KR102101879B1 - Multivalent vaccine composition against streptococcus - Google Patents

Abstract

Pneumococcal ( Streptococcus pneumoniae ) vaccine composition, more specifically, (i) capsular polysaccharide (capsular polysaccharide)-protein conjugate, (ii) 2-phenoxyethanol (2-Phenoxyethanol, 2- PE), and (iii) a vaccine composition comprising formaldehyde (HCHO) and a method of manufacturing the same are provided.

Description

Multivalent pneumococcal vaccine composition {MULTIVALENT VACCINE COMPOSITION AGAINST STREPTOCOCCUS}

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

Streptococcus pneumonia (Pneumococcus) is a gram-positive and hemolytic streptococci that is a major causative agent of meningitis, pneumonia and severe invasive infectious diseases in infants, children and the elderly worldwide. More than 1.6 million people die each year from pneumococcal disease (2008, International Health Organization data). In particular, the incidence of invasive infectious diseases caused by pneumococcal disease in children under 5 years of age and elderly people over 65 years of age is low. high.

Streptococcus pneumoniae is classified into over 90 serotypes according to the structural and immunological characteristics of the capsular polysaccharide, which is the main pathogenic factor surrounding the outer (cell membrane). It is said to be associated with 80 to 90% of pathogenicity. The only host of pneumococci is humans, and they usually exist in colonies in the nasopharynx of healthy normal people (20-40% in infants, 5-10% in adults). In 2005, the Centers for Disease Control and Prevention (CDC) reported that approximately 2.1 million children under the age of 5 died from pneumonia annually in developing countries, and 1.2 million of them died from pneumococcal infection. It has been reported, and in the United States, it has been reported that about 30,000 and 50,000 cases of meningitis and sepsis caused by pneumococcus occur annually (Peters TR, Poehling KA et al. Invasive pneumococcaldisease. JAMA 2007; 297: 1825-6). In addition, pneumoACTION, a database of pneumococcal disease, showed that 24,047 cases of pneumococcal infections occurred in children in Korea in 2000, of which 47 were killed (www.pneumoadip.org). Furthermore, according to a recent study on the analysis of pneumococcal serotypes in children and adolescents by the Korea Centers for Disease Control and Prevention, pneumococcus is the most common (43.7%) causative agent of invasive infection in infants between 3 months and 59 months. appear. In pneumococci causing invasive infectious diseases worldwide, multi-drug resistant bacteria that are resistant to not only penicillin, but also three or more agents are increasing, further increasing the difficulty of treating pneumococcal infectious diseases.

Therefore, there is a continuous need to inoculate pneumococcal vaccines for children and the elderly who are at high risk for pneumococcal infection.

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

On the other hand, vaccine dose injections should use preservatives to prevent contamination of microorganisms. For multi-dose vaccine products exported to underdeveloped countries through the United Nations, multi-dose products including preservatives are preferred due to the environment, distribution method, and cost of the country of use. Preservatives used in vaccine products include thimerosal, phenoxyethanol (2-PE), phenol (Phenol), and the amount of preservatives commonly used in the art. For example, chimerosal is used at a concentration of 10 μg / mL, 2-PE is used at a concentration of 5 mg / mL, and multi-dose vaccine products including this are EP-B (European Pharmacopoeia B category) or USP (US Pharmacopeia). It can be commercialized by passing the standard antiseptic test.

Chimerosal (Thimerosal, Thiomersal, merthiolate) is an ethyl mercury derivative compound that has been used as a preservative for multi-dose vaccine injections since the early 1930s. Chimerosal has been used for the purpose of preventing the growth of contaminated microorganisms and maintaining aseptic conditions when storing or using vaccine products, and a number of 5-valent liquid mixed vaccines (D, T, P, Hib) that have obtained WHO PQ (Prequalified) , HBsAg) contains chimerosal as a preservative.

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

However, the amount in which these preservative components are used to sufficiently achieve the antiseptic power of the vaccine may cause toxicity and / or side effects, and thus it is necessary to develop a technique that provides sufficient antiseptic power while reducing their use.

An example is (i) the capsular polysaccharide-protein conjugate of Streptococcus pneumoniae , (ii) 2-Phenoxyethanol (2-PE), and (iii) formaldehyde. It provides a pneumococcal vaccine composition comprising (Formaldehyde, HCHO). The vaccine composition may be a multiple dose vaccine composition for multiple doses.

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

Another example is preparing a capsular polysaccharide-protein conjugate of Streptococcus pneumoniae , and the capsular polysaccharide-protein conjugate, and 2-phenoxyethanol (2-PE). And a method for preparing a pneumococcal vaccine composition having improved stability and / or preservative power (or antiseptic power) or a pneumococcal vaccine stability and / or preservative power, comprising mixing formaldehyde (HCHO). ) Provide a method of enhancement. The vaccine composition may be a multiple dose vaccine composition for multiple doses. The preparing of the capsular polysaccharide-protein conjugate may include connecting the capsular polysaccharide and the carrier protein through -OC (NH) -NH- by performing a cyanylation method.

An example is (i) the capsular polysaccharide-protein conjugate of Streptococcus pneumoniae , (ii) 2-phenoxyethanol (2-PE), and (iii) formaldehyde, HCHO It provides a pneumococcal vaccine composition or pneumococcal immunogenic composition comprising.

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

As described herein, “comprising (comprising a predetermined component)” may mean comprising or consisting essentially of a component other than the component described. .

