.5 mg (with preservatives); Succinate buffer solution about 295 쨉 g; And about 3 mg of 2-phenoxyethanol and about 60 μg of formaldehyde, for example, 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, Examples 14 to 19, 14 to 17, or 14 to 15 include pneumococcal vaccine compositions.

In another specific example of the present invention, a serum type specific IgG concentration higher than that of Prevenar 13 was confirmed in rabbit serum inoculated with the vaccine composition (Table 1). In addition, the functional immunogenicity test (Opsonophagocytic assay) showed a superior effect to that of Prevenar 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 aspect of the invention is a pharmaceutical composition comprising 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19 capsular polysaccharide- , 14 to 17, or 14 to 15 species of an immunogenic composition for S. pneumoniae. The conjugate and the pneumococci have been described above.

13 to 24, 13 to 13, 13 to 15, 14 to 24, 14 to 19, 14 to 17 species of the capsular polysaccharide-transport protein conjugate of the present invention , Or from 14 to 15, 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, and 14 To 17, or 14 to 15 different serotypes of Streptococcus pneumoniae, and when they are administered into the body, they are recognized as antigens and can be used for the production of antibodies thereto. The immunological response can be used as an immunogenic composition against S. pneumoniae.

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

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

Vaccine composition, immunogenic composition, and prevention of pneumococcal disease have been described above.

Another aspect of the present invention is a method for the detection of thirteen isolated cloven membranes derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F Each of the capsular polysaccharides is conjugated using a cyanylation method so that the capsular polysaccharide and the carrier protein CRM197 have a structure linked to the -OC (NH) -NH- group.

Immunogenic compositions and methods for their preparation have been described above.

The polyvalent pneumococcal vaccine composition according to the present invention comprises a polyvalent conjugate of a specific conjugated polysaccharide-protein and the combination and the amount of 2-phenoxyethanol and formaldehyde optimized as described above as a preservative, And is characterized by being excellent in stability and / or preserving power. Therefore, the vaccine composition and the immunogenic composition according to the present invention can be more safely and usefully used for preventing disease caused by S. pneumoniae in 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.

Manufacturing example  1: Preparing the capsular polysaccharide

1-1. Preparation of cell bank

Streptococcus pneumoniae with 16 different serotypes (1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 12F and 15B) USA from the Center for Disease Control and Prevention (US), and a cell bank was prepared as follows.

Streptococcus pneumoniae strains were plated on blood agar medium to identify pneumococci and to remove the existing medium components. After selecting a single colony with a good growth rate among 10 or more single colonies, the cells are inoculated and cultured in a liquid medium (soybean (Kerry Bio-Science) or yeast extract (Bio-springer) , And synthetic glycerol was added to prepare a research cell bank (Research Cell Bank, RCB) containing synthetic glycerol.

One of the vials of the study cell bank having confirmed the expression of the polysaccharide having the inherent serotype was taken out, and the cell was grown in a liquid medium not containing an animal-derived component, and synthetic glycerol was added to prepare a master cell bank. One vial of the bank was taken out to proliferate the cells in a liquid medium containing no animal-derived components, and synthetic glycerol was added to prepare a cell bank for production.

The cell bank thus prepared was stored in an ultra-frozen state at -70 캜 or lower and used in the following test.

1-2. Fermentation and polysaccharide separation

One vial of the cell bank for production was thawed and inoculated into a liquid medium containing no animal-derived components. The seed culture was carried out at 37 ± 2 ° C in an untreated semi-solid state until the optical density (OD 600) was reached. After the contamination of the cultured medium was confirmed, the cells were inoculated into a fermenter containing a liquid medium (Soybean (Kerry Bio-Science) or Yeast extract (Bio-springer) -mediated medium) containing no animal-derived components (OD600 = 2.5 ± 0.2).

Thereafter, the culture was performed while maintaining the pH of the medium at 7.2 ± 0.2 using a sterilized potassium hydroxide solution at 37 ± 2 ° C with minimum stirring. From 2 hours after the initiation of the culture, samples were sampled to measure the cell concentration in the culture medium and the glucose concentration contained in the medium. The culture was terminated at the time when the glucose in the medium was depleted.

