CN110520154A - Multivalent streptococcus pneumoniae vaccine compositions - Google Patents

Abstract

The present invention relates to a Streptococcus pneumoniae (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 methods of making the vaccine compositions are provided.

Description

Multivalent pneumococcal vaccine composition

technical field

The present invention relates to a vaccine composition against pneumococcus (Streptococcus pneumoniae), more specifically, to a vaccine composition comprising (i) a capsular polysaccharide-protein conjugate, (ii) 2 - phenoxyethanol (2-PE) and (iii) formaldehyde (HCHO), and a process for the preparation of said vaccine composition.

Background technique

Pneumococcus (Streptococcus pneumoniae) is a Gram-positive, hemolytic strain of Streptococcus and is the leading 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, International Health Organization data), especially, among immunocompromised children under 5 years of age and the elderly over 65 years of age, caused by pneumococcal High incidence of invasive infectious disease.

Pneumococci are classified into approximately more than 90 serotypes based on the structural and immunological characteristics of capsular polysaccharides that surround their exterior (cell membrane) as a major virulence factor, and about 20 of them are known to be similar to 80 to 80 serotypes in humans. 90% pathogenicity associated. The only host of pneumococci is humans, and they usually colonize the healthy normal nasopharynx (20-40% of infants, 5-10% of adults). In 2005, the Centers for Disease Control and Prevention (CDC) reported that in developing countries alone, approximately 2.1 million children under the age of 5 died of pneumonia each year, 1.2 million of them from pneumonia pneumococcal infections, and about 3,000 cases of meningitis and 50,000 cases of sepsis due to pneumococci occur annually in the United States (Peters TR, Poehling KA et al., Invasive pneumococcal disease, JAMA 2007;297:1825- 6). Furthermore, according to the pneumoACTION database which is a database of pneumococcal diseases, in 2000, the incidence of pneumococcal infection among children in Korea was 24,047 cases per year, and 47 of them died (www.pneumoadip.org). In addition, according to the latest "study on the serotype analysis of pneumococcus in children and adolescents in the country" released by the Centers for Disease Control and Prevention ), pneumococcus was the most common (43.7%) cause of invasive infection in infants between 3 months and 59 months. For pneumococcus, which causes invasive infectious diseases worldwide, multidrug-resistant bacteria that exhibit resistance to more than three drugs in addition to penicillin have increased, and thus make the treatment of pneumococcal infectious diseases more difficult .

Therefore, there is an ongoing need for vaccination against pneumococcus in children and the elderly who are at high risk for pneumococcal infectious disease.

For the prevention of pneumococcal infectious disease, polyvalent pneumococcal polysaccharide vaccines have been developed and licensed since 1977, and these capsular polysaccharide vaccines have been shown to be useful for preventing pneumococcal disease in the elderly and high-risk patients of. However, since the immune system is less mature in infants and children than in adults, it is difficult to expect the effect of a vaccine when only polysaccharide vaccines are administered because the immune system cannot recognize polysaccharide antigens as external invasive factors . In order to solve such a problem that polysaccharide vaccines reduce immunogenicity in infants and children, etc., a 7-valent pneumococcal conjugate vaccine (7vPnC, ), a capsular polysaccharide-protein conjugate vaccine that conjugates an immunogenicity-enhancing carrier protein to a polysaccharide antigen, and has been reported in many references to be effective in preventing middle ear infections in infants and children. Effective for invasive disease and inflammation. However, the use of the 7-valent vaccine caused a decrease in invasive disease caused by the vaccine serotype used for the vaccine, but in addition showed a relative increase in pneumococcal disease caused by some non-vaccine serotypes. Therefore, a 10-valent capsular polysaccharide-protein conjugate vaccine has been developed and is commercially available and to 13-valent pneumococcal conjugate vaccine with 6 serotypes added to the basic serotype But for some of the serotypes included, the possibility of their insufficient efficacy as vaccines has been reported [Andrews NJ et al., (2014) Lancet Infec Dis(14) 839; EMEA Assessment Report of Prevenar 13 (EMEA Assessment Report for Prevenar 13), (2009) EMA/798877/2009], therefore, there is always a need for the development of new vaccine formulations showing higher and more stable thresholds.

At the same time, preservatives should be used in vaccine injection formulations to prevent microbial contamination. For mixed vaccine products exported from UN etc. to underdeveloped countries, multi-dose products containing preservatives are preferred due to the country's environment, delivery method, cost, etc. Thimerosal, 2-phenoxyethanol (2-PE), phenol, etc. are present as preservatives for vaccine products and are present in amounts commonly used in the art. For example, thimerosal is used at a concentration of 10 μg/mL and 2-PE is used at a concentration of 5 mg/mL, and multi-dose vaccine products containing Bactericidal ability tests can be commercialized.

Merthiolate, an ethylmercury-derived compound, has been used as a preservative in multiple-dose vaccine injections since the early 1930s. Thimerosal has been used for the purpose of preventing the growth of contaminating microorganisms and maintaining sterile conditions during the storage and use of vaccine products, and many 5-valent liquid mixed vaccines (including D, T, P, Hib , HBsAg) contains this thimerosal as a preservative.

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

However, since the amounts of these preservative components used to achieve bactericidal activity in vaccines may be sufficient to cause toxicity and/or side effects, there is a need for the development of technologies that reduce their use and provide adequate bactericidal activity .

Contents of the invention

[technical problem]

One example provides a pneumococcal vaccine composition comprising (i) a capsular polysaccharide-protein conjugate of Streptococcus pneumoniae, (ii) 2-phenoxyethanol (2-PE), and (iii) Formaldehyde (HCHO). The vaccine composition may be a multi-dose vaccine composition for multiple administrations.

Another example provides a pharmaceutical composition for preventing or treating pneumococcal infection disease, which comprises the pneumococcal vaccine composition. The pharmaceutical composition for prevention or treatment may be a multi-dose pharmaceutical composition for multiple administrations.

Other examples provide a method for preparing a pneumococcal vaccine composition with improved stability and/or preservation (or bactericidal activity), the method comprising preparing the capsular polysaccharide of Streptococcus pneumoniae (Streptococcuspneumoniae)- The step of protein conjugate, and the step of mixing said capsular polysaccharide-protein conjugate with 2-phenoxyethanol (2-PE) and formaldehyde (HCHO); Methods of stability and/or preservative capacity (or bactericidal activity). The vaccine composition may be a multi-dose vaccine composition for multiple administrations. The step of preparing a capsular polysaccharide-protein conjugate may include the step of performing a cyanation method to link the capsular polysaccharide and protein via -O-C(NH)-NH-.

[Technical solutions]

One embodiment provides a pneumococcal vaccine composition or pneumococcal immunogenic composition comprising or consisting essentially of:

(i) capsular polysaccharide-protein conjugates, wherein the capsular polysaccharide is derived from Streptococcus pneumoniae (Streptococcuspneumoniae),

(ii) 2-phenoxyethanol (2-PE), and

(iii) Formaldehyde (HCHO).

Herein, a pneumococcal immunogenic composition means a composition that induces an immune response against pneumococcus and is used in the same meaning as a pneumococcal vaccine composition unless otherwise indicated.

The pneumococcal vaccine composition provided herein may be an immunogenic composition with improved stability and/or preservation (bactericidal ability) by including capsular polysaccharide of Streptococcus pneumoniae and as a preservative The efficacy (immunogenicity) of inducing an immune response against pneumococcus was maintained for a long time with 2-phenoxyethanol and formaldehyde.

The capsular polysaccharide may be capsular polysaccharide derived from Streptococcus pneumoniae. Specifically, the capsular polysaccharide can be derived from 2 or more serotypes of Streptococcus pneumoniae, such as 5 or more, 7 or more, 9 or more, 11 or more, 13 or more or 14 Capsular polysaccharides of the above serotypes. In one embodiment, the capsular polysaccharide may comprise 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 derived from Streptococcus pneumoniae serotypes Capsular polysaccharides of 1 to 24, 14 to 19, 14 to 17, or 14 to 15 serotypes. For example, the capsular polysaccharide may comprise a polysaccharide derived from the group consisting of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24 of 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F , capsular polysaccharides of 14 to 19, 14 to 17 or 14 to 15 serotypes. In one embodiment, the capsular polysaccharide may comprise polysaccharides derived from Streptococcus pneumoniae (Streptococcuspneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F 13 capsular polysaccharides derived from 14 capsules of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F Polysaccharides, derived from 15 capsular polysaccharides of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2 and 12F, or 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 Seventeen capsules derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F polysaccharides, but not limited thereto.

The capsular polysaccharide-protein conjugate can be the above-mentioned capsular polysaccharides derived from two or more serotypes that are separately coupled to proteins by common methods such as covalent bonds. United things. In one embodiment, the conjugate may have a cyanation method in which each capsular polysaccharide (specifically, the hydroxyl group of the polysaccharide) and the protein (specifically, the amino group of the protein) are passed through -O-C(NH )-NH-linked structure ([polysaccharide]-O-C(NH)-NH-[protein]). When the capsular polysaccharide and protein are conjugated using a cyanation method, it can advantageously maintain immunogenicity after conjugation since capsular polysaccharides are less likely to occur compared to other methods (e.g. reductive amination methods, etc.) Structural modification of membrane polysaccharides. For example, it has been reported that although some serotypes (19F etc.) This problem is not possible when it is coupled to the protein by the cyanation method.