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

The capsular polysaccharide may be a capsular polysaccharide derived from Streptococcus pneumoniae . Specifically, the capsular polysaccharide is derived from two or more of Streptococcus pneumoniae serotypes, such as 5 or more, 7 or more, 9 or more, 11 or more, 13 or more, or 14 or more serotypes It can be a capsular polysaccharide. In one example, the capsular polysaccharide, among the Streptococcus pneumoniae serotype, 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 serotype-derived capsular polysaccharides. For example, the capsular polysaccharide is Streptococcus pneumoniae serotype 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 selected from the group consisting of 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19 Species, 14 to 17, or 14 to 15 serotype-derived capsular polysaccharides. In one embodiment, the capsular polysaccharide is a streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 13Fs derived from 23F Polysaccharides; 14 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F; 15 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 12F; And / or 15 capsular polysaccharides derived from Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 15B; And / or 17 stenosis from Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F Polysaccharides may be included, but are not limited thereto.

In the capsular polysaccharide-protein conjugate, two or more serotype-derived capsular polysaccharides described above may be individually conjugated to each other through a protein and a conventional method, such as covalent bonding. In one example, the conjugate has a structure in which each capsular polysaccharide (specifically, the hydroxyl group of the polysaccharide) and the protein (specifically, the amino group of the protein) are linked through -OC (NH) -NH- using a cyanation method. ([Polysaccharide] -OC (NH) -NH- [protein]). In the case of conjugating the protein with the capsular polysaccharide using the cyanylation method as described above, compared to other methods (for example, a reduction amination method, etc.), the degree of structural modification of the capsular polysaccharide is low, and thus immunogenicity is maintained even after conjugation. It may be more advantageous. For example, when a certain serotype (19F, etc.) is conjugated with a protein by a reductive amination method, a structure in which a hexasaccharide ring structure is cleaved and opened may occur depending on the reaction combination, which may lead to a decrease in immunogenicity. On the other hand, there are reports that this problem is unlikely to occur when conjugated with a protein by the 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 a sufficient amount and purity. The threatening polysaccharides from various serotypes can each be conjugated with the same or different proteins, such as the same protein.

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

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

Other carrier proteins that can be used

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

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

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

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

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

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

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

In one embodiment, the pneumococcal vaccine composition is

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

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

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

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

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

Can be

Multivalent pneumococcal vaccine compositions provided herein, such as 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to The pneumococcal vaccines of 19, 14 to 17, or 14 to 15 have pneumococcal vaccines that have significantly better potency than conventional multivalent vaccines (e.g., 13-valent vaccines), and prevent and / or prevent pneumococcal infection disease. Very good effect can be expected for treatment.

The pneumococcal vaccine composition may be a composition formulated as a single dose for multiple doses or a multiple dose (multiple doses) for multiple doses. As used herein, “multidose” refers to a vaccine dose that may be administered (inoculated) more than once (eg, 2 or more) to a single dose (inoculation) subject or once or 1 to a dose of 2 or more (inoculation) individuals. It may mean a formulation unit comprising a vaccine dose that can be administered (inoculated) more than once (eg, two or more times).

The pneumococcal vaccine composition contains preservatives in addition to pneumococcal capsular polysaccharide-protein conjugates of two or more serotypes. Preservatives that can be used in the vaccine composition (e.g., injectable formulations) include 2-phenoxyethanol, formaldehyde, chlorobutanol, m-cresol, methylparaben, propylparaben, benzethonium chloride, benzalkonium chloride, benzoic acid, and benzyl alcohol , Phenol, chimerosal, phenyl mercury nitrate, or the like, or one or more selected from the group consisting of mercury nitrate.

In one example, the preservative, the multivalent pneumococcal vaccine composition described above, for example, 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 is a form optimized for the pneumococcal vaccine composition, and may include 2-phenoxyethanol and formaldehyde. In this way, by including 2-phenoxyethanol as a preservative together with formaldehyde, the amount of 2-phenoxyethanol is reduced, thereby reducing toxicity and / or side effects, while achieving an improved antiseptic effect by synergism of the two components. can do.

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

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

The vaccine composition is about 3 months or more, about 6 months or more, about 1 year or more, about 1.5 years or more under normal storage conditions, for example, at a temperature of 2 ° C to 8 ° C, 20 ° C to 25 ° C, or about 37 ° C It is characterized by being stable for about 2 years or more, or about 2.5 years or more. The stability of the vaccine composition herein refers to that the vaccine composition maintains its original antigenicity (immunogenicity) at an equal level, and / or each component is maintained without degradation or loss, and / or infection of bacteria, viruses, etc. It can mean none.

Another example provides a pharmaceutical composition for the prevention or treatment of pneumococcal infection or pneumococcal infection disease comprising the multivalent pneumococcal vaccine composition described above. Another example is the prevention of pneumococcal infection or pneumococcal infection disease comprising administering a pharmaceutically effective amount of the multivalent pneumococcal vaccine composition described above to an individual in need thereof for the prevention or treatment of pneumococcal infection or pneumococcal infection disease. Provide a method of treatment. The pneumococcal vaccine composition contained in the pharmaceutical composition or the pneumococcal vaccine composition used in the prevention or treatment method may be a single dose pharmaceutical composition for single administration or a multiple dose pharmaceutical composition for multiple administration.

The pneumococcal infection disease refers to all diseases caused by pneumococcal infection, and may be pneumonia, otitis media, sinusitis, bacteremia, and the like. "Pneumonia" is a type of acute inflammatory disease of the lung parenchyma. The infectious agents 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 appear, and sputum is often a blood clot. Complications that can cause pleurisy, meningitis, endocarditis, peritonitis, etc. ( Diagn.Microbiol.Infect.Dis., 2001, 39: 181-185).

The term "pneumococcus" in the present invention refers to Streptococcus pneumoniae and is a commensal organism that generally colonizes the mucosal surface of the human nasopharynx. If the host's factor allows access to the organism's lower respiratory tract, a vigorous inflammatory reaction follows, which causes dense consolidation when the alveolar space fills the exudate, resulting in pneumonia. Can cause 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. Accordingly, when a vaccine preparation is prepared using pneumococcal polysaccharide, the immune response may be different depending on the type of the capsular polysaccharide, that is, the serotype of pneumococcal pneumococcus derived.