After the culture was completed, an appropriate amount of sterilized 12% (w / v) sodium deoxycholate was prepared to a final concentration of 0.12% (w / v) To release the polysaccharide bound thereto.

1-3. Purification of capsular polysaccharides

Phosphoric acid was added to the sample treated with sodium deoxycholate obtained in Preparation Example 1-3 for a long time, and the pH was adjusted to 3.5 ± 0.3 with 85% phosphoric acid. The mixture was allowed to stand for 15 hours ± 3 hours, xG, room temperature, 1 hour). The recovered supernatant was passed through a depth filter (0.55 - 9.0 μm), concentrated, and buffer exchanged with a phosphate buffer solution.

After the buffer exchange, the sample was passed through an activated carbon filter, and then the impurities were removed by the following two methods:

1) 14 serotypes of serotypes 1, 2, 3, 4, 5, 6A, 6B, 9V, 12F, 15B, 18C, 19A, 19F and 23F are capable of ionic bonding with CTAB (cetyltrimethylammonium bromide) Specifically, a 10 wt% CTAB solution was treated in an amount of 1.5 wt% (in the case of 23F) or 2.5% (in the case of 23F) based on the weight of the process solution and reacted for 1 hour ± 10 minutes. The pellet was then recovered by centrifugation at a rate of 17,000 x G for 1 hour. Thereafter, the pellet recovered through the CTAB treatment was resuspensioned with 200 to 300 mM sodium chloride (NaCl) solution to treat sodium chloride (NaCl) and sodium iodide (NaI), and 50% Sodium (NaI) was used to remove CTAB ions. However, in case of serotype 3, a 350 mM NaCl solution was used. After treatment with sodium chloride and sodium iodide, the supernatant was recovered by centrifugation and used for further processing. 2) For two serotypes 7F and 14 serotypes not reacting with CTAB, the reaction was carried out by adding a solution of aluminum phosphate (Algel) and then treating the solution with 10 wt% Lt; / RTI &gt; The supernatant obtained via centrifugation (centrifugation at 17,000 xg centrifugation) was collected and used for further processing.

After the two types of impurity removal processes were completed, the samples were subjected to a depth filter and an ultrafiltration (UF / DF) process, and then adjusted to the amount of ethanol and sodium chloride.

Manufacturing example  2: Streptococcus Pneumoniae  Preparation of the capsular polysaccharide-protein conjugate

2-1: Reducing Aminolysis Cyanylation  Comparison of bonding methods

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. The concrete bonding process is as follows.

2-1-1. Reducing Amination method

11.7 mg of sodium periodate was added to the polysaccharide solution of the serotype 9 V and the polysaccharide was activated with stirring at 21 to 25 ° C. The oxidized polysaccharide was concentrated and dialyzed against 100 KDa ultrafiltration filter and WFI (water for injection), and the residual polysaccharide was mixed with CRM197 protein and saccharide / CRM 197 = 0.5 (weight ratio) and lyophilized. The lyophilized complex was thawed and stabilized (equilibrated) at 21-25 ° C. The equilibrated complex was incubated (37 ± 2 ° C) in a sodium phosphate (Na ₃ PO ₄ ) buffer solution at a concentration of 0.1 M per 20 g of saccharide and dissolved. Cyanoborohydride (100 mg / To initiate the conjugation reaction between sugars. After incubation at 37 ± 2 ° C for about 44 to 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) is added to give 1.8 to 2.2 molar equivalents of sodium borohydride per 1 mol of saccharide and the resulting reaction mixture is incubated with stirring at 23 +/- 2 DEG C, Any unreacted aldehyde was reduced. To the resulting saccharide-protein conjugation mixture was added 5 mL of 0.9% (w / v) sodium chloride aqueous solution to dilute and the diluted conjugation 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 polysaccharide solution of serum type 9V prepared without hydrolysis. To activate the polysaccharide, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) solution was added to the serotype 9V polysaccharide solution so as to have a concentration of 0.5% (w / w) relative to the polysaccharide and stirred for 15 minutes to activate the polysaccharide Respectively. Sodium hydroxide solution was added to the obtained reaction mixture to raise the pH to 9.5 占 0.1 占 폚, and the mixture was stirred for 3 minutes so that the hydroxyl group of the polysaccharide was sufficiently activated by CDAP. CRM197 was added to the polysaccharide solution after the polysaccharide activation process so that the ratio of CRM197 to the polysaccharide was 1.0% (w / w) (CRM197 weight / polysaccharide weight) and the reaction was allowed to proceed at room temperature for 1 hour.