The protein may be a carrier protein, eg, may be a protein that exhibits non-toxicity and non-reactivity and that can be collected in sufficient quantity and purity. Capsular polysaccharides derived from various serotypes may be coupled to the same or different proteins, eg, the same protein, respectively.

In one embodiment, the protein may be a CRM197 protein. The CRM197 protein is an avirulent variant of diphtheria toxin, a toxoid, isolated from the culture medium of Corynebacterium diphtheria strain C7 (CRM197, e.g. grown on casamino acid and yeast extract basal medium) . The CRM197 protein can be purified from the C7 medium of Corynebacterium diphtheria strain by ultrafiltration, ammonium sulfate precipitation, and ion-exchange chromatography, or obtained recombinantly by common methods, e.g. see US Patent No. 5,614,382 (incorporated herein by reference) methods disclosed in . In one embodiment, the CRM197 protein may comprise the amino acid sequence of GenBank accession number 1007216A, or have more than 90%, more than 95%, more than 98%, more than 99%, more than 99.5% or more than 99.9% of the amino acid sequence. source amino acid sequence.

In addition, toxoids derived from Bacillus diphtheria other than Corynebacterium diphtheria strain C7 may be used as carrier proteins.

In addition, the available carrier protein can be at least one selected from the following:

Inactivated bacterial toxoids, such as tetanus toxoid, pertussis toxoid, cholera toxoid (such as disclosed in International Patent Application Publication No. WO2004/083251), derived from Escherichia coli (Escherichia coli) (Escherichia coli) .coli) LT, Escherichia coli (Escherichia coli) ST and exotoxin A of Pseudomonas aeruginosa (Psendomonasaeruginosa);

Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porin, transferrin-binding protein, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesion protein (PsaA), derived from C5a peptidase from group A or group B streptococci, Haemophilus influenza protein D;

Ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), purified protein derivative of tuberculin (PPD);

Variants (toxoids) of diphtheria toxin such as CRM197, CRM173, CRM228, CRM45 and the like.

The capsular polysaccharide-protein conjugate may be a multivalent polysaccharide-protein conjugate, including two or more such as five or more, seven or more, or nine derived from Streptococcus pneumoniae serotypes A conjugate in which each of the capsular polysaccharides of the above, 11 or more, 13 or more or 14 serotypes is coupled to the above-mentioned carrier protein such as CRM197 protein. In one embodiment, the capsular polysaccharide-protein conjugate can be 13-24 valent, 13-19 valent, 13-17 valent, 13-15 valent, 14-24 valent, 14 valent to 19 valent, 14 to 17 valent or 14 to 15 valent polysaccharide-protein conjugates, wherein 13 to 24, 13 to 19, 13 serotypes of Streptococcus pneumoniae Each of the capsular polysaccharides of to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17, or 14 to 15 serotypes is associated with the carrier protein, e.g. CRM197 protein coupling. For example, the capsular polysaccharide-protein conjugate may be 13-24 valent, 13-19 valent, 13-17 valent, 13-15 valent, 14-24 valent, 14-19 valent, 14-valent to 17-valent or 14- to 15-valent polysaccharide-protein conjugates, which are derived from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, and 8 of Streptococcus pneumoniae , 13 to 24, 13 to 19, 13 to 17, 13 Each of the capsular polysaccharides of 1 to 15, 14 to 24, 14 to 19, 14 to 17, or 14 to 15 serotypes are coupled to the carrier protein, eg, CRM197. In one embodiment, the capsular polysaccharide-protein conjugate may be: a 13-valent polysaccharide-protein conjugate comprising serotypes 1, 3, 4, 5, and 3 derived from Streptococcus pneumoniae. Each of the 13 capsular polysaccharides of 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F and a carrier protein such as CRM197; a 14-valent polysaccharide-protein conjugate comprising Each of the 14 capsular polysaccharides of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F and a carrier protein such as CRM197 ; 15-valent polysaccharide-protein conjugate, which comprises derived from Streptococcus pneumoniae (Streptococcuspneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2 Each of the 15 capsular polysaccharides and 12F and a carrier protein such as CRM197; a 15-valent polysaccharide-protein conjugate comprising serotypes 1, 3, 4, 5, 3, 4, 5, Each of the 15 capsular polysaccharides of 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 15B and a carrier protein such as CRM197; or a 17-valent polysaccharide-protein conjugate comprising Seventeen capsules derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F Each of polysaccharides and carrier proteins such as, but not limited to, CRM197.

The pneumococcal vaccine composition provided herein is a pneumococcal conjugate vaccine (PCV) comprising the capsular polysaccharide-protein conjugate. The pneumococcal vaccine composition may be a multivalent pneumococcal vaccine composition, which comprises two or more such as five or more, seven or more, or nine serotypes derived from Streptococcus pneumoniae A polysaccharide-protein conjugate in which each of the capsular polysaccharides of the above, 11 or more, 13 or more or 14 serotypes is coupled to the above-mentioned carrier protein, such as CRM197 protein. In one embodiment, the pneumococcal vaccine composition may be 13-valent to 24-valent, 13-valent to 19-valent, 13-valent to 17-valent, 13-valent to 15-valent, 14-valent to 24-valent, 14-valent to 19-valent , 14-valent to 17-valent or 14-valent to 15-valent pneumococcal vaccine composition, said composition comprising 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 polysaccharide-protein conjugates, of which 13 to 24, 13 to 15 derived from Streptococcus pneumoniae serotypes Each of the capsular polysaccharides of 19, 13-17, 13-15, 14-24, 14-19, 14-17, or 14-15 serotypes is associated with The carrier protein such as CRM197 is coupled. For example, the pneumococcal vaccine composition can be valenced from 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17 Valence or 14-valent to 15-valent pneumococcal vaccine composition, said composition comprises wherein is derived from and is selected from Streptococcus pneumoniae (Streptococcus pneumoniae) serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8 , 13 to 24, 13 to 19, 13 to 17, 13 Each of the capsular polysaccharides of 1 to 15, 14 to 24, 14 to 19, 14 to 17, or 14 to 15 serotypes coupled to the carrier protein, such as the CRM197 protein Polysaccharide-protein conjugates.

In one embodiment, the pneumococcal vaccine composition may be:

(1) A 13-valent pneumococcal vaccine composition comprising 13 polysaccharide-protein conjugates derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V Each of the 13 capsular polysaccharides of , 14, 18C, 19A, 19F and 23F is coupled to the carrier protein such as CRM197 protein;

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

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

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

(5) 17-valent pneumococcal vaccine composition, which comprises 17 polysaccharide-protein conjugates, which are derived from Streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V Each of the 17 capsular polysaccharides of , 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F is coupled to the carrier protein such as the CRM197 protein.

Compared with conventionally developed multivalent vaccines (such as 13-valent vaccines), the multivalent pneumococcal vaccine compositions provided herein, for example, 13-valent to 24-valent, 13-valent to 19-valent, 13-valent to 17-valent, 13-valent 15-valent, 14-valent to 24-valent, 14-valent to 19-valent, 14-valent to 17-valent, or 14-valent to 15-valent pneumococcal vaccines have remarkably excellent threshold values and can be expected to be very good at preventing and/or treating pneumonia Effects of bacterial infectious diseases.

The pneumococcal vaccine composition may be a single dose for single administration or a multi-dose composition for multiple administrations. As used herein, "multiple doses" may mean comprising vaccine doses that may be administered (vaccinated) to 1 individual administered (vaccinated) more than once (e.g., more than 2 times) or may be administered (vaccinated) to 2 A formulation unit for administering (vaccinating) more than one or more than one (eg more than two) vaccine doses to an individual.

In addition to the pneumococcal capsular polysaccharide-protein conjugates of more than two serotypes, the pneumococcal vaccine composition may further contain a preservative. The preservative that can be used in the vaccine composition (such as injection preparation) can be selected from 2-phenoxyethanol, formaldehyde, chlorobutanol, m-cresol, methylparaben, propylparaben At least one of esters, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thimerosal, phenylmercuric nitrate, and the like.

In one embodiment, as the above-mentioned multivalent pneumococcal vaccine composition, for example, the above-mentioned 13-valent to 24-valent, 13-valent to 19-valent, 13-valent to 17-valent, 13-valent to 15-valent, 14-valent to In the optimized form of 24-valent, 14-valent to 19-valent, 14-valent to 17-valent or 14-valent to 15-valent pneumococcal vaccine composition, the preservative may comprise 2-phenoxyethanol and formaldehyde. Likewise, by including 2-phenoxyethanol and formaldehyde as preservatives, the amount of 2-phenoxyethanol used is reduced, thereby reducing toxicity and/or side effects and achieving greater Improved anti-corrosion effect.