Since the capsular polysaccharide is recognized as an antigen when administered in the body and is capable of producing an antibody against it, a vaccine composition for prevention of pneumococcus (or pneumococcal infection disease) can be prepared using this. The term "antigen" in the present invention refers to a substance capable of specifically inducing an immune response when a substance invades the body. In one embodiment Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C , 19A, 19F, 20, 22F, 23F, and 13F to 13F derived capsular polysaccharides can each act as antigens.

Another example provides a method for preparing a pneumococcal vaccine composition with improved stability and / or preservative (or antiseptic) or a method for enhancing the stability and / or preservative (or preservative) of a pneumococcal vaccine. The above method

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

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

It may include.

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

In the above 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 reduced in size through hydrolysis to reduce viscosity and induce effective immunogenicity.

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

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

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

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

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

It may include.

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

In a specific embodiment of the present invention, Streptococcus pneumoniae having 13 or 15 different serotypes were each dissolved using sodium deoxycholate and freed of polysaccharides bound to cells. Polysaccharides derived from 21 serotypes 1, 2, 3, 4, 5, 6A, 6B, 9V, 8, 9N, 10A, 11A, 12F, 15B, 17F, 18C, 19A, 19F, 20, 22F, and 23F In the case of ionic bonding with CTAB (cetyltrimethylammonium bromide), the CTAB process was performed to purify, and three serotypes 7F, 14, and 33F that did not react with CTAB were purified using aluminum phosphate gel (Algel) solution. The reaction with the CTAB is, for example, in an amount of 0.5 to 5% by weight or 1 to 3% by weight of CTAB in the reaction, for example, 1 to 20% (w / v) or 5 to 15% (w / v). In the case of polysaccharides derived from 23F, 2 to 3% by weight, and in the case of polysaccharides derived from serotypes other than 23F, 1 to 2% by weight) may be added, but is not limited thereto. After the reaction with the CTAB, after centrifugation, any one or more of pellet recovery, resuspension of the pellet using a sodium chloride solution (eg, about 100 to 500 mM), and removal of CTAB ions using sodium iodide may be additionally performed. However, it is not limited thereto. The aluminum phosphate gel reaction may be performed by adding the aluminum phosphate gel solution to the reactants in an amount of 1 to 20% by weight or 5 to 15% by weight, but is not limited thereto.

In the case of using only the capsular polysaccharide as a vaccine composition, infants and children whose immune system is less than an adult may not recognize it as an antigen, and thus an immune response may not occur. In the present invention, a conjugate form of a carrier protein and a capsular polysaccharide is combined. It was prepared and used.

The "carrier protein" refers to a protein capable of increasing the immunogenicity of a polysaccharide antigen by being covalently conjugated with a capsular polysaccharide, and its specific type is as described above. CRM197 can be used in one embodiment. The carrier protein may be conjugated to the stenosis polysaccharide through a standard conjugation method, and the stenosis polysaccharide-carrying protein conjugate formed therethrough may be one or more stenosis polysaccharide conjugated to one carrier protein.

All known methods for preparing a conjugate of a capsular polysaccharide and a carrier protein can be included in the scope of the present invention, wherein the conjugate is a cyanide polysaccharide and a carrier protein linked by a -OC (NH) -NH- group It has a structure. The cyanylation method may be appropriately performed by a person skilled in the art through a known method, and may be performed using, for example, CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr, but is not limited thereto.

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

The obtained capsular polysaccharide-carrying protein conjugate can be purified by various methods. Examples of these methods include concentration / dialysis filtration processes, column chromatography and multilayer filtration. Purified polysaccharide-protein conjugates can be formulated by mixing each with the vaccine composition of the present invention. Formulation of the vaccine composition of the present invention can be performed using methods recognized in the art. For example, 13 individual capsular polysaccharide-carrying protein conjugates can be formulated with a physiologically acceptable vehicle to produce a composition. Examples of such vehicles may include, but are not limited to, water, buffered saline, water for injection, polyols (eg glycerol, propylene glycol, liquid polyethylene glycol) or dextrose solutions.

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

In the present invention, the term "vaccine" refers to an immunogen or antigenic substance that is immune to a living body by administering it to a human or animal for the prevention of infectious diseases as a biological agent containing an antigen that immunizes the living body.

The vaccine composition may further include one or more selected from the group consisting of adjuvants, preservatives, buffers, cryoprotectants, salts, divalent cations, nonionic detergents and free radical oxidation inhibitors.

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

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

(2) Oil-in-water emulsion formulations (with or without other specific immune stimulants such as muramil peptide (defined below) or bacterial cell wall components), e.g. (a) MF59 (No. WO90 / 14837) : Contains 5% (w / v) squalene, 0.5% (w / v) Tween 80 and 0.5% (w / v) span 85 (optionally containing various amounts of MTP-PE Can further contain), formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidizer, Microfluidics, Newton, MA), (b) SAF: 10% (w / v ) Squalene, contains 0.4% (w / v) Tween 80, 5% (w / v) pluronic-block polymers L121 and thr-MDP (see below), microfluidized with submicron emulsion ( microfluidization), or vortexing to form large particle size emulsions, and (c) Ribi ™ Adjuvant System (RAS) (Corixa, Hamilton, MT): 2% (w / v) squalene, 0.2% ( w / v) consisting of Tween 80 and 3-O-deacylated monophosphoryl lipid A (MPL ™) (Corixa), trehalose dimycholate (TDM) and cell wall framework (CWS) as described in U.S. Patent No. 4,912,094 Contains one or more bacterial cell wall components from the group, preferably MPL + CWS (Detox ™);

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

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

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

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

(7) Wild-type cholera toxin (CT) or, for example, a mutated cholera toxin (GW) in which glutamic acid at amino acid position 29 is substituted with another amino acid, specifically histidine, according to WO-2000 / 18434 -2002/098368 and WO-2002 / 098369), pertussis toxin (PT), or E. coli heat-labile toxin (LT), especially LT-K63, LT-R72, CT-S109, PTK9 / G129 (WO -93/13302 and WO-92 / 19265) detoxified mutants of bacterial ADP-ribosylating toxins; And

(8) Complement component Complement like C3d trimer.