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

As a result, it was confirmed that the yield of the conjugate prepared by the cyanylation method was four times or more as compared with the conjugate prepared by the reductive amination method. Thus, the present inventors prepared conjugates using the cyanylation method with the capsular polysaccharide and CRM197.

2-2. Junction and purification of the conjugate

The preparation of the conjugate of Streptococcus pneumoniae of the collapsible polysaccharide and CRM197 was carried out through the following steps.

Step 1. Dissolving and hydrolyzing the capsular polysaccharide

The capsular polysaccharide originating from each serotype was dissolved in the injection water to give a final concentration range in the ranges described below, and filtered through a 0.45 mu m filter:

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

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

3) Serotypes 6A, 12F and 19A ranged from 8 to 12 mg / ml, and

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

The solution was incubated at the pH and temperature ranges described below for each serotype:

1) For serotype 1, 2, 4, 5, 6B, 7F, 14 and 23F,

2) for serotypes 6A and 19F, for from 0.5 to 4 hours at 70 to 80 &lt; 0 &gt; C,

3) For serotypes 3, 9V, 12F and 18C, incubation is carried out at pH 2.0 and 65 to 80 ° C for 0.5-4 hours using a phosphate solution.

4) Hydrolysis is not performed for serotype 15B, 19A.

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

Step 2. Capsular polysaccharide and Of CRM197  Bonding reaction process

A 2M NaCl polysaccharide solution was prepared by adding sodium chloride powder to all serotypes. For each serum, a solution of appropriate CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) in a ratio of 100 mg of CDAP per 1 ml of 1/1 acetonitrile / water solution (v / v) . In detail

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

2) Serum type 2 and 4, compared with polysaccharides, CDAP 2 (w / w),

(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) Serum type 12F was dissolved in the ratio of CDAP 5 (w / w) to polysaccharide and added to each polysaccharide solution.

Then, sodium hydroxide solution was added to raise the pH to 9.5, and then the hydroxyl group of the polysaccharide was agitated for 3 to 7 minutes to be fully activated by CDAP. CRM197 was added to each serotype polysaccharide solution in an amount of CRM197 0.75 (w / w) versus polysaccharide, and the conjugation reaction proceeded for 2 hours. After that, the reaction conversion rate was measured using SE-HPLC, and CDAP was added as needed.

Step 3. End of bonding reaction

For all serotypes, a glycine solution of 3 to 6 molar equivalents based on 1 molar equivalent of the added CDAP was added and the pH was adjusted to 9.0 to terminate the reaction. The bonding solution was agitated at 21 캜 to 24 캜 for one hour and then stored at a low temperature of 2 to 8 캜 overnight.

Step 4. Ultrafiltration

The diluted conjugate mixture was concentrated and dialyzed against an ultrafiltration filter using a minimum of 20 volumes of buffer (5 mM Succinate buffer (pH 5.8) containing 150 mM NaCl). Here, a buffer solution containing 0.9% (w / v) sodium chloride was used, maintaining a pH range of 5.5 to 6.5 with a buffer solution. The cutoff molecular weight of the ultrafiltration filter was 300 kDa in all serotypes and the permeate was discarded.

Step 5. Sterilization  percolation

The residual solution after dialysis filtration was diluted to a concentration of less than 0.4 g / L based on the polysaccharide content concentration using a buffer (5 mM Succinate buffer (pH 5.8) containing 150 mM NaCl) and filtered through a 0.22 탆 filter. The filtered product was subjected to control (saccharide content, residual DMAP) during the manufacturing process. The filtered residue was subjected to control during the manufacturing process to determine whether further concentration, dialysis filtration and / or dilution was necessary.