The content of 2-phenoxyethanol contained in the multivalent pneumococcal vaccine composition of the present invention may be less than 10 mg/ml, less than 7 mg/ml, less than 6 mg/ml or less than 5 mg/ml, for example more than 4 mg/ml And less than 10mg/ml, 4mg/ml to 7mg/ml, more than 4mg/ml and less than 7mg/ml, 4 to 6mg/ml, 4.5mg/ml to 6mg/ml, 5mg/ml to 6mg/ml, 4 to 5 mg/ml or 4.5 mg/ml to 5 mg/ml (when used herein, the term "to" used in a numerical range refers to a range between above the lower limit value and below the upper limit value). The content of 2-phenoxyethanol in the vaccine composition is preferably above the lower limit of the range to exhibit a significant antiseptic effect, and is preferably less than the upper limit of the range or the upper limit of the range To ensure no or acceptable toxicity and/or side effects.

The content of formaldehyde contained in the multivalent pneumococcal vaccine composition of the present invention may be 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 contained in the vaccine composition is preferably above the lower limit of the range to exhibit a significant antiseptic effect, and is preferably below the upper limit of the range to ensure no or acceptable toxicity and/or side effects.

The vaccine composition is characterized in that it is stable for about 3 months or more, about 6 months or more, about 1 More than a year, more than about 1.5 years, more than about 2 years, or more than about 2.5 years. Here, the stability of the vaccine composition may mean that the vaccine composition maintains the original antigenicity (immunogenicity) at the same level, and/or each component is maintained without degradation or loss, and/or No infection such as bacteria/virus etc.

Another embodiment provides a pharmaceutical composition for preventing or treating pneumococcal infection or pneumococcal infectious disease, which comprises the above-mentioned multivalent pneumococcal vaccine composition. Other embodiments provide a method for preventing or treating pneumococcal infection or pneumococcal infectious disease, the method comprising administering a pharmaceutically effective dose to an individual in need of prevention or treatment of pneumococcal infection or pneumococcal infectious disease The above-mentioned polyvalent pneumococcal vaccine composition. The pneumococcal vaccine composition contained in the pharmaceutical composition or the pneumococcal vaccine composition used in the prophylactic or therapeutic method may be a single-dose pharmaceutical composition for a single administration or for multiple administrations multi-dose pharmaceutical composition.

Pneumococcal infectious disease means all diseases caused by pneumococcal infection, and may be pneumonia, inflammation in the middle ear, sinusitis, bacteremia, and the like. "Pneumonia" is an acute infectious disease of the lung parenchyma, and its infection sources are mainly Streptococcus pneumoniae and Klebsiellapneumoniae. Specifically, pneumococcal pneumonia accounts for approximately 50% of all pneumonias, and is a disease in which severe chills, fever, cough, and chest pain occur with often bleeding sputum, and may cause complications such as pleurisy, meningitis, endocardial Inflammation, peritonitis, etc. (Diagn. Microbiol. Infect. Dis., 2001, 39: 181-185).

As used herein, the term "pneumococcus" refers to Streptococcus pneumoniae, and in general, a commensal organism that colonizes the mucosal surface of the nasopharynx of humans. When host factors allow the organism to gain access to the lower respiratory tract, a strong inflammatory response ensues and thus causes a dense consolidation as the alveolar spaces fill with exudate, causing pneumonia. Pneumococcus can synthesize more than 90 capsular polysaccharides with unique structures, and according to these structural and immunological characteristics of capsular polysaccharides, the serotypes of pneumococci are classified. Therefore, when a preparation for a vaccine is prepared using the capsular polysaccharide of pneumococcus, different immune responses may be exhibited depending on the kind of capsular polysaccharide, that is, the serotype of pneumococcus from which the capsular polysaccharide is derived.

Since the capsular polysaccharide is recognized as an antigen when administered into the body, allowing the production of antibodies against it, it allows its use to produce vaccine compositions for the prevention of pneumococci (or pneumococcal infectious diseases). Herein, the term "antigen" means a substance that can specifically elicit an immune response when invaded into the body. In one embodiment, derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, Thirteen to twenty-four capsular polysaccharides of 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F can serve as antigens, respectively.

Other embodiments provide a method for preparing a pneumococcal vaccine composition with improved stability and/or preservation (or bactericidal ability), or a method for improving the stability and/or preservation of a pneumococcal vaccine ability (or bactericidal ability) method. The method can include:

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

(2) A step of mixing the capsular polysaccharide-protein conjugate with 2-phenoxyethanol (2-PE) and formaldehyde (HCHO).

Capsular polysaccharide, protein, conjugate, and 2-phenoxyethanol and formaldehyde contents were the same as described above.

In the method, the capsular polysaccharide can be prepared by standard techniques known to those skilled in the art, and is not particularly limited to the method. The capsular polysaccharide can be hydrolyzed to reduce its size to reduce viscosity and induce potent immunogenicity.

The step (1) of preparing the capsular polysaccharide-protein conjugate may include the step of linking the capsular polysaccharide and protein via -O-C(NH)-NH- by performing a cyanation method.

In one embodiment, the step (1) of preparing the capsular polysaccharide-protein conjugate may include:

(i) a step of isolating or isolating and purifying capsular polysaccharide from Streptococcus pneumoniae;

(ii) a step of dissolving and/or hydrolyzing the isolated capsular polysaccharide; and

(iii) a step of linking the capsular polysaccharide to a protein via -O-C(NH)-NH- by performing a cyanation method.

In other embodiments, the step (1) of preparing the capsular polysaccharide-protein conjugate may be followed by the step of linking the capsular polysaccharide and the protein via -O-C(NH)-NH- by performing a cyanation method Afterwards, one or more steps selected from the ultrafiltration step, the sterilizing filtration step and the adsorption step are further performed. The cyanation method can be performed using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr.

In a specific example of the present invention, sodium deoxycholate was used to lyse Streptococcus pneumoniae with 13 or 15 respective different serotypes, respectively, and release polysaccharides attached to cells. Then, purified from 21 serotypes 1, 2, 3, 4, 5, 6A, 6B, 9V, 8, 9N, 10A, 11A by performing the CTAB (cetyltrimethylammonium bromide) method , 12F, 15B, 17F, 18C, 19A, 19F, 20, 22F, and 23F polysaccharides, because ionic bonds can be formed between them and CTAB, and aluminum phosphate gel (Algel) was used to purify 3 polysaccharides that did not react with CTAB. serotypes 7F, 14 and 33F. The reaction with CTAB can be performed by adding 0.5 to 5% by weight or 1 to 3% by weight to the reaction (e.g. 2 to 3% by weight for polysaccharides derived from 23F, 2 to 3% by weight for polysaccharides derived from serotypes other than 23F For example, 1 to 2% by weight) in an amount of 1 to 20% (w/v) or 5 to 15% (w/v) of CTAB, but not limited thereto. After the reaction with CTAB, one or more steps of recovering the precipitate after centrifugation, resuspending the precipitate using a sodium chloride solution (for example, about 100 to 500 mM) and removing CTAB ions using sodium iodide may be further carried out, but not limited to this. The aluminum phosphate gel reaction may be performed by adding an aluminum phosphate gel solution in an amount of 1 to 20% by weight or 5 to 15% by weight to a reactant, but is not limited thereto.

Since infants and children with a lower immune system compared to adults cannot recognize it as an antigen when using a vaccine composition utilizing only the capsular polysaccharide, an immune response may not occur, so the preparation of And a form of a conjugate in which a carrier protein is combined with the capsular polysaccharide is used.

"Carrier protein" refers to a protein that can increase the immunogenicity of the polysaccharide antigen by covalently binding to the capsular polysaccharide, and the specific type is the same as above. In a particular embodiment, CRM197 can be used. The carrier protein can be coupled to the capsular polysaccharide by standard conjugation methods, and the capsular polysaccharide-carrier protein conjugate formed by it can be one or more capsular polysaccharides coupled to a carrier protein. Capsular polysaccharide-carrier protein conjugates.

Known methods for the preparation of capsular polysaccharide and carrier protein conjugates are all included within the scope of the present invention, and the conjugates have wherein the capsular polysaccharide and carrier protein are passed through Structures linked by -O-C(NH)-NH- groups. The cyanation method can be suitably carried out by a method known to those skilled in the art, for example, it can be carried out by using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr, but Not limited to this.

As an example for the preparation of capsular polysaccharide and carrier protein conjugates, saccharides can be formed by chemically activating purified capsular polysaccharides and coupling each chemically activated capsular polysaccharide to the carrier protein one by one. Conjugates. Cyanolation activity using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) treatment can change the hydroxyl group of the capsular polysaccharide into a cyanate group, and by using it to interact with The amino groups of the carrier protein CRM197 form a covalent bond. The cyanation reaction by CDAP can specifically be terminated by adding 3 molar equivalents of glycine solution compared with 1 molar equivalent of CDAP and adjusting the pH to 9.0, but not limited thereto, and those skilled in the art can according to the purpose The reaction solution and reaction conditions are appropriately controlled.

The collected capsular polysaccharide-carrier protein conjugates can be purified by various methods. Examples of those methods include concentration/diafiltration methods, column chromatography and multilayer filtration. The vaccine composition of the present invention is formulated and used by mixing the purified polysaccharide-protein conjugates separately. For example, compositions can be prepared by formulating 13 individual capsular polysaccharide-carrier protein conjugates with a physiologically acceptable vehicle. Examples of such vehicles may be water, buffered saline solution, water for injection, polyols (eg glycerol, propylene glycol, liquid polyethylene glycol) or dextrose solution, but are not limited thereto.