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

The aluminum salt adjuvant may be an aluminum-precipitated vaccine or an aluminum-adsorbed vaccine. Aluminum salts include hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, alhydrogels, superfos, amphogels, aluminum hydroxide, aluminum hydroxyphosphate sulfate (APA), Amorphous alumina and the like may be included, but is 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 hydroxyphosphate sulfate precipitates. Using a high shear mixer, the size of the precipitate is 2 to 8 μm, then dialyzed with physiological saline. It can be prepared by sterilization. In one embodiment, the protein is adsorbed using commercially available Al (OH) 3 (eg Alhydrogel or Superfos). 50-200 g of protein can be adsorbed per mg of aluminum hydroxide, and this ratio is dependent on the pI of the protein and the pH of the solvent. Proteins with low pI bind more strongly than proteins with high pI. Aluminum salts can form antigenic reservoirs that slowly release antigens for 2-3 weeks, non-specifically activating macrophage, complement, and innate immune mechanisms.

The term "preservative (or preservative)" in the present invention refers to a substance having an anti-viral and / or antibacterial action that inhibits the growth of microorganisms in the vaccine composition, for example, thimerosal, phenoxyethanol ( 2-phenoxyethanol), formaldehyde, or a mixture thereof, but is not limited thereto, and any conventional preservative used in the art may be used.

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

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

In addition, the vaccine composition of the present invention may include a nonionic detergent. For example, polysorbate 20 and polysorbate 80 among polyoxyethylene sorbitan esters (commonly called Tweens); Copolymers of ethylene oxide (EO), propylene oxide (PO), butylene oxide (BO) (eg DOWFAXTM); Ethoxynols having different repeat numbers of the ethoxy (oxy-1,2-ethanediyl) group, in particular, osthoxynol-9 (Triton-100); Ethylphenoxypolyethoxyethanol (IGEPAL CA-630 / NP-40); Phospholipids such as lecithin; Nonylphenol ethoxylates such as NP series; Polyoxyethylene fatty acid ethers derived from lauryl, cetyl, stearyl, oleyl alcohol (Brij surfactants), in particular triethylene glycol monolauryl ether (Brij 30); Sorbitan ethers known as SPANs, in particular surfactants such as sorbitan trioleate (Span 85) and sorbitan monolaurate, but are not limited thereto. Tween 80 can be included in emulsions, and mixtures of nonionic detergents such as Tween 80 / Span 85 can also be used. A combination of a polyoxyethylene sorbitan ester such as Tween 80 and octocinol such as Triton X-100 is also suitable, and a combination of Laureth 9 and Tween, or octocinol is also useful. Specifically, polyoxyethylene sorbitan ester (eg, Tween 80) is 0.01% (w / v) to 1% (w / v), particularly 0.1% (w / v); Octylphenoxy polyoxyethanol or nonylphenoxy polyoxyethanol (e.g. Triton X-100) is 0.001% (w / v) to 0.1% (w / v), especially 0.005% (w / v) to 0.02% ( w / v); Polyoxyethylene ethers (e.g. laureth 9) are 0.1% (w / v) to 20% (w / v), preferably 0.1% (w / v) to 10% (w / v), especially 0.1% (w / v) to 1% (w / v) or about 0.5% (w / v).

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

As used herein, “multi-dose dose” or “multi-dose” refers to a vaccine dose that may be administered more than once (inoculation) to a single dose (inoculation) individual, or one or more doses to two or more administration (inoculation) individuals. It may mean a vaccine dose that can be administered (inoculated) more than once.

Physiologically acceptable carriers used in liquid formulations are aqueous or non-aqueous solvents, suspensions, emulsions and oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, ethyl oleate. Aqueous carriers include water, alcohol / aqueous solvents, emulsions or suspensions, physiological saline, buffer solutions. Examples of oils are vegetable or animal oils, peanut oil, soybean oil, olive oil, sunflower oil, synthetic oils such as liver oil and marine oil, and lipids 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 is basically isotonic, but is not limited thereto. On the other hand, isotonic or hypertonic properties may be beneficial for storage of the composition. If the vaccine composition is hypertonic, it can be diluted to isotonic prior to administration. Isotonic agents for dilution may be ionic isotonic agents such as salts or nonionic isotonic agents such as carbohydrates. Ionic isotonic agents include, but are not limited to, sodium chloride, calcium chloride, potassium chloride, magnesium chloride, and the like. Nonionic isotonic agents include, but are not limited to, sorbitol, glycerol, and the like.

The amount of the conjugate in each vaccine dose can be selected as an amount that induces an immune protective response without significant side effects, and this amount can vary depending on the serotype of pneumococcus. Specifically, the vaccine composition, compared to the weight of the capsular polysaccharide derived from serotype 1 (i.e., 1 part by weight), serotype 2, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A , 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F derived capsular polysaccharides in a weight ratio of 0.8 to 1.2 or 0.9 to 1.1, respectively (i.e., 0.8 to 1.2 parts by weight or 0.9 to 1.1 parts by weight) Parts by weight), and in the case of a capsular polysaccharide derived from serotype 6B, a weight ratio of 1.6 to 2.4, 1.8 to 2.2, or 1.9 to 2.1 (that is, 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) Part), but is not limited thereto.

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

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

The optimal amount of ingredients for a particular vaccine can be confirmed by standard studies involving observation of a suitable immune response in the subject. For example, the results of animal experiments can be extrapolated to determine the vaccination dose in humans. In addition, those skilled in the art can empirically determine the dose as needed.