Step 6. Adsorption

An aluminum salt (aluminum phosphate) was added to the bactericidal filtrate so as to have a final concentration of 1 mg / mL based on the aluminum ion, adsorbed, and an extra salt was added so as to maintain the pH range of 5.5 to 6.5. After the adsorption was completed, the quality of the stock solution was checked for quality, and the solution was stored at 2 to 8 ° C until used.

Example  1. Formulation of vaccine against polyvalent pneumococcal conjugate

The capsular polysaccharides derived from Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 12F and 15B prepared in Preparation Example 2 And the CRM197 protein, the following combinations of polyvalent pneumococcal polysaccharide-protein conjugates were prepared:

(1) 13 is a conjugate: serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F;

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

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

The required volume of the conjugate bulk solution was calculated based on the batch volume and the polysaccharide concentration of the conjugate stock solution. After addition of aluminum phosphate and addition of 0.85% (w / v) physiological saline and 5 mM succinate buffer (pH 5.8), as in step 6 of preparation example 2, was added to the bulk solution of the conjugates of each serotype, A bulk solution of each conjugate containing 0.85% sodium chloride, 5 mM succinate buffer (pH 5.8) and 1 mg / mL aluminum element (concentration of aluminum element in aluminum phosphate) was prepared. 10, 25, 40, 50, 80, or 100 mg / ml) as a preservative, and finally with thiorerol (85 or 100 ug / ml) or 2-phenoxyethanol , 100, 120, or 170 ug / ml) were added and mixed with the serotype combinations described above to prepare a formulated vaccine composition of S. pneumoniae conjugate. At this time, the concentration of each serogroup polysaccharide of the polyvalent vaccine composition is 4.4 / / mL (however, Type 6B is 8.8 / / mL). The pH was checked and adjusted to pH 5.8 if necessary. The resulting formulated final vaccine composition stock solution was filled into Type 1 borosilicate glass vials. The filled vaccine composition was stored at 2-8 [deg.] C.

Example  2: Assessment of immunogenicity of vaccine against polyvalent pneumococcal conjugate

Of the vaccine compositions prepared in Example 1, 13 vaccines (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) In 0.5 mL, 2.2 [mu] g each of the capsular polysaccharides (4.4 [mu] g for serotype 6B capsular polysaccharide); About 29.3 [mu] g CRM197 transport protein; 0.5 mg of aluminum element (2 mg aluminum phosphate) adjuvant; About 4.25 mg of sodium chloride; Succinate buffer solution about 295 쨉 g; About 3 mg of 2-phenoxyethanol, and about 60 占 퐂 of formaldehyde were selected and evaluated for their ability to induce an immune response in a rabbit. This immunogenicity was confirmed by antigen-specific ELISA for serum IgG concentration and by opsonophagocytic assay (OPA) for antibody function.

The vaccine composition LBVE013 or Prevenar 13® used as a positive control was administered at the planned human clinical dose (2.2 μg each polysaccharide, exception: 6B 4.4 μg) at 0, 2, and 4 weeks in New Zealand White ) Rabbit muscle, and serum was collected at intervals of 2 weeks after inoculation. The results of IgG measurement using ELISA for collected serum are shown in Table 1. This will be described in detail as follows.

2-1. Serotype specific IgG  Concentration measurement

Clarified polysaccharides for 13 serotypes were applied to 96-well plates at 5 ㎍ / well and coated at room temperature for 16 hours. To minimize the nonspecific antigen-antibody response of the sera, sera of each individual were taken in equal amounts and pooled between the same groups. The serum pool was adsorbed to 333.3 ㎍ / mL of cell wall polysaccharide (SSI) and 333.3 ㎍ / mL of serous polysaccharide (PnPs22F) of serum type 22F for 30 minutes at room temperature , And diluted with an appropriate dilution buffer (1: 100 ~ 1: 40000) with an antibody diluent buffer containing Tween 20. The coated plates were washed four times with wash buffer and 50 ul of preadsorbed and diluted serum was added to the coated well-plates and allowed to react for 1 hour at room temperature. The reacted well plate was washed 4 times by the same method, and a goat anti-rabbit IgG-HRP conjugate (1: 20000) was added to each well, followed by reaction at room temperature for 30 minutes . Plates were washed 4 times in the same manner as above and 100 μl of TMB substrate solution (3,3 ', 5,5'-Tetramethylbenzidine; Sigma, St. Louis, Mo., USA) stabilized at room temperature was added to each well. For 15 minutes. 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 the reference wavelength. Blood samples obtained by immunizing Prevenar 13® as a control for objective immunogenicity assays were analyzed by the same method.