In a specific example of the present invention, 13 capsular polysaccharide-carrier conjugates were prepared by performing the following steps: 1) dissolution and hydrolysis of 13 capsular polysaccharides, 2) by using CDAP (1-cyano- 4-dimethylaminopyridinium tetrafluoroborate) for the coupling reaction process of each capsular polysaccharide and CRM197, 3) termination of the coupling reaction, 4) ultrafiltration, 5) sterile filtration and 6 ) adsorption step.

As used herein, the term "vaccine" is an antigen-containing biological agent that provides immunity to a living body, and means an immunogen or antigen for preventing infectious diseases that produces immunity in a living body by administration to humans or animals sexual substance.

The vaccine composition may further comprise one or more selected from adjuvants, preservatives, buffers, cryoprotectants, salts, divalent cations, non-ionic detergents and free radical oxidation inhibitors.

In this context, the term "adjuvant" means a substance used to increase the immunogenicity of the immunogenic composition of the invention. Typically, such adjuvants are provided to enhance the immune response and are well known to those skilled in the art. Adjuvants suitable for improving the efficacy of the vaccine composition of the present invention may include at least one selected from:

(1) Aluminum salt (alum) (such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.);

(2) Emulsion formulations of the water-in-oil type (with or without other specific immunostimulators such as cell wall peptides (defined hereinafter) or bacterial cell wall components), for example (a) MF59 (No.WO90/14837): Containing 5% (w/v) squalene, 0.5% (w/v) Tween 80 and 0.5% (w/v) Span 85 (randomly also containing various amounts of MTP-PE), and using micro A jet machine such as 110Y micro jet machine (Microfluidics, Newton, MA) is formulated into submicron particles, (b) SAF: containing 10% (w/v) squalene, 0.4% (w/v) Tween 80, 5 % (w/v) Pluronic block polymer L121 and thr-MDP (see below) with large particle size emulsions formed into submicron emulsions or cavities by microfluidics, and (c) Ribi ^TM adjuvant System (RAS) (Corixa, Hamilton, MT): Contains one or more bacterial cell wall components from the following: 2% (w/v) squalene, 0.2% (w/v) Tween 80 and 3-O-deacylated monophosphoryl lipid A (MPL ^™ ) (Corixa), trehalose dipycolate (TDM) and cell wall skeleton (CWS) disclosed in U.S. Patent No. 4,912,094, preferably MPL+CWS ( Detox ^™ );

(3) Saponin adjuvants such as Quil A or STIMULON ^™ QS-21 (Antigenics, Framingham, MA, US Patent No. 5,057,540) or particles formed therefrom (eg ISCOM (immunostimulatory complex));

(4) Bacterial lipopolysaccharides, synthetic lipid A homologues (for example: aminoalkylglucosamine phosphate compounds (AGP)) or derivatives or homologues thereof (this can be purchased from Corixa and disclosed in U.S. Patent No., 6,113,918; an example of the AGP is 2-[(R)-3-tetradecanoyloxytetradecylamino]ethyl 2-deoxy-4-O-phosphono-3-O-[(R) -3-tetradecyloxytetradecanoyl]-2-[(R)-3-tetradecyloxytetradecylamino]-b-D-glucopyranoside, and it is also known as 529 (formerly known as RC529), and it is formulated as an aqueous form or as a stable emulsion;

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

(6) Cytokines, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL 12, IL-15, IL-18, etc.), interferon ( Such as gamma interferon), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (MCSF), tumor necrosis factor (TNF), co-stimulatory molecules B7-1 and B7-2, etc.;

(7) Wild-type cholera toxin (CT) or mutated cholera toxin such as in which glutamic acid located in the 29th amino acid position is replaced by other amino acids, especially histidine (WO-2002/098368 and WO-2002/098369) , pertussis toxin (PT) or Escherichia coli thermolabile toxin (LT), especially detoxified variants of bacterial ADP-ribosylating toxins such as LT-K63, LT-R72, CT-S109, PTK9/G129 (WO-93/13302 and WO-92/19265); and

(8) a trimer of complement such as complement component C3d,

But not limited to this.

The muramyl peptide may comprise N-acetyl-muramoyl-L-threonyl-D-isoglucosamine (thr-MDP), N-acetyl-muramoyl-L-alanine-2 -(1'-2'-dipalmitoyl-sn-glyceryl-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc., but not limited thereto.

The aluminum salt adjuvant can be an alum-precipitated vaccine or an alum-adsorbed vaccine. The aluminum salt may comprise alumina hydrate, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, aluminum gel, syperfos, aluminum hydroxide gel, aluminum hydroxide, aluminum hydroxyphosphate sulfate ( aluminum phosphate adjuvant (APA)), amorphous alumina, etc., but not limited thereto. APA means suspension of aluminum hydroxyphosphate. It can be prepared by mixing aluminum chloride and sodium phosphate in a ratio of 1:1 (by volume) to precipitate aluminum hydroxyphosphate sulfate, and to make the size of the precipitate 2-8 μm, which is then washed with saline solution Dialyzed and sterilized. As an embodiment, commercially available Al(OH) ₃ (such as aluminum glue or superfos) is used to adsorb proteins. 50-200 g of protein can be adsorbed per 1 mg of aluminum hydroxide, and the ratio depends on the pi of the protein and the pH of the solvent. Proteins with a low pi bind more strongly than proteins with a high pi. The aluminum salt can non-specifically activate macrophages, complement and innate immune mechanisms by forming an antigen storage point that slowly releases antigens for 2-3 weeks.

Herein, the term "antibacterial agent (or preservative)" means an antiviral and/or antimicrobial substance that inhibits the proliferation of microorganisms in the vaccine composition, and may be, for example, thimerosal, 2-phenoxyethanol, formaldehyde or a mixture thereof, but not limited thereto, and all common preservatives used in the art can be used.

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

In particular, when the vaccine composition of the present invention is intended for parenteral administration, the buffer may be selected among buffers suitable for USP. For example, the buffering agent may be selected from 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, At least one of acid, tartaric acid, base such as ammonia, diethanolamine, glycine, triethanolamine, TRIS.

In addition, the vaccine compositions of the present invention may contain non-ionic detergents. For example, it may contain surfactants such as polysorbate 20 and polysorbate 80, especially polyoxyethylene sorbitan ester (commonly known as Tween); ethylene oxide (EO), propylene oxide ( PO) and butylene oxide (BO) copolymers (such as DOWFAXTM); oxtoxinols with different repeating numbers of ethoxy (oxy-1,2-ethanediyl) groups, especially oxtoxinol-9 (Triton -100); ethylphenoxypolyethoxyethanol (IGEPAL CA-630/NP-40); nonylphenol ethoxylates such as NP series; produced from lauryl alcohol, cetyl alcohol, stearyl alcohol, oleyl alcohol Polyoxyethylene fatty acid ethers (Brij surfactants), especially triethylene glycol monolauryl ether (Brij 30); sorbitan ethers known as SPAN, especially sorbitan trioleate Esters (Span 85), but not limited thereto.

Tween 80 can be included in the emulsion and a mixture with a non-ionic detergent such as Tween 80/Span 85 can be used. Combinations of polyoxyethylene sorbitan esters such as Tween 80 and oxtoxinols such as Triton X-100 are suitable, and combinations of Laureth 9 with Tween or oxtoxinol are also useful. Specifically, 0.01% (w/v) to 1% (w/v), especially 0.1% (w/v) polyoxyethylene sorbitan ester (such as Tween 80); 0.001% (w /v) to 0.1% (w/v), especially 0.005% (w/v) to 0.02% (w/v) of octylphenoxypolyoxyethanol or nonylphenoxypolyoxyethanol (such as Triton X-100); and 0.1% (w/v) to 20% (w/v), preferably 0.1% (w/v) to 10% (w/v), especially 0.1% (w/v) to 1% (w/v) or about 0.5% (w/v) of polyoxyethylene ether (such as laureth9).

The vaccine compositions of the present invention may be formulated in single-dose vials, multi-dose vials, or prefilled syringes. Accordingly, one embodiment provides a vial or prefilled syringe comprising a single dose or multiple doses, eg more than one dose, of said vaccine composition. The vaccine composition may also include a physiologically acceptable carrier.

Herein, "multiple doses" may mean that more than one dose of the vaccine agent may be administered (vaccinated) to 1 administered (vaccinated) individual, or may be administered (vaccinated) once to 2 or more administered (vaccinated) individuals above vaccines.

As physiologically acceptable carriers for liquid preparations, there are aqueous or nonaqueous solvents, suspensions, emulsions and oils. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, and ethyl oleate. Such aqueous carriers include water, alcoholic/aqueous solvents, emulsions or suspensions, saline solutions and buffered solutions. Examples of oils include synthetic oils such as vegetable or animal oils, peanut oil, soybean oil, sunflower oil, liver oil and marine animal oil, and lipids obtained from milk or eggs. The vaccine composition of the present invention may be isotonic, hypertonic or hypotonic, and the pharmaceutical composition administered by infusion or injection may preferably be isotonic, but not limited thereto. Also, isotonic or hypertonic may be advantageous for storage of the composition. When the vaccine composition is hypertonic, it may be diluted prior to administration so as to become isotonic. The isotonic agent may be an ionic isotonic agent such as a salt or a nonionic isotonic agent such as a sugar. The ionic isotonic agent includes sodium chloride, calcium chloride, potassium chloride, magnesium chloride, etc., but is not limited thereto. The non-ionic isotonic agent includes sorbitol, glycerin, etc., but is not limited thereto.