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 an individual susceptible to pneumococcal disease and prevent pneumococcal disease by administering a pharmaceutically effective amount by a systemic or mucosal route. The term "prevention" of the present invention refers to all actions to suppress or delay infection by the pneumococcus by administration of the vaccine composition of the present invention. “Pharmaceutically effective amount” as defined in the present invention refers to a dosage required to induce antibodies to a degree capable of significantly reducing the probability or severity of infection with pneumococcus. The term "administration" of the present invention refers to the introduction of a given substance into a subject in any suitable way. The vaccine composition of the present invention may be administered by oral, nasal, rectal, transdermal or aerosol inhalation routes, and may be administered by bolus or slowly injected, but is not limited thereto. The administration may include intramuscular, intraperitoneal, intradermal or subcutaneous routes of injection; Or mucosal administration to the oral / digestive tract, airway tract, or genitourinary tract. As an embodiment, intranasal administration may be used for the treatment of pneumonia or otitis media, in which case it is possible to more effectively prevent the nasopharyngeal carrier of pneumococci, thereby weakening the infection at an early stage.

The "object" to which the vaccine composition or pharmaceutical composition of the present invention is administered may mean a living organism to which a pathogen may be infected or a cell, tissue, or culture separated therefrom, and the organism to be a higher vertebrate. It may be, 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 inoculation, or may be administered twice, three times, four times or more at appropriate intervals, but is not limited thereto. For example, routine immunization plans for infants and infants for invasive diseases caused by Streptococcus pneumoniae may be 2, 4, 6 and 12 to 15 months of age.

In addition, the composition may further include at least one protein derived from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for inclusion may include all of the proteins identified in WO-2002 / 083855 as well as the proteins described in WO-2002 / 053761.

In a specific embodiment of the present invention, in the total 0.5 mL of the vaccine composition, 2.2 μg of each polysaccharide, except that 6B-derived polysaccharide was 4.4 μg; About 29.3 μg of CRM197 transport protein; 0.5 mg aluminum element (2 mg aluminum phosphate) adjuvant; Sodium chloride about 4.25 mg (without preservative) or about 3.5 mg (with preservative); About 295 μg of succinate buffer; 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 A, 14 to 19, 14 to 17 or 14 to 15 pneumococcal vaccine compositions are exemplified.

In another specific embodiment of the present invention, in the serum of rabbits inoculated with the vaccine composition, serotype specific IgG concentrations higher than Privena 13® were confirmed (Table 1). In addition, it was confirmed that the functional immunogenicity test for this (Opsonophagocytic assay) shows a better effect than Privena 13® (Table 2). Thus, it can be seen that the vaccine composition of the present invention has a very good effect for the prevention of pneumococcal disease.

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

Capsular polysaccharide-carrying protein conjugates of the present invention 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17 Or, the composition comprising 14 to 15 species is 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 It contains a capsular polysaccharide derived from Streptococcus pneumoniae having 17 or 17 or 15 different serotypes, and when administered to the body, it can be recognized as an antigen to produce antibodies against it. As it causes an immune response, it can be used as an immunogenic composition against pneumococcus.

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

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

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

Another aspect of the present invention is a Streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 13F and 13F isolated membranes derived from It is a method for producing the immunogenic composition, comprising the step of conjugating each of the capsular polysaccharides to a structure in which the capsular polysaccharide and the transport protein CRM197 are linked by -OC (NH) -NH- groups using a cyanylation method.

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

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

Hereinafter, the present invention will be described in more detail by 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: Preparation of 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) It was obtained from the Center for Disease Control and Prevention (CDC) of the United States, and a cell bank was prepared by the following method.

Streptococcus pneumoniae strains were smeared on blood agar medium to identify pneumococcus and remove existing media components. After selecting a single colony with good growth among 10 or more single colonies, inoculate and incubate in a liquid medium (Soytone (Kerry Bio-Science) or Yeast extract (Bio-springer) derived medium) that does not contain animal-derived ingredients and grow. After the preparation, synthetic glycerol was added to prepare a research cell bank (RCB) containing synthetic glycerol.

A master cell bank was prepared by adding a synthetic glycerol after proliferating the cells in a liquid medium containing no animal-derived components by taking one vial out of the research cell bank for which polysaccharide expression with unique serotype was confirmed. After taking out one vial from the bank, cells were grown in a liquid medium containing no animal-derived ingredients, and then synthetic glycerol was added to prepare a cell bank for production.

The prepared cell bank was stored in an ultra-freezing state of -70 ° C or lower and used in the following test.

1-2. Fermentation and polysaccharide separation

One vial of the production cell bank was thawed and inoculated into a liquid medium containing no animal-derived ingredients. The seed culture was carried out at 37 ± 2 ° C. in an unstirred state until a constant cell density (Optical Density, OD 600) was reached. After confirming whether the culture of the culture in which the seed culture was completed was contaminated, it was inoculated into a fermenter containing a liquid medium (Soytone (Kerry Bio-Science) or Yeast extract (Bio-springer) derived medium) that does not contain animal-derived ingredients (OD600 = 2.5 ± 0.2).

Then, this 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 minimal stirring. Sampling was started from 2 hours after initiation of culture to measure the concentration of cells in the culture medium and the concentration of glucose contained in the medium. The culture was terminated when glucose in the medium was depleted.