The results are shown in Table 1 below.

LBVE013 Prevenar13® Serotype IgG ([mu] g / mL) Serotype IgG ([mu] g / 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 confirmed that the immunogenicity pattern of the serotype of the polyvalent (13a) pneumococcal vaccine composition (LBVE013) is significantly different from that of Prevenar 13®. Rabbits inoculated with the 13-valent vaccine composition (LBVE013) according to this example showed higher levels of titer than those of Prevena 13® in all serotypes compared to Prevenar 13®, and serotypes 1, 6B, 7F, 9V 14 and 19F showed 2 to 6 times better results than Prevenar 13®.

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

Serum from rabbits was subjected to OPA analysis to assess the function of antibodies induced by serotype.

Specifically, the same amount of serum was taken for each individual, and the serum was pooled between the same groups. Streptococcus pneumoniae was cultured in THD medium (Todd-Hewitt Broth w / 2% Yeast Extract) for each serotype and diluted with an osmosis buffer to 200-300 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. Next, 50 μl of a mixture of HL-60 cells and complement (cell: complement = 4: 1), which had been differentiated in advance, was added and reacted for 45 minutes in a CO 2 incubator (37 ° C).

The phagocytosis was stopped by lowering the temperature, and 10 ㎕ of the reaction solution was spread on a dried agar medium for 30 to 60 minutes in advance. Then, the cells were cultured in a CO ₂ incubator (37 ° C) for 12 to 18 hours, and the number of clusters was counted. OPA was expressed as a dilution factor with 50% death.

The obtained results 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 9V 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, an OPA titer of 2187 indicates that the activity is extremely high when the 50% level of the most diluted portion is not reached, compared with the negative control. From the above test results, it was confirmed that the polyvalent pneumococcal vaccine composition according to the present example had significantly higher serum IgG titer as compared to Prevnar 13®. Therefore, the polyvalent pneumococcal vaccine composition according to the present embodiment can be very useful for preventing diseases caused by pneumococci.

Reference example : vaccine Buoyancy  Test protocols and standards

In this example, the vaccine buoyancy test was conducted according to the EP-B standard required for the vaccine product in the USP and the European Pharmacopoeia (EP) by the World Health Organization (WHO).

The contents of the test will be described in detail. Two types of bacteria, Pseudomonas aeruginosa (ATCC No. 9027, PA), Staphylococcus aureus (ATCC No. 6538, SA), yeast Candida albicans (ATCC No. 10231, CA) and fungus Aspergillus niger 10 &lt; ⁵ &gt; to 10 &lt; ⁶ &gt; CFU / mL (CFU; Colony forming units) After 24 hours, 7 days, 14 days, and 28 days, the cells were sampled and cultured in a solid medium, and the number of colonies was counted on days 3 to 5.

Table 3 shows EP-B and the buoyant force standards of each country's pharmacopeia for results according to the above method.

Pharmacy standard Log Reduction (decrease in log CFU / mL) 6 hours 24 hours 7 days 14 days 28th 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, EP requirements are more stringent than USP or JPP and are classified into categories A and B according to the formulation, and the level required for vaccine products in WHO is EP-B (EP 5.1.3 Efficacy of antimicrobial perserbation, USP 37-51, Antimicrobial effectiveness testing.

 In the present example, in developing a vaccine against a polyvalent pneumococcal polysaccharide-protein conjugate, anti-flushing test was conducted by adding timesorosal and 2-PE, which are vaccine preservatives commonly used in the art, It was confirmed that the buoyancy criterion was not satisfied.