The amount of conjugate in each vaccine dose can be selected to induce an immune protective response without significant side effects, and this amount can vary according to the serotype of the pneumococcus. In particular, the vaccine composition may comprise 0.8 to 1.2 or 0.9 to 1.1 parts by weight (ie 0.8 to 1.2 parts by weight or 0.9 to 1.1 parts by weight) based on the weight of capsular polysaccharide derived from serotype 1 ) derived from serotypes 2, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F and may comprise 1.6 to 2.4, 1.8 to 2.2 or 1.9 to 2.1 weight ratios (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) of serotype 6B derived Capsular polysaccharides, but not limited thereto.

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

Furthermore, most particularly, polysaccharides other than those derived from serotype 6B, that is to say from serotypes 1, 2, 3, 4, 5, 6A, 7F, 8, 9V, 9N , 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, the capsular polysaccharides may be contained in an amount of 2 to 2.4 μg or 2.1 to 2.3 μg, for example about 2.2 μg , and the capsular polysaccharide derived from serotype 6B may be included in an amount of 4 to 4.8 μg, 4.2 to 4.6 μg or 4.3 to 4.5 μg, for example about 4.4 μg, but not limited thereto.

Optimal amounts of components for certain vaccines can be demonstrated by standard studies involving observation of appropriate immune responses in subjects. For example, vaccination doses for humans can be determined by extrapolating the results of animal experiments. In addition, those skilled in the art can empirically determine the dosage according to the requirements of the field.

The vaccine composition may also include aluminum and sodium chloride, but not limited thereto.

The vaccine compositions of the present invention are useful for protecting individuals at risk of pneumococcal infection and preventing pneumococcal disease by administering a pharmaceutically effective amount via systemic or mucosal routes. The term "prevention" of the present invention means all actions of inhibiting or delaying the infection caused by pneumococcus by the administration of the vaccine composition of the present invention. "Pharmaceutically effective amount" defined in the present invention means a dose required to produce antibodies at a level that significantly reduces the possibility of infection by pneumococcus or the severity of infection. The term "administering" in the present invention means introducing a prescribed substance into an individual by some suitable method. The vaccine composition of the present invention can be administered through oral, nasal, rectal, transdermal or aerosol inhalation routes, but not limited thereto. The administration may be selected from injection via intramuscular, intraperitoneal, intradermal or subcutaneous routes, or via the mucosa of the oral/digestive, airway or genitourinary tract. As an embodiment, intranasal administration can be used to treat pneumonia or inflammation in the middle ear, and in this case, the inflammation can be attenuated at an early stage by more effective prevention of nasopharyngeal pneumococcal vectors.

"Individual" to whom the vaccine composition or pharmaceutical composition of the present invention is administered may mean a living organism in which pathogenic bacteria can infect or a cell, tissue or culture medium isolated therefrom, and said organism It may be a higher vertebrate, more specifically a mammal such as a human, etc., but is not particularly limited thereto.

In other embodiments of the present invention, the composition of the present invention may be administered by a single inoculation, or administered two, three, four or more times at appropriate intervals, but is not limited thereto. For example, a regular vaccination schedule for infants and newborns for invasive disease caused by Streptococcus pneumoniae 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. As examples of Streptococcus pneumoniae proteins suitable for inclusion, not only the proteins disclosed in WO-2002/053761 but also the proteins identified in WO-2002/083855 may be included within the scope of the present invention Inside.

In a specific example of the present invention, a polyvalent such as 13-24, 13-19, 13-17, 13-15, 14-24, 14-19 valent, 14-valent to 17-valent or 14-valent to 15-valent pneumococcal vaccine composition, which contained 2.2 μg of each polysaccharide in a total of 0.5 mL of vaccine composition, but the polysaccharide derived from 6B was 4.4 μg, about 29.3 μg CRM197 Carrier protein, 0.5 mg elemental aluminum (2 mg aluminum phosphate) adjuvant, about 4.25 mg (without preservatives) or about 3.5 mg (with preservatives) sodium chloride, about 295 μg succinate buffer solution, along with about 3 mg of 2-phenoxyethanol and about 60 μg of formaldehyde.

In another specific example of the present invention, in the serum of rabbits inoculated with the vaccine composition, the serotype-specific IgG concentration ratio higher levels (Table 1). Furthermore, it was confirmed that it exhibits more Better results, even in a functional immunogenicity confirmatory test (opsonophagocytosis assay) performed on it (Table 2). Therefore, it can be seen that the vaccine composition of the present invention has a very excellent effect for use in the prevention of pneumococcal disease.

Other aspects of the invention are an immunogenic composition against pneumococcus comprising 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 capsular polysaccharide-carrier protein conjugates. The conjugate and pneumococcus were the same as described above.

The present invention comprises 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 The composition of the capsular polysaccharide-carrier protein conjugate comprises 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 Capsular polysaccharides of Streptococcus pneumoniae to 19, 14 to 17, or 14 to 15 serotypes each different, and recognize it as an antigen when administered in vivo, thereby eliciting an immune response in order to generate antibodies against it, and thus it can be used as an immunogenic composition against pneumococcus.

A further aspect of the invention is a method of preventing pneumococcal disease by administering said vaccine composition or immunogenic composition to an individual in need thereof.

Other aspects of the present invention provide the use of a composition comprising 13 capsular polysaccharide-carrier protein conjugates in the preparation of a vaccine composition for preventing pneumococcal disease, wherein the 13 conjugates are derived from Covalently conjugated to each of the 13 capsular polysaccharides of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F A conjugate to the carrier protein, and the carrier protein is a CRM197 protein, and the conjugate has wherein the capsular polysaccharide and the carrier protein are passed through -O-C(NH)-NH- using a cyanation method Group-linked structures.

Vaccine composition, immunogenic composition and prevention of pneumococcal disease etc. are the same as described above.

Another aspect of the present invention is a method for preparing the immunogenic composition, which method comprises the preparation of serotypes 1, 3, 4, 5, 6A, 6B, The step of coupling each of the 13 isolated capsular polysaccharides of 7F, 9V, 14, 18C, 19A, 19F and 23F to the carrier protein CRM197 such that the capsular polysaccharide and carrier protein have )-NH-group-linked structure.

The immunogenic composition and its method of preparation are the same as described above.

[beneficial effect]

The multivalent pneumococcal vaccine composition of the present invention comprises a capsular polysaccharide-protein multivalent conjugate having a unique coupling structure, and includes an optimized combination of 2-phenoxyethanol and formaldehyde as preservatives and content, thereby maintaining excellent immunogenicity and having excellent stability and/or preservation. Therefore, the vaccine composition and immunogenic composition of the present invention can be used more safely and usefully for the prevention of diseases caused by pneumococcus in infants, children and adults.

[mode of the present invention]

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are given for the purpose of illustration and are not intended to limit the scope of the invention.

Preparation Example 1: Preparation of Capsular Polysaccharide

1-1. Preparation of cell bank

Obtain 16 different serotypes (1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F) from the commissioned organization US CDC (United States Centers for Disease Control and Prevention). , 12F and 15B) Streptococcus pneumoniae (Streptococcuspneumoniae), and the cell bank was prepared by the following method.

Streptococcus pneumoniae strains were plated on blood agar media to confirm pneumococci and remove media components present. After selecting a well-growing single colony of more than 10 single colonies, it was inoculated in a liquid medium (soybean peptone (Kerry Bio-Science) or yeast extract (Bio-springer) source) that did not contain animal-derived components therein. culture medium), culture and proliferate, and then add synthetic glycerol to prepare a research cell bank (RCB) containing the synthetic glycerol.

After removing 1 vial of a research cell bank whose expression of a polysaccharide with an inherent serotype has been confirmed and proliferating the cells in a liquid medium free of components of animal origin, synthetic glycerol was added, thereby preparing a master cell bank, and After taking out 1 bottle of the master cell bank and proliferating the cells in a liquid medium free of animal-derived components, synthetic glycerol was added, thereby preparing a cell bank for preparation.

The prepared cell bank was stored in a super-frozen state at -70°C or lower to use it for the following experiments.

1-2. Fermentation and polysaccharide separation

One vial of the cell bank used for preparation was thawed and inoculated in liquid medium free of animal-derived components. The seed culture was performed at 37±2° C. without agitation until a constant cell concentration (optical density, OD 600 ) was reached. After confirming whether the culture medium in which the seed culture was completed was contaminated, it was inoculated in a liquid medium containing no animal-derived components (soybean peptone (Kerry Bio-Science) or yeast extract (Bio-springer) source medium) in the fermentor (OD600=2.5±0.2).

The main culture was then maintained at 37±2°C with sterile potassium hydroxide solution to maintain the pH of the medium to 7.2±0.2 under minimal agitation. The concentration of cells in the culture solution and the concentration of glucose contained in the medium were measured by sampling from 2 hours after the initiation of culture. Cultivation was complete when the glucose in the medium was depleted.