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

1-3. Purification of the capsular polysaccharide

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

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

1) For 14 serotypes of serotypes 1, 2, 3, 4, 5, 6A, 6B, 9V, 12F, 15B, 18C, 19A, 19F, and 23F, ion binding with CTAB (cetyltrimethylammonium bromide) is possible, The CTAB process was performed, and specifically, 10% by weight of the CTAB solution was treated with an amount of 1.5% by weight (other than 23F) or 2.5% (for 23F) relative to the weight of the process solution, and reacted for 1 hour ± 10 minutes. Then, the pellet was recovered by centrifugation at 17,000 xG for 1 hour. Thereafter, to treat sodium chloride (NaCl) and sodium iodide (NaI), 200-300 mM sodium chloride (NaCl) solution was used to resuspension the pellets recovered through the CTAB treatment, and iodization corresponding to 50% by weight of CTAB usage CTAB ions were removed using sodium (NaI). However, in the case of serotype 3, a 350 mM NaCl solution was used. After treatment with sodium chloride and sodium iodide, the supernatant was recovered through centrifugation and used in a subsequent process. 2) In the case of two serotypes of serotypes 7F and 14 that do not react with CTAB, aluminum phosphate gel (Algel) solution was added to react, followed by treatment with an aluminum phosphate gel solution of 10% by weight compared to the total process solution 1 Time reaction. The supernatant obtained through centrifugation (1centrifugation at 17,000 xG conditions) was recovered and used in a subsequent process.

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

Manufacturing example  2: Streptococcus New Monae  Preparation of capsular polysaccharide-protein conjugates

2-1: reductive Amination method Cyanylated  Bonding method comparison

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) Then, the bonding yields of the two bonding methods were compared. The specific bonding process is as follows.

2-1-1. Reductive Amination method

11.7 mg of sodium periodate was added to the serotype 9V polysaccharide stock solution to activate polysaccharide while stirring at 21 to 25 ° C. After the oxidized polysaccharide was concentrated and dialyzed using a 100 KDa ultrafiltration filter and water for injection (WFI), the remaining residual polysaccharide was lyophilized by mixing CRM197 protein with saccharide / CRM197 = 0.5 (weight ratio). The lyophilized complex was thawed and stabilized (equilibrated) at 21-25 ° C. After the equilibrated complex is dissolved by incubating (37 ± 2 ° C) in sodium phosphate (Na ₃ PO ₄ ) buffer solution at a rate of 0.1 M per 20 g of saccharide, cyanoborohydride (100 mg / mL) is added and protein and The conjugation reaction between sugars was initiated. 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) was added so that sodium borohydride 1.8 to 2.2 molar equivalents per mole of saccharide was added, and the resulting reaction mixture was incubated with stirring at 23 ± 2 ° C. Any unreacted aldehyde was reduced. The obtained saccharide-protein conjugate mixture was diluted by adding 5 mL of 0.9% (w / v) aqueous sodium chloride solution, and the diluted conjugate mixture was dialyzed and filtered using a 100 kDa MWCO membrane.

2-1-2. Cyanylation method

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

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

As a result, it was confirmed that the yield of the conjugate produced by the cyanylation method was 4 times or more compared to the conjugate produced by the reductive amination method. Therefore, the present inventors prepared a conjugate using a cyanide method with capsular polysaccharide and CRM197.

2-2. Conjugation and purification of conjugates

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

Step 1. Dissolution and hydrolysis of capsular polysaccharide

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

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

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

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

4) For serotypes 3, 7F, 14, 15B and 23F, ranges from 2 to 4 mg / ml.

For each serotype, the process of incubating the solution at the pH and temperature ranges described below was performed:

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

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

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

4) For serotypes 15B and 19A, hydrolysis was not performed.

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

Step 2. Capsular polysaccharide and CRM197  Junction reaction process

A sodium chloride powder was added to all serotypes to prepare a 2M NaCl polysaccharide solution. For each serum, the appropriate solution of CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) dissolved in a ratio of 100 mg of CDAP per 1 ml of 1/1 acetonitrile / injectable water (v / v) solution is used in the amount described below for each serotype. Was added. In detail

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

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

3) CDAP 3 (w / w) compared to polysaccharides for serotypes 1, 3, 7F, 15B, 19F and 19A, 4) CDAP 4 (w / w) compared to polysaccharides for serotypes 5, 6B, 18C, 23F, and

5) For serotype 12F, it was dissolved at a ratio of CDAP 5 (w / w) to polysaccharide and added to each polysaccharide solution.

Subsequently, the sodium hydroxide solution was added to raise the pH to 9.5, and then stirred for 3 to 7 minutes so that the hydroxyl group of the polysaccharide was sufficiently activated by CDAP. CRM197 was added to each serotype polysaccharide solution in an amount of CRM197 0.75 (w / w) compared to polysaccharide, and a conjugation reaction was performed for 2 hours. Thereafter, the reaction conversion rate was measured using SE-HPLC, and CDAP was additionally added as necessary.

Step 3. Terminating the conjugation reaction

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

Step 4. Ultrafiltration

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

Step 5. Sanitization  percolation

The residual liquid after dialysis filtration was diluted with a buffer (5 mM Succinate buffer solution containing 150 mM NaCl (pH 5.8)) to less than 0.4 g / L based on the polysaccharide content concentration and filtered through a 0.22 µm filter. The filtered product was subjected to control (sugar content, residual DMAP) during the manufacturing process. The filtered residue was subjected to control during the manufacturing process to determine if additional concentration, dialysis filtration and / or dilution was required.

Step 6. Adsorption

The aluminum salt (aluminum phosphate) was added to the antibacterial filter solution so that the final concentration was 1 mg / mL based on aluminum ions and adsorbed, and an extra salt was added to maintain a pH range of 5.5 to 6.5. After the adsorption is completed, the quality of the stock solution is verified by performing a quality inspection, and stored at 2 to 8 ° C. until refrigeration.

Example  1. Formulation of multivalent pneumococcal conjugate vaccine

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

(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.