In addition, when a high concentration of 2-PE (7 mg / mL or more) was used, a patent was already filed by a third party and could not be used in the company.

Therefore, the inventors of the present invention developed a new composition that can increase the preservative effect while minimizing the content of 2-PE in order to satisfy the buoyancy criteria without infringing the patents of other companies as a preservative of the polyvalent pneumococcal conjugation vaccine As a result, the following results were obtained.

Example  3: polyvalent pneumococcal polysaccharide-protein conjugation vaccine Buoyancy  Screening

Two species of bacteria Pseudomonas aeruginosa (ATCC No. 9027, PA), Staphylococcus aureus (ATCC No. 6538, SA), yeast Candida albicans Staphylococcus aureus (ATCC No. 6538, SA), which is well known to not die well in 2-PE, among four species of fungi (ATCC No. 10231, CA) and fungus Aspergillus niger (ATCC No. 16404, AN) Were selected. The sample was sampled 24 hours later and the flotation force was evaluated to select the combination of 2-PE and formaldehyde satisfying EP-B. Accordingly, the test was carried out according to the test method of EP-B.

Specifically, referring to the method for producing a polyvalent pneumococcal polysaccharide-protein conjugated vaccine of Example 1, tiermolysin, or 2-PE and / or formaldehyde were added as shown in Table 2 below to prepare polyvalent pneumococcal protein conjugates of vaccine compositions 1 to 12 A vaccine composition was prepared, and Staphylococcus aureus (ATCC No. 6538, SA) was inoculated with 10 ⁵ to 10 ⁶ CFU / mL (CFU = colony forming unit) at 0 hour. Then, the samples were sampled at 0 hour and 24 hours, cultured in a solid medium, and the number of colonies was counted on days 3 to 5 to calculate the log reduction of the colonies. The results are shown in Table 4 below.

2-PE concentration
(mg / mL) Formaldehyde concentration (占 퐂 / mL) Tymolosate Concentration (/ / mL) SA buoyancy (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 shown in Table 4, the flotation power of SA passed the EP-B standard from the combination of 5.0 mg / mL of 2-PE and 100 μg / mL of formaldehyde. Based on these results, it can be confirmed that a combination of about 5.0 mg / mL of 2-PE and 100 μg / mL or more of formaldehyde is appropriate as a preservative for polyvalent streptococcal polysaccharide-protein conjugated vaccine.

Example  4: polyvalent pneumococcal polysaccharide-protein conjugation vaccine Buoyancy  Test 1

The buoyancy test was carried out according to the test method of EP-B. The vaccine compositions 13 to 15 of the polyclonal antibiotics conjugated vaccine preparation were mixed with two bacterium Pseudomonas Four strains of bacteria such as aeruginosa (ATCC No. 9027, PA), Staphylococcus aureus (ATCC No. 6538, SA), yeast Candida albicans (ATCC No. 10231, CA) and fungus Aspergillus niger (ATCC No. 16404, AN) To 10 &lt; ⁵ &gt; to 10 &lt; ⁶ &gt; CFU / mL (CFU = colony forming units). Thereafter, the bacteria were sampled at 0 hours, 24 hours, 7 days, and 28 days, cultured in a solid medium, and the number of colonies was counted on days 3 to 5 to calculate the log reduction of the colonies. The fungi and yeast were sampled at 0 hours, 14 days, and 28 days, cultured in a solid medium, and counted on the 3rd to 5th days to calculate the log reduction of the colonies. The results are shown in Table 5.