After the completion of the culture, 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, thereby lysing the cells and release the polysaccharides bound to the cells.

1-3. Purification of capsular polysaccharide

After adding phosphoric acid to the sodium deoxycholate-treated sample obtained in Preparation 1-3 for a long time and titrating the pH to 3.5 ± 0.3, and then allowing it to react by standing for 15 hours ± 3 hours, the supernatant was collected by centrifugation solution (17,000xG, room temperature, 1 hour). The collected supernatant was passed through a depth filter (0.55–9.0um) and concentrated, and subjected to buffer exchange with phosphate buffer.

After the buffer replacement, the sample was passed through a charcoal filter, followed by removal of impurities by the following two methods:

1) Since 14 serotypes of serotypes 1, 2, 3, 4, 5, 6A, 6B, 9V, 12F, 15B, 18C, 19A, 19F and 23F are different from CTAB (hexadecyltrimethyl Ionic bonding of ammonium bromide) is available, so the CTAB method was carried out, specifically, with a 10 wt % CTAB solution at 1.5 wt % (in cases other than 23F) or 2.5 % by weight of the process solution (in the case of 23F) was treated so that it was reacted for 1 hour ± 10 minutes. The precipitate was then collected by centrifugation at 17,000xG for 1 hour. Subsequently, for treatment with sodium chloride (NaCl) and sodium iodide (NaI), the precipitate collected by CTAB treatment was resuspended using 200-300 mM sodium chloride (NaCl) solution, and the An amount of 50% by weight sodium iodide (NaI) removes CTAB ions. However, for serotype 3, a 350 mM NaCl solution was used. After treatment with sodium chloride and sodium iodide, the supernatant was collected by centrifugation and used in subsequent processes. 2) For serotypes 7F and 14, which do not react with CTAB, add aluminum phosphate gel (Algel) solution and react, and then use 10% by weight of aluminum phosphate gel solution based on the total process solution Process and react for 1 hour. The supernatant obtained from centrifugation (1 hour at 17,000xG) was collected and used in subsequent processes.

The samples after the two types of impurity removal methods were passed through the process of depth filter and ultrafiltration (UF/DF), and then they were manufactured into powder form by controlling the amount of ethanol and sodium chloride and stored.

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

2-1: Comparison of coupling methods between reductive amination and cyanation

使用血清型9V的纯化多糖和CRM197载体蛋白(GenBank登记号1007216A；SEQ IDNO：1；mgaddvvdssk sfvmenfssy hgtkpgyvds iqkgiqkpks gtqgnydddw kefystdnkydaagysvdne nplsgkaggv vkvtypgltk vlalkvdnae tikkelglsl teplmeqvgt eefikrfgdgasrvvlslpf aegsssveyi nnweqakals veleinfetr gkrgqdamye ymaqacagnr vrrsvgsslscinldwdvir dktktkiesl kehgpiknkm sespnktvse ekakqyleef hqtalehpel selktvtgtnpvfaganyaa wavnvaqvid setadnlekt taalsilpgi gsvmgiadga vhhnteeiva qsialsslmvaqaiplvgel vdigfaaynf vesiinlfqv vhnsynrpay spghktqpfl hdgyavswnt vedsiirtgfqgesghdiki taentplpia gvllptipgk ldvnkskthi svngrkirmr craidgdvtf crpkspvyvgngvhanlhva fhrsssekih sneissdsig vlgyqktvdh tkvnsklslf feiks)，尝试还原胺化和氰基化的偶联方法，以比较两种偶联方法的偶联得率。 The specific coupling method is as follows.

2-1-1. Reductive amination

11.7 mg of sodium periodate was added to the undiluted solution of the polysaccharide of serotype 9V and stirred at 21 to 25°C to activate the polysaccharide. After the oxidized polysaccharide was concentrated and diafiltered using a 100 KDa ultrafiltration filter and WFI (water for injection), the remaining residual polysaccharide was mixed with CRM197 protein at a sugar/CRM197=0.5 ratio (weight ratio) and freeze-dried. The lyophilized complex was thawed and stabilized (equilibrium) at 21 to 25°C. After the equilibrated complex was cleaved by isothermally treating it in sodium phosphate (Na ₃ PO ₄ ) buffer at a ratio of 0.1 M/20 g sugar (37±2° C.), cyanoborohydride ( 100 mg/mL), thus triggering the coupling reaction between the protein and the sugar. After isothermal treatment 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) was added to give 1.8 to 2.2 molar equivalents of sodium borohydride per mole of sugar, and the resulting reaction mixture was treated isothermally with stirring, thereby reducing any untreated sugar present in the sugar. Reactive aldehydes. To the resulting glycoprotein coupling mixture was added 5 mL of 0.9% (w/v) sodium chloride solution to dilute it, and the diluted coupling mixture was dialyzed and filtered using a 100 kDa MWCO membrane.

2-1-2. Cyanolation

Sodium chloride powder was added to the undiluted solution of the polysaccharide of serotype 9V prepared without the hydrolysis process to prepare a 2M NaCl polysaccharide solution. To activate the polysaccharide, a CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) solution was added to the serotype 9V polysaccharide solution so that the concentration was 0.5% (w/ After w), the polysaccharide activation reaction was induced by stirring for 15 minutes. After adding sodium hydroxide solution to the resulting reacted mixture and increasing the pH to 9.5±0.1, it was stirred for 3 minutes so that the hydroxyl groups of the polysaccharide were fully activated by CDAP. CRM197 was added to the polysaccharide passing through the polysaccharide activation process so that the ratio of CRM197 to polysaccharide was 1.0% (w/w) (CRM197 weight/polysaccharide weight), whereby coupling reaction was performed at room temperature for 1 hour.

The coupling reaction was accomplished by adding 3 molar equivalents of 2M glycine solution to 1 molar equivalent of CDAP, adjusting the pH to 9.0 and incubating it overnight. The completed conjugate was concentrated in an ultrafiltration filter and diafiltered through a buffer solution containing 0.9% (w/w) sodium chloride.

As a result, it was confirmed that the yield of the conjugate prepared by the cyanation method was more than 4 times that of the conjugate prepared by the reductive amination method. Therefore, the inventors of the present invention prepared conjugates using capsular polysaccharide and CRM197 by using the cyanation method.

2-2. Conjugation and purification of the conjugate

The preparation of the conjugate of the capsular polysaccharide of Streptococcus pneumoniae and CRM197 was carried out by the following process steps.

Step 1. Solubilization and hydrolysis of capsular polysaccharide

Capsular polysaccharide powder derived from each serotype was dissolved in water for injection so that the final concentration range was within the range mentioned below, and filtered through a 0.45 μm filter:

1) in the case of serotypes 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/ml,

3) serotypes 6A, 12F and 19A are in the range of 8 to 12 mg/ml, and

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

The process of isothermal treatment of the solution was carried out within the pH and temperature ranges mentioned below by serotype:

1) In the case of serotypes 1, 2, 4, 5, 6B, 7F, 14 and 23F, overnight at 70 to 80°C,

2) In the case of serotypes 6A and 19F, 0.5 to 4 hours at 70 to 80°C,

3) In the case of serotypes 3, 9V, 12F and 18C, the isothermal treatment process is carried out using phosphoric acid solution at pH 2.0 and 65 to 80°C for 0.5 to 4 hours,

4) In the case of serotypes 15B, 19A, no hydrolysis is performed.

Then, it was cooled to 21°C to 24°C and sodium hydroxide was added to the target pH, thereby stopping the hydrolysis.

Step 2. Coupling reaction process of capsular polysaccharide and CRM197

Sodium chloride powder was added to all serotypes, thereby preparing 2M NaCl polysaccharide solutions. Dissolve CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) appropriate for each serotype at a ratio of 100 mg CDAP per 1 ml of 1/1 acetonitrile/water for injection (v/v) solution , the solution was added in the amounts mentioned below by serotype. describe in detail,

Dissolve it in the following proportions

1) In the case of serotypes 6A, 9V and 14, CDAP is 1 to 1.5 (w/w) compared to polysaccharide,

2) In the case of serotypes 2 and 4, CDAP was 2 (w/w) compared to polysaccharides,

3) In the case of serotypes 1, 3, 7F, 15B, 19F and 19A, CDAP was 3 (w/w) compared to polysaccharide,

4) In the case of serotypes 5, 6B, 18C, 23F, CDAP is 4 (w/w) compared to polysaccharide, and

5) In the case of serotype 12F, CDAP is 5 (w/w) compared to polysaccharide,

and added to each polysaccharide solution.

Subsequently, sodium hydroxide solution was added and the pH was raised to 9.5, and then it was stirred for 3 to 7 minutes so that the hydroxyl groups of the polysaccharide were fully activated by CDAP. CRM197 was added to the polysaccharide solution of each serotype in an amount of 0.75 (w/w) of CRM197 compared to the polysaccharide, whereby a coupling reaction was performed for 2 hours. Then, the conversion of the reaction was measured using SE-HPLC, and further CDAP was added if necessary.

Step 3. Termination of Coupling Reaction

For all serotypes, the reaction was terminated by adding 3 to 6 molar equivalents of glycine solution compared to 1 molar equivalent of CDAP added and adjusting the pH to 9.0. After stirring the coupling solution at 21°C to 24°C for 1 hour, it was stored overnight at a low temperature of 2 to 8°C.