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

Example  2: Evaluation of the immunogenicity of the multivalent pneumococcal conjugate vaccine

Among the vaccine compositions prepared in Example 1, 13 valent (serum type 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) vaccine, total vaccine formulation In 0.5 mL, 2.2 μg of each capsular polysaccharide (only 4.4 μg of serotype 6B capsular polysaccharide); About 29.3 μg of CRM197 transport protein; 0.5 mg aluminum element (2 mg aluminum phosphate) adjuvant; Sodium chloride about 4.25 mg; About 295 μg of succinate buffer; A multivalent pneumococcal vaccine composition (referred to as LBVE013) containing about 3 mg of 2-phenoxyethanol and about 60 μg of formaldehyde was selected to evaluate whether it has the ability to induce an immune response in rabbits. This immunogenicity was confirmed by antigen-specific ELISA in the case of serum IgG concentration, and through opsonophagocytic assay (OPA) for antibody function.

New Zealand White at Week 0, Week 2, and Week 4 at the planned human clinical dose (2.2 μg each polysaccharide, 4.4 μg 6B each) of Prevenar13® used as the vaccine composition LBVE013 or positive control. ) Rabbits were immunized into the muscle and serum was collected at 2 weeks intervals after inoculation. The results of IgG measurement using ELISA for the collected serum are shown in Table 1. The details are as follows.

2-1. Serotype specific IgG  Concentration measurement

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

The results are shown in Table 1 below.

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

As shown in Table 1, it can be confirmed that the immunogenic response pattern for each serotype of the multivalent (13-valent) pneumococcal vaccine composition (LBVE013) is significantly different from that of Privena 13®. In the rabbits inoculated with the 13-valent vaccine composition (LBVE013) according to the present embodiment, higher levels of titers were observed in all serotypes compared to Privena13®, especially serotypes 1, 6B, 7F, and 9V. At 14 and 19F, it showed 2 to 6 times better effect than Privena 13®.

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

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

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

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

The results obtained above 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 titer is very high even when the most diluted section does not reach the 50% level compared to the negative control. Through the test results, it was confirmed that the multivalent pneumococcal vaccine composition according to the present embodiment has a remarkably excellent serum IgG titer compared to Privena 13®. Therefore, the multivalent pneumococcal vaccine composition according to the present embodiment can be very usefully used to prevent diseases caused by pneumococcus.

Reference example : vaccine Antiseptic  Test protocol and standards

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

Specifically, two types of bacteria Pseudomonas aeruginosa (ATCC No. 9027, PA), Staphylococcus aureus (ATCC No. 6538, SA), yeast Candida albicans (ATCC No. 10231, CA), and four fungi of the fungus Aspergillus niger (ATCC No. 16404, AN) were each added to the vaccine composition. At the time, 10 ⁵ to 10 ⁶ CFU / mL (CFU; Colony forming units) were inoculated. Then, samples were sampled on the 24th, 7th, 14th, and 28th days, and cultured in a solid medium to count colonies on the 3rd to 5th days.

The antiseptic power standards of EP-B and pharmacopoeia of each country are shown in Table 3 below.

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

* NR: No Recovery ** NI: No Increase

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

 In this example, in the development of a multivalent pneumococcal polysaccharide-protein conjugated vaccine, antiseptic testing was performed by adding chimerosal and 2-PE, a vaccine preservative commonly used in the art, and chimerosal or low concentration of 2-PE. When used alone, it was confirmed that the criterion for preservation was not satisfied.

In addition, when using high concentration of 2-PE (7mg / mL or more), the company had already applied for a patent from a third party and could not use it.

Accordingly, the inventors of the present invention experimented to develop a new composition capable of increasing the antiseptic effect while minimizing the content of 2-PE in order to satisfy the antiseptic power standards without infringement of other companies' patents as a preservative for the multivalent pneumococcal protein conjugate vaccine. As a result of proceeding, the following results were obtained.

Example  3: Multivalent pneumococcal polysaccharide-protein conjugate vaccine Antiseptic  Screening

Bacterial species 2 Pseudomonas aeruginosa (ATCC No. 9027, PA), Staphylococcus aureus (ATCC No. 6538, SA), Yeast Candida albicans (ATCC No. 10231, CA), among the four fungi of the fungus Aspergillus niger (ATCC No. 16404, AN), Staphylococcus aureus (ATCC No. 6538, SA), which is known to not die well in 2-PE, for antiseptic screening It was selected as a fungus. The purpose was to select a 2-PE and formaldehyde combination that satisfies EP-B by evaluating the antiseptic force with a sample sampled at 24 hours. Accordingly, the test was conducted according to the test method of EP-B.

Specifically, referring to the preparation method of the polyvalent pneumococcal polysaccharide-protein conjugate vaccine of Example 1, as shown in Table 2 below, chimerosal, or 2-PE and / or formaldehyde is added to form the polyvalent pneumococcal protein conjugate 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 hours. Then, samples were sampled at 0 hours 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 colonies. The results are shown in Table 4 below.

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

As can be seen from Table 4, the antiseptic 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 2-PE about 5.0 mg / mL and formaldehyde 100 μg / mL or more is suitable as a preservative for the multivalent pneumococcal polysaccharide-protein conjugate vaccine.

Example  4: Multivalent pneumococcal polysaccharide-protein conjugate vaccine Antiseptic  Test 1

The antiseptic force test was conducted according to the test method of EP-B. Two bacterial Pseudomonas in the multivalent pneumococcal protein conjugate vaccine formulation of vaccine composition 13-15 Four species of aeruginosa (ATCC number 9027, PA), Staphylococcus aureus (ATCC number 6538, SA), yeast Candida albicans (ATCC number 10231, CA), and fungus Aspergillus niger (ATCC number 16404, AN) at 0 hours each Was inoculated with 10 ⁵ to 10 ⁶ CFU / mL (CFU = colony forming unit). After that, 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 colonies. Fungi and yeast were sampled at 0 hours, 14 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 colonies. Table 5 shows the results.