sampling
time Log Reduction (decrease in log CFU / mL) Germ Fungi and yeast P.A. S.A. EP-B standard C.A. A.N. EP-B standard Vaccine composition 13:
13
(2-PE 5.1 mg / mL + formaldehyde 102 [mu] g / mL) 24 hours 1.7 2.2 1 or more - - - 7 days NR NR 3 or more - - - 14 days - - - NR 1.2 1 or more 28th NR NR NI NR NR NI Vaccine composition 14:
15 A
(2-PE 5.1 mg / mL + formaldehyde 102 [mu] g / mL) 24 hours NR 2.1 1 or more - - - 7 days NR NR 3 or more - - - 14 days - - - NR NR 1 or more 28th NR NR NI NR NR NI Vaccine composition 15: 15 is B
(2-PE 5.1 mg / mL + formaldehyde 102 [mu] g / mL) 24 hours NR 1.9 1 or more - - - 7 days NR NR 3 or more - - - 14 days - - - NR NR 1 or more 28th NR NR NI NR NR NI

* NR: No Recovery ** NI: No Increase

As shown in Table 5, two bacterial species Pseudomonas aeruginosa (ATCC No. 9027, PA) in the polyvalent pneumococcal polysaccharide-protein conjugate vaccine compositions 13 to 15 containing the serotypes 13, 15, , Staphylococcus aureus (ATCC No. 6538, SA), yeast Candida As a result of examining the antifungal effect of four strains of albicans (ATCC No. 10231, CA) and fungus Aspergillus niger (ATCC No. 16404, AN), 13 strains, 15 strains A and 15 strains, regardless of serotype, The buoyant force against species bacterium met EP-B criteria. As a result, it was confirmed that the preservative of the polyvalent pneumococcal polysaccharide-protein conjugation vaccine satisfies EP-B flotation when the amount of 2-PE is about 5 mg / mL and formaldehyde is about 100 μg / mL .

In the art, pneumococcal protein conjugation vaccine generally uses more than 7 mg / mL of 2-PE as a preservative. When the formaldehyde is added in an amount of 100 μg / mL or more in combination with the result of Example 3, -PE content to 5 mg / mL, EP-B's flotation power can be achieved. Thus, the present invention provides a powerful and effective antifungal agent with an optimized combination of 2-phenoxyethanol (2-PE) and formaldehyde (HCHO) as a preservative for the polyvalent streptococcal polysaccharide-protein conjugate vaccine Lt; RTI ID = 0.0 &gt; multi-dose &lt; / RTI &gt;

Example  5: Stability of polyvalent streptococcal polysaccharide-protein conjugate vaccine composition

Based on the results of Example 4 above, a new composition of polyvalent pneumococcal polysaccharide-protein conjugated vaccine composition 16 was prepared. Specifically, the vaccine composition 16 was prepared by the following method: 14 kinds of 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 the aluminum concentration) were added and stirred to be mixed well. To the distilled water for injection, 5 mM succinic acid and 0.85% (w / v) sodium chloride were dissolved and the buffer was mixed with the filtered bacteria. Finally, 2-PE was added at a concentration of 6.0 mg / mL and formaldehyde was added at a concentration of 120 μg / mL to homogenize and the final pH was adjusted to 5.8. The concentration of each serotype in the vaccine composition thus prepared is 4.4 / / mL (however, 6B is 8.8 ㎍ / mL).

When the prepared vaccine composition 16 was stored at 2 to 8 캜 for 6 months, the content of the preservative was tested. As a result, the recovery rate of the contents of 2-PE and formaldehyde was maintained in a range of 90 to 110% Respectively.

In summary, the combination of 2-PE and formaldehyde exemplified in the examples of the present invention is an excellent composition which can effectively maintain the flushing ability of the polyclonal polyclonal polysaccharide-protein conjugated vaccine for a long period of time, and the polysaccharide- It has been confirmed that the present invention can be usefully used for providing a multi-dose composition in which the buoyancy of the vaccine is improved or the preservation period is extended.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> LG CHEM, LTD. <120> MULTIVALENT VACCINE COMPOSITION AGAINST STREPTOCOCCUS <130> DPP20180202KR <150> KR 10-2017-0032643 <151> 2017-03-15 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 536 <212> PRT <213> Artificial Sequence <220> <223> CRM197 protein <400> 1 Met Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu   1 5 10 15 Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile              20 25 30 Gln Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp          35 40 45 Asp Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala      50 55 60 Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly  65 70 75 80 Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys                  85 90 95 Val Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr             100 105 110 Glu Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe         115 120 125 Gly Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly     130 135 140 Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu 145 150 155 160 Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln                 165 170 175 Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val             180 185 190 Arg Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp         195 200 205 Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His     210 215 220 Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser 225 230 235 240 Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu                 245 250 255 Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro             260 265 270 Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Ala Gln         275 280 285 Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala     290 295 300 Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly 305 310 315 320 Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu                 325 330 335 Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val             340 345 350 Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu         355 360 365 Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly     370 375 380 His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn 385 390 395 400 Thr Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly                 405 410 415 His Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly             420 425 430 Val Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys         435 440 445 Thr His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala     450 455 460 Ile Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro Val Tyr Val 465 470 475 480 Gly Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser                 485 490 495 Ser Glu Lys Ile His Ser Asn Glu Ile Ser Ser Ser Ser Ser Gle Val             500 505 510 Leu Gly Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn Ser Lys Leu         515 520 525 Ser Leu Phe Phe Glu Ile Lys Ser     530 535