Step 4. Ultrafiltration

The diluted coupling mixture was concentrated in an ultrafiltration filter and diafiltered with a minimum of 20 volumes of buffer solution (5 mM succinate buffer solution (pH 5.8) containing 150 mM NaCl). Here, a buffer solution maintaining a pH range of 5.5 to 6.5 and containing 0.9% (w/w) sodium chloride was used as the buffer solution. Ultrafiltration filters with a fragment molecular weight of 300 kDa were used for all serotypes and the dialysis solution was disrupted.

Step 5. Sterile Filtration

Dilute the solution remaining after diafiltration with a buffer solution (5 mM succinate buffer solution (pH 5.8) containing 150 mM NaCl) to a concentration of polysaccharide content below 0.4 g/L, and then pass it through a 0.22 μm filter filter. The filtered product was controlled during preparation (sugar content, residual DMAP). The filtered residual solution is controlled during preparation to determine if additional concentration, diafiltration and/or dilution is required.

Step 6. Adsorption

Aluminum salt (aluminum phosphate) was added to the sterile-filtered solution to a final concentration of 1 mg/mL as aluminum ion and adsorbed, with additional salt added to maintain a pH range of 5.5 to 6.5. Quality suitability was confirmed by quality checking the undiluted solution after adsorption was complete, and it was refrigerated at 2 to 8°C until use.

Example 1. Preparation of multivalent pneumococcal conjugate vaccine

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

(1) 13-valent 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.

Conjugate bulk solution requirements are calculated based on the batch volume and the polysaccharide concentration of the conjugate undiluted solution. Same as step 6 of Preparation Example 2, after adding aluminum phosphate to the conjugate bulk solution of each serotype, add 0.85% (w/w) saline solution and 5mM succinate buffer solution (pH 5.8), thus Bulk solutions of each conjugate were prepared containing 0.85% sodium chloride, 5 mM succinate buffer solution (pH 5.8), and 1 mg/mL aluminum (concentration of aluminum in aluminum phosphate). Finally, as preservatives, thimerosal (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 170ug/ml) and slowly mixed as the above-mentioned combination of serotypes, thereby preparing a formulated multivalent pneumococcal conjugate vaccine composition. At this time, the concentration of capsular polysaccharide of each serotype of the multivalent vaccine composition was 4.4 μg/mL (only serotype 6B was 8.8 μg/mL). Check the pH and adjust to pH 5.8 if necessary. The prepared undiluted solution of the formulated final vaccine composition was filled into Type 1 borosilicate glass vials. The bottled vaccine composition was stored at 2 to 8°C.

Example 2: Estimation of Immunogenicity of Multivalent Pneumococcal Conjugate Vaccines

Choose to contain 2.2 μg of each capsular polysaccharide (4.4 μg for serotype 6B capsular polysaccharide only), about 29.3 μg CRM197 carrier protein, 0.5 mg elemental aluminum (2 mg aluminum phosphate) adjuvant, about 4.25 mg sodium chloride, about 295 μg succinate buffer solution, about 3 mg 2-phenoxyethanol and about 60 μg formaldehyde polyvalent pneumococcal vaccine composition (designated as LBVE013) as in the vaccine composition prepared in Example 1 13-valent (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) vaccine to confirm whether it has the ability to induce an immune response in rabbits. This immunogenicity was confirmed by antigen-specific ELISA in the case of serotype IgG concentration and by opsonized phagocytosis assay (OPA) in the case of antibody function.

The vaccine composition LBVE013 or used as positive control group The planned human clinical doses (2.2 μg of each polysaccharide, with the exception of 4.4 μg for 6B) were immunized in the muscle of New Zealand white rabbits at weeks 0, 2 and 4, and at 2 weeks post-vaccination Serum was collected at intervals. IgG measurements obtained for the collected sera using ELISA are shown in Table 1. The detailed description is as follows.

2-1. Measurement of serotype-specific IgG concentration

A total of 13 capsular polysaccharides of each serotype were treated in an amount of 5 µg/well on a 96-well plate, whereby it was coated at room temperature for 16 hours. In order to minimize the non-specific antigen-antibody reaction of the sera, the sera of each individual were collected in the same amount, thereby pooling the sera of the same group. After reacting the serum pool with 333.3 μg/mL C-PS (cell wall polysaccharide; SSI (staten Serum Institute)) and 333.3 μg/mL capsular polysaccharide of serotype 22F (PnPs22F) at room temperature for 30 minutes to adsorb it , which is diluted with a buffer solution for antibody dilution containing Tween 20 at an appropriate dilution ratio (about 1:100 to 1:40000). The coated plate was washed 4 times with a washing buffer solution, and 50 μl of the absorbed and diluted serum was placed in the coated well plate, which was then reacted at room temperature for 1 hour. The reacted plate wells were washed 4 times by the same method, and goat anti-rabbit IgG antibody-HRP conjugate (1:20000) was placed in each well, and then it was reacted at room temperature for 30 minutes. The plate was washed 4 times by the same method, and 100 μl of stabilized TMB substrate solution (3,3',5,5'-tetramethylbenzidine; Sigma, St.Louis, MO, USA) was incubated at room temperature It was placed in each well, and then it was reacted at room temperature for 15 minutes. After stopping the reaction by placing 100 µl of 1N sulfuric acid solution, absorbance at 450 nm was measured using 650 nm as a reference wavelength. In order to objectively estimate the immunogenicity, the same method was used to analyze the Collected blood samples.

The results are disclosed in Table 1 below.

[Table 1]

As shown in Table 1, it was confirmed that the immunogenic response pattern according to the serotype of the multivalent (13-valent) pneumococcal vaccine composition (LBVE013) was definitely different from that of mode. and In contrast, in rabbits inoculated with the 13-valent vaccine composition (LBVE013) of this example, higher threshold levels were shown in all serotypes, especially serotypes 1, 6B, 7F, 9V, 14 and 19F showed Compare The effect is 2 to 6 times higher.

2-2. Functional immunogenicity confirmation test (opsonocyte phagocytosis assay, OPA)

The function of antibodies induced by serotypes was assessed by OPA analysis of sera obtained from rabbits.

Specifically, the same amount of sera was collected from each individual, thereby pooling the sera of the same group. Each serotype of Streptococcus pneumoniae was cultured in THY medium (Todd-Hewitt broth, containing 2w% yeast extract), and diluted to 200 to 300 CFU/10 μl using conditioning buffer. 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 differentiated HL-60 cells and complement (cell:complement=4:1) was added, and reacted in CO ₂ medium (37° C.) for 45 minutes.

Cell phagocytosis was stopped by lowering the temperature, and 10 μl of the reaction solution was spread on the agar medium that had been dried for 30 to 60 minutes. Then, it was cultured in a CO ₂ medium (37° C.) for 12 to 18 hours, and the number of colonies was counted. The OPA threshold is expressed as the dilution rate at which 50% mortality is observed.

The results obtained are shown in Table 2 below:

[Table 2]

In Table 2, for example, the OPA titer expressed as 2187 is a case where the 50% level was not reached even in the most dilute fraction compared with the negative control group, and it means that the threshold was significantly high. Through the above test results, it has been confirmed that the multivalent pneumococcal vaccine composition of the present embodiment is compatible with Compared with significantly better serum IgG threshold. Therefore, the multivalent pneumococcal vaccine composition of this example can be very usefully used to prevent diseases caused by pneumococcus.

Reference example: Procedures and standards for vaccine bactericidal ability test

The bactericidal ability test of the vaccine was carried out according to the EP-B standard, which is the standard required by the World Health Organization (WHO) of the United States Pharmacopoeia (USO) and European Pharmacopoeia (EP) for vaccine products in this example.

If the content of the test is specifically outlined, two bacteria, Pseudomonas aeruginosa (ATCC No.9027, PA), Staphylococcus aureus (ATCC No.6538, SA), yeast albino 10 ⁵ to 10 ⁶ CFU/mL (CFU; colony forming unit) of four pathogens including Candida albicans (ATCC No.10231, CA) and fungus Aspergillus niger (ATCC No.16404, AN) The vaccine composition was inoculated at 0 hours respectively. Colony numbers were counted at 3 to 5 days by taking samples at 24 hours, 7 days, 14 days and 28 days and culturing them on solid medium.

For the results according to the above method, EP-B and the standard of bactericidal ability of each country's pharmacopoeia are shown in Table 3 below.

[table 3]

*NR: not recycled

**NI: not increased

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

In this example, in order to develop a polyvalent pneumococcal polysaccharide-protein conjugate vaccine, thimerosal and 2-PE, commonly used vaccine preservatives in this field, were added to thereby perform a bactericidal ability test, and it was confirmed that thimerosal alone Or in the case of low concentrations of 2-PE, the criteria for the bactericidal ability were not met.

Also, since other companies have filed patent applications for the case of using high-concentration 2-PE (7mg/mL or more), it cannot be used in our company.

Therefore, as a result of ongoing experiments, the inventors of the present invention obtained the following results to develop a bactericidal effect that can minimize the content of 2-PE and increase the bactericidal effect so as to meet the bactericidal ability standard without violating other companies' standards. patented novel composition as a preservative for the multivalent pneumococcal protein conjugate vaccine.