sampling
time Log Reduction (log CFU / mL reduction) Germ Fungi and yeast P.A. S.A. EP-B standard C.A. A.N. EP-B standard Vaccine composition 13:
13th
(2-PE 5.1 mg / mL + Formaldehyde 102 μg / 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 28 days NR NR NI NR NR NI Vaccine composition 14:
15A
(2-PE 5.1 mg / mL + formaldehyde 102 μg / mL) 24 hours NR 2.1 1 or more - - - 7 days NR NR 3 or more - - - 14 days - - - NR NR 1 or more 28 days NR NR NI NR NR NI Vaccine composition 15: 15 B
(2-PE 5.1 mg / mL + formaldehyde 102 μg / mL) 24 hours NR 1.9 1 or more - - - 7 days NR NR 3 or more - - - 14 days - - - NR NR 1 or more 28 days NR NR NI NR NR NI

* NR: No Recovery ** NI: No Increase

As shown in Table 5, in the multivalent pneumococcal polysaccharide-protein conjugated vaccine compositions 13 to 15, including each serotype of 13, 15, and 15, B, two bacterial Pseudomonas aeruginosa (ATCC No. 9027, PA) , Staphylococcus aureus (ATCC No. 6538, SA), yeast Candida As a result of confirming the antiseptic ability against 4 fungi of albicans (ATCC No. 10231, CA) and fungus Aspergillus niger (ATCC No. 16404, AN), 4 in all 13, 15, and 15-valent B regardless of serotype. The preservative power of the species bacteria met the EP-B standard. As a result of this, it was confirmed that the preservative of the multivalent pneumococcal polysaccharide-protein conjugated vaccine satisfies the preservative power of EP-B when 2-PE is contained in an amount of about 5 mg / mL and formaldehyde in an amount of about 100 μg / mL. .

In the art, pneumococcal protein-conjugated vaccine typically uses 2-PE 7 mg / mL or more as a preservative. In addition to the results of Example 3, when the result of adding formaldehyde in an amount of 100 μg / mL or more, 2 -It shows that even if the PE content is lowered to 5 mg / mL, the antiseptic power of EP-B can be achieved. Thus, in the present specification, a strong and effective antiseptic power is applied by applying an optimized combination of 2-phenoxyethanol (2-PE) and formaldehyde (HCHO) as a preservative for a multivalent pneumococcal polysaccharide-protein conjugate vaccine. It became possible to provide a multiple dose composition to maintain.

Example  5: Stability of the polyvalent pneumococcal polysaccharide-protein conjugated vaccine composition

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

As a result of testing the content of the preservative after storing the prepared vaccine composition 16 at 2 to 8 ° C for 6 months, the recovery rate of 2-PE and formaldehyde was maintained in a stable range without loss of the preservative in the range of 90 to 110%. Confirmed.

In summary, the combination of 2-PE and formaldehyde exemplified in the Examples of the present invention is an excellent composition capable of effectively maintaining the antiseptic power of a multivalent pneumococcal polysaccharide-protein conjugate vaccine for a long period of time, and multivalent polysaccharide-protein conjugate It has been confirmed that the antiseptic power of the vaccine is improved or can be usefully used to provide a multi-dose composition having an extended antiseptic power retention period.

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

<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 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             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) capsular polysaccharide-carrying protein conjugate,
(ii) 2-Phenoxyethanol (2-PE) 4 mg / mL or more and less than 7 mg / mL, and
(iii) 90 to 200 μg / mL of formaldehyde (HCHO),
The capsular polysaccharide is a multivalent pneumococcal vaccine composition comprising 13 to 24 types of Streptococcus pneumoniae serotype-derived capsular polysaccharides. The method of claim 1, wherein the capsular polysaccharide is Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F. , 18C, 19A, 19F, 20F, 22F, 23F, and 33F, comprising 13 to 24 selected from capsular polysaccharides. The method of claim 2, wherein the capsular polysaccharide
13 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F;
14 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F;
15 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 12F; 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 17 capsular polysaccharides from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F
Multivalent pneumococcal vaccine composition comprising a. The multivalent pneumococcal vaccine composition of claim 1, wherein the carrier protein is CRM197 protein. The multivalent pneumococcal vaccine composition according to claim 1, wherein the capsular polysaccharide-carrying protein conjugate has a structure in which the capsular polysaccharide and the transport protein are linked by -O-C (NH) -NH- groups using a cyanylation method. The multivalent pneumococcal vaccine composition according to claim 5, wherein the cyanylation method is performed using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr. According to claim 1, with respect to 1 part by weight of the capsular polysaccharide derived from serotype 1,
The capsular polysaccharides derived from serotypes 2, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 23F, 12F and 15B are 0.9 to 1.1 parts by weight, and
1.8 to 2.2 parts by weight of the capsular polysaccharide from serotype 6B
Multivalent pneumococcal vaccine composition comprising a. The multi-dose multivalent pneumococcal vaccine composition according to any one of claims 1 to 7. A pharmaceutical composition for the prevention or treatment of pneumococcal infection or pneumococcal infection disease comprising the multivalent pneumococcal vaccine composition of any one of claims 1 to 7. The pharmaceutical composition of claim 9, wherein the pneumococcal infection disease is pneumonia. (1) preparing a capsular polysaccharide-protein conjugate of Streptococcus pneumoniae , and
(2) mixing the capsular polysaccharide-protein conjugate with 2-phenoxyethanol (2-PE) and formaldehyde (HCHO)
Including,
The capsular polysaccharides include 13 to 24 types of Streptococcus pneumoniae serotype-derived capsular polysaccharides,
The amount of the 2-phenoxyethanol is 4 mg / mL or more and less than 7 mg / mL, and the amount of formaldehyde is 90 to 200 μg / mL,
Method for preparing a multivalent pneumococcal vaccine composition. 12. The method of claim 11, wherein the step of preparing the (1) capsular polysaccharide-protein conjugate comprises a step of connecting a capsular polysaccharide and a protein through -OC (NH) -NH- by performing a cyanation method. The method of producing a multivalent pneumococcal vaccine composition.