Claims (12)

(i) a capsular polysaccharide-transfer protein conjugate,
(ii) 4 mg / mL to less than 7 mg / mL of 2-phenoxyethanol, 2-PE, and
(iii) 90 to 200 占 퐂 / mL of formaldehyde (HCHO)
Wherein the capsular polysaccharide comprises 13 to 24 species of the capsular polysaccharide derived from Streptococcus pneumoniae serotype. The method of claim 1, wherein the capsular polysaccharide is selected from the group consisting of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F , 18C, 19A, 19F, 20, 22F, 23F, and 33F, of the capsular polysaccharides of the polyvalent pneumococcal vaccine composition. 3. The composition of claim 2, wherein the snack polysaccharide comprises
Thirteen kinds of capsular polysaccharides derived from Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F;
14 clathrin polysaccharides derived from Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F;
Fifteen kinds of snack polysaccharides derived from Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2 and 12F; or
15 clathrin polysaccharides derived from Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2 and 15B; Or 17 kinds of 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
Wherein the vaccine composition is a vaccine composition. 2. The vaccine composition according to claim 1, wherein the carrier protein is CRM197 protein. 2. The polyvalged pneumococcal vaccine composition according to claim 1, wherein the capsular polysaccharide-carrier protein conjugate has a structure in which the capsular polysaccharide and the carrier protein are linked by -O-C (NH) -NH- group using a cyanylation method. The polyvalged pneumococcal vaccine composition according to claim 5, wherein the cyanylation method is performed using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr. The pharmaceutical composition according to claim 1, wherein, relative to 1 part by weight of the capsular polysaccharide derived from serotype 1,
The capsular polysaccharides from serotypes 2, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 23F, 12F and 15B,
1.8-2.2 parts by weight of a somatic polysaccharide derived from serotype 6B
Wherein the pneumococcal vaccine composition comprises a polyclonal antibody. 8. The multi-dose pneumococcal vaccine composition according to any one of claims 1 to 7, A pharmaceutical composition for preventing or treating a pneumococcal infection or a pneumococcal infection disease comprising the polyvalent pneumococcal vaccine composition according to any one of claims 1 to 7. 10. The pharmaceutical composition according to claim 9, wherein the pneumococcal infection disease is pneumonia. (1) preparing a capsular polysaccharide-protein conjugate of Streptococcus pneumoniae , and
(2) mixing the above-mentioned snack polysaccharide-protein conjugate with 2-phenoxyethanol (2-PE) and formaldehyde (HCHO)
Lt; / RTI &gt;
The capsular polysaccharide comprises 13 to 24 species of the capsular polysaccharide derived from Streptococcus pneumoniae serotype,
Wherein the amount of 2-phenoxyethanol is less than 7 mg / mL and the amount of formaldehyde used is 90 to 200 μg / mL,
A method for producing a polyvalent pneumococcal vaccine composition. 12. The method of claim 11, wherein the step (1) of preparing the capsular polysaccharide-protein conjugate comprises the step of performing a cyanylation method to link the capsular polysaccharide and the protein through -OC (NH) -NH- Lt; RTI ID = 0.0 &gt; pneumococcal &lt; / RTI &gt; vaccine composition.