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

In two kinds of bacteria, Pseudomonas aeruginosa (ATCC No.9027, PA), Staphylococcus aureus (ATCC No.6538, SA), yeast Candida albicans (ATCC No.10231, CA) and the fungus Aspergillus niger (Aspergillus niger) (ATCC No.16404, AN) were four kinds of pathogens, the well-known Staphylococcus aureus (Staphylococcusaureus) (ATCC No. .6538, SA) as bacteria for screening bactericidal ability. The purpose of conducting the experiment was to select the combination of 2-PE and formaldehyde satisfying EP-B by estimating the bactericidal ability using samples taken at 24 hours. Therefore, the test was carried out according to the test method of EP-B.

Specifically, by referring to the preparation method of the multivalent pneumococcal polysaccharide-protein conjugate vaccine in Example 1, the multivalent vaccine compositions 1 to 12 were prepared by adding thimerosal or 2-PE and/or formaldehyde according to Table 2. pneumococcal protein conjugate vaccine composition, and 10 ⁵ to 10 ⁶ CFU/mL (CFU = colony forming unit) Staphylococcus aureus (Staphylococcus aureus) (ATCC No.6538, SA) bacteria were inoculated into these preparations. The number of colonies was counted at 3 to 5 days by taking samples at 0 hours and 24 hours and culturing on a solid medium, thereby calculating the logarithm (Log) of colony reduction. The results are shown in Table 4 below.

[Table 4]

As can be seen in Table 4, the bactericidal ability of SA from the combination of 2-PE 5.0 mg/mL and formaldehyde 100 μg/mL passed the EP-B standard. On the basis of the results, it was confirmed that the combination of about 5.0 mg/mL 2-PE and more than 100 μg/mL formaldehyde is suitable as a preservative for multivalent pneumococcal polysaccharide-protein conjugate vaccines.

Example 4: Bactericidal ability test of multivalent pneumococcal polysaccharide-protein conjugate vaccine 1

The bactericidal test is carried out according to the test method of EP-B. Two kinds of bacteria, Pseudomonas aeruginosa (ATCC No.9027, PA), Staphylococcus aureus (ATCC No.6538, SA), and yeast Candida albicans (ATCC No. No.10231, CA) and the fungus Aspergillus niger (Aspergillus niger) (ATCC No.16404, AN) total of 10 ⁵ to 10 ⁶ CFU/mL (CFU; colony forming unit) were inoculated into the vaccine combination at 0 Multivalent pneumococcal protein conjugate vaccine formulations of objects 13 to 15. The log reduction of colonies was calculated by taking samples at 0 hours, 24 hours, 7 days and 28 days and culturing them on solid medium, counting the number of colonies at 3 to 5 days. Fungi and yeast were sampled at 0 hours, 14 days and 28 days and cultured on solid media, and then the number of colonies was counted at 3 to 5 days, thereby calculating the logarithm of colony reduction. The results are shown in Table 5.

[table 5]

*NR: not recycled

**NI: not increased

As shown in Table 5, 2 kinds of bacteria P. Pseudomonas aeruginosa (ATCC No.9027, PA), Staphylococcus aureus (ATCC No.6538, SA), yeast Candida albicans (ATCC No.10231, CA) and The results of the bactericidal ability of four pathogens of the fungus Aspergillus niger (ATCC No.16404, AN), regardless of the serotype, in all 13-valent, 15-valent A and 15-valent B against 4 kinds of bacteria Bacterial ability meets EP-B standard. From the results, it was confirmed that in the case where the preservative of the polyvalent pneumococcal polysaccharide-protein conjugate vaccine contained about 5 mg/mL 2-PE and about 100 μg/mL formaldehyde, the bactericidal ability of the EP-B standard was satisfied.

In this field, pneumococcal protein conjugate vaccines generally use 7 mg/mL or more of 2-PE as a preservative, but the results together with the results of Example 3 show that the addition of formaldehyde in an amount of 100 μg/mL or more In some cases, the content of 2-PE is as low as 5mg/mL, and the bactericidal ability of EP-B can also be achieved. Therefore, in the present invention it is possible to provide a multi-dose composition in which an optimized combination of 2-phenoxyethanol (2-PE) and formaldehyde (HCHO) is used as a preservative for the polyvalent pneumococcal polysaccharide-protein conjugate vaccine agent, and maintained a strong and effective bactericidal ability.

Example 5: Stability of multivalent pneumococcal polysaccharide-protein conjugate vaccine composition

Based on the results of Example 4, a new composition of multivalent pneumococcal polysaccharide-protein conjugate vaccine composition 16 was prepared. Specifically, the vaccine composition 16 was prepared by the following method: placing 14 kinds of polysaccharide-protein conjugates (serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F) and 1 mg/mL aluminum phosphate (as aluminum concentration) and stir to mix well. The sterile-filtered buffer was mixed by dissolving 5 mM succinate and 0.85% (w/v) sodium chloride in distilled water for injection. After final homogenization by addition of 2-PE at a concentration of 6.0 mg/mL and formaldehyde at a concentration of 120 μg/mL, the final pH was adjusted to 5.8. The concentration of each serotype in the prepared vaccine composition was also 4.4 μg/mL (8.8 μg/mL only for 6B).

As a result of testing the content of preservatives after storing the prepared vaccine composition 16 at 2 to 8° C. for 6 months, it was confirmed that the content recoveries of 2-PE and formaldehyde were stably maintained in the range of 90 to 110% Inside, no loss of preservatives.

In summary, it was confirmed that the combination of 2-PE and formaldehyde described in the examples of the present invention is an excellent composition for effectively maintaining the bactericidal activity of the multivalent pneumococcal polysaccharide-protein conjugate vaccine for a long time, and it It can be usefully used to provide multi-dose compositions in which the bactericidal ability of the multivalent polysaccharide-protein conjugate vaccine is increased or the length of time for which the bactericidal ability is maintained is prolonged.

From the above description, those skilled in the art should understand that the present invention can be embodied in other specific forms without departing from its technical spirit or essential attributes. In this regard, the above-described embodiments should be considered in all respects as illustrative and not restrictive. The scope of the invention should be indicated by the appended claims rather than the above description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

<110> LG Chem, Ltd. (LG CHEM, LTD.)

<120> Multivalent Streptococcus pneumoniae vaccine composition

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<151> 2017-03-15

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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 Val 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 Asp Ser Ile Gly Val

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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)

1. A multivalent vaccine composition against pneumococcus comprising: (i) capsular polysaccharide-carrier protein conjugate, (ii) more than 4 mg/mL and less than 7 mg/mL of 2-phenoxyethanol (2-PE), and (iii) 90 μg/mL to 200 μg/mL formaldehyde (HCHO), Wherein the capsular polysaccharide comprises 13 to 24 capsular polysaccharides derived from serotypes of Streptococcus pneumoniae. 2. The multivalent vaccine composition against pneumococcus according to claim 1, wherein said capsular polysaccharide comprises 13 to 24 capsular polysaccharides selected from the group consisting of: derived from 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. 3. The multivalent vaccine composition against pneumococcus according to claim 2, wherein said capsular polysaccharide comprises Thirteen 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 Seventeen 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. 4. The multivalent vaccine composition against pneumococcus according to claim 1, wherein said carrier protein is CRM197 protein. 5. The multivalent vaccine composition against pneumococcus according to claim 1, wherein said capsular polysaccharide-carrier protein conjugate has a compound wherein said capsular polysaccharide and carrier protein are passed through -O-C(NH) using a cyanation method Structures linked by -NH- groups. 6. The multivalent vaccine composition against pneumococcus according to claim 5, wherein the cyanylation method is carried out using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr. 7. The multivalent vaccine composition against pneumococcus of claim 1, comprising: Based on 1 part by weight of capsular polysaccharide derived from serotype 1, 0.9 to 1.1 parts by weight of each capsular polysaccharide derived from serotypes 2, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 23F, 12F, and 15B, and 1.8 to 2.2 parts by weight of capsular polysaccharide derived from serotype 6B. 8. The multivalent vaccine composition against pneumococcus according to any one of claims 1 to 7 for multiple administration. 9. A pharmaceutical composition for preventing or treating pneumococcal infection or pneumococcal infection disease, comprising the multivalent vaccine composition against pneumococcus according to any one of claims 1 to 7. 10. The pharmaceutical composition of claim 9, wherein the pneumococcal infectious disease is pneumonia. 11. A method for preparing a multivalent vaccine composition against pneumococcus, the method comprising: (1) preparing the capsular polysaccharide-protein conjugate of Streptococcus pneumoniae, and (2) mixing the capsular polysaccharide-protein conjugate with 2-phenoxyethanol (2-PE) and formaldehyde (HCHO), wherein said capsular polysaccharide comprises 13 to 24 capsular polysaccharides derived from Streptococcus pneumoniae serotypes, and 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 μg/mL to 200 μg/mL. 12. The method for preparing a multivalent vaccine composition against pneumococcus according to claim 11, wherein the step (1) of preparing a capsular polysaccharide-protein conjugate comprises performing a cyanation method to combine the capsular polysaccharide and Proteins go through the -O-C(NH)-NH-linkage